List of Suggested Reviewers or Reviewers Not To Include (optional)

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1 List of Suggested Reviewers or Reviewers Not To Include (optional) SUGGESTED REVIEWERS: Kumar Sudesh, Universiti Sains Malysia, expertise on PHA production. David L. Kaplan, Tufts University, expertise on biomaterials. Brian F. Pfleger, University of Wisconsin, Madison, expertise in bioengineering Matthew P. DeLisa, Cornell University, expertise in bioengineering, REVIEWERS NOT TO INCLUDE: Guoqiang Chen, Tsinghua University, Direct competitor Sang Yup Lee, KAIST, Direct competitor

2 COVER SHEET FOR PROPOSAL TO THE NATIONAL SCIENCE FOUNDATION PROGRAM ANNOUNCEMENT/SOLICITATION NO./CLOSING DATE/if not in response to a program announcement/solicitation enter NSF 11-1 PD /18/12 FOR CONSIDERATION BY NSF ORGANIZATION UNIT(S) (Indicate the most specific unit known, i.e. program, division, etc.) CBET - Cellular & Biochem Engineering FOR NSF USE ONLY NSF PROPOSAL NUMBER DATE RECEIVED NUMBER OF COPIES DIVISION ASSIGNED FUND CODE DUNS# (Data Universal Numbering System) FILE LOCATION 9/17/ CBET /3/217 9:4am S EMPLOYER IDENTIFICATION NUMBER (EIN) OR TAXPAYER IDENTIFICATION NUMBER (TIN) NAME OF ORGANIZATION TO WHICH AWARD SHOULD BE MADE SUNY College of Environmental Science and Forestry AWARDEE ORGANIZATION CODE (IF KNOWN) NAME OF PRIMARY PLACE OF PERF SUNY-ESF SHOW PREVIOUS AWARD NO. IF THIS IS A RENEWAL AN ACCOMPLISHMENT-BASED RENEWAL IS THIS PROPOSAL BEING SUBMITTED TO ANOTHER FEDERAL AGENCY? YES NO IF YES, LIST ACRONYM(S) ADDRESS OF AWARDEE ORGANIZATION, INCLUDING 9 DIGIT ZIP CODE SUNY College of Environmental Science and Forestry PO Box 9 Albany, NY ADDRESS OF PRIMARY PLACE OF PERF, INCLUDING 9 DIGIT ZIP CODE SUNY-ESF 1 Forestry Dr. Syracuse,NY, ,US. IS AWARDEE ORGANIZATION (Check All That Apply) SMALL BUSINESS MINORITY BUSINESS (See GPG II.C For Definitions) FOR-PROFIT ORGANIZATION WOMAN-OWNED BUSINESS TITLE OF PROPOSED PROJECT SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics REQUESTED AMOUNT $ 483,89 PROPOSED DURATION (1-6 MONTHS) 36 months REQUESTED STARTING DATE 6/1/13 IF THIS IS A PRELIMINARY PROPOSAL THEN CHECK HERE SHOW RELATED PRELIMINARY PROPOSAL NO. IF APPLICABLE CHECK APPROPRIATE BOX(ES) IF THIS PROPOSAL INCLUDES ANY OF THE ITEMS LISTED BELOW BEGINNING INVESTIGATOR (GPG I.G.2) HUMAN SUBJECTS (GPG II.D.7) Human Subjects Assurance Number DISCLOSURE OF LOBBYING ACTIVITIES (GPG II.C.1.e) Exemption Subsection or IRB App. Date PROPRIETARY & PRIVILEGED INFORMATION (GPG I.D, II.C.1.d) INTERNATIONAL COOPERATIVE ACTIVITIES: COUNTRY/COUNTRIES INVOLVED HISTORIC PLACES (GPG II.C.2.j) (GPG II.C.2.j) EAGER* (GPG II.D.2) RAPID** (GPG II.D.1) VERTEBRATE ANIMALS (GPG II.D.6) IACUC App. Date HIGH RESOLUTION GRAPHICS/OTHER GRAPHICS WHERE EXACT COLOR PHS Animal Welfare Assurance Number REPRESENTATION IS REQUIRED FOR PROPER INTERPRETATION (GPG I.G.1) PI/PD DEPARTMENT PI/PD POSTAL ADDRESS Chemistry PI/PD FAX NUMBER Syracuse, NY 1321 United States NAMES (TYPED) High Degree Yr of Degree Telephone Number Electronic Mail Address PI/PD NAME Christopher Nomura PhD ctnomura@esf.edu CO-PI/PD CO-PI/PD CO-PI/PD CO-PI/PD Page 1 of 2

3 CERTIFICATION PAGE Certification for Authorized Organizational Representative or Individual Applicant: By signing and submitting this proposal, the Authorized Organizational Representative or Individual Applicant is: (1) certifying that statements made herein are true and complete to the best of his/her knowledge; and (2) agreeing to accept the obligation to comply with NSF award terms and conditions if an award is made as a result of this application. Further, the applicant is hereby providing certifications regarding debarment and suspension, drug-free workplace, lobbying activities (see below), responsible conduct of research, nondiscrimination, and flood hazard insurance (when applicable) as set forth in the NSF Proposal & Award Policies & Procedures Guide, Part I: the Grant Proposal Guide (GPG) (NSF 11-1). Willful provision of false information in this application and its supporting documents or in reports required under an ensuing award is a criminal offense (U. S. Code, Title 18, Section 11). Conflict of Interest Certification In addition, if the applicant institution employs more than fifty persons, by electronically signing the NSF Proposal Cover Sheet, the Authorized Organizational Representative of the applicant institution is certifying that the institution has implemented a written and enforced conflict of interest policy that is consistent with the provisions of the NSF Proposal & Award Policies & Procedures Guide, Part II, Award & Administration Guide (AAG) Chapter IV.A; that to the best of his/her knowledge, all financial disclosures required by that conflict of interest policy have been made; and that all identified conflicts of interest will have been satisfactorily managed, reduced or eliminated prior to the institution s expenditure of any funds under the award, in accordance with the institution s conflict of interest policy. Conflicts which cannot be satisfactorily managed, reduced or eliminated must be disclosed to NSF. 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Submission of this certification is a prerequisite for making or entering into this transaction imposed by section 1352, Title 31, U.S. Code. Any person who fails to file the required certification shall be subject to a civil penalty of not less than $1, and not more than $1, for each such failure. Certification Regarding Nondiscrimination By electronically signing the NSF Proposal Cover Sheet, the Authorized Organizational Representative is providing the Certification Regarding Nondiscrimination contained in Exhibit II-6 of the Grant Proposal Guide. 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By electronically signing the NSF Proposal Cover Sheet, the Authorized Organizational Representative or Individual Applicant located in FEMA-designated special flood hazard areas is certifying that adequate flood insurance has been or will be obtained in the following situations: (1) for NSF grants for the construction of a building or facility, regardless of the dollar amount of the grant; and (2) for other NSF Grants when more than $25, has been budgeted in the proposal for repair, alteration or improvement (construction) of a building or facility. Certification Regarding Responsible Conduct of Research (RCR) (This certification is not applicable to proposals for conferences, symposia, and workshops.) By electronically signing the NSF Proposal Cover Sheet, the Authorized Organizational Representative of the applicant institution is certifying that, in accordance with the NSF Proposal & Award Policies & Procedures Guide, Part II, Award & Administration Guide (AAG) Chapter IV.B., the institution has a plan in place to provide appropriate training and oversight in the responsible and ethical conduct of research to undergraduates, graduate students and postdoctoral researchers who will be supported by NSF to conduct research. The undersigned shall require that the language of this certification be included in any award documents for all subawards at all tiers. AUTHORIZED ORGANIZATIONAL REPRESENTATIVE SIGNATURE DATE NAME William J Nicholson Electronic Signature Sep :36AM TELEPHONE NUMBER fm127rrs-7 * EAGER - EArly-concept Grants for Exploratory Research ** RAPID - Grants for Rapid Response Research ELECTRONIC MAIL ADDRESS wjnichol@esf.edu Page 2 of 2 FAX NUMBER

4 Project Summary Plastics are ubiquitous in modern society. However, modern uses of plastic materials present two major problems: (i) The major precursors for the chemical synthesis of plastics are petroleum products, which are non-renewable resources and (ii) petroleum-based plastics are inherently non-biodegradable, presenting a litany of environmental problems post disposal. An attractive alternative is the biosynthesis of biodegradable materials from renewable carbon feedstocks (for example, sugars and fatty acids). Although there are many types of biobased materials available, such as polysaccharides (starches, cellulose, etc.), these materials do not possess the physical or chemical properties that would allow them to substitute for common petroleum-based plastics such as polyethylene or polypropylene. Thus, alternative approaches must be found in order to produce biobased and biodegradable materials with these desirable material properties. This proposal seeks to take the bacterial production of polyhydroxyalkanoate (PHA)-based biodegradable plastics to unprecedented heights via engineering synthetic pathways for producing novel polymers in Escherichia coli. Intellectual Merit of the proposed work. Polyhydroxyalkanoates (PHAs) are microbially produced, biobased, and biodegradable polymers that have a wide array of uses ranging from substitutes for nonbiodegradable, petroleum-based plastics in bulk commodity products to biomedical applications. One of the factors that limits the widespread use of PHAs for more specialized applications is the lack of control over repeating unit compositions and by extension the inability to produce polymers with desired physical properties. The long-term goal of this research project is to genetically engineer E. coli for producing expanded classes of PHA materials with desirable material properties. The material properties of PHA polymers are dictated by the composition of their repeating units. PHAs comprised of repeating units of 3-5 carbon atoms are known as short-chain-length PHAs (SCL-PHAs) and tend to be thermoplastic in nature but lack toughness, while PHAs comprised of repeating units of 6-14 carbon atoms are known as medium-chain-length PHAs (MCL-PHAs) and tend to be elastomeric. The material properties of SCL-MCL PHA copolymers depend upon their mol ratio of SCL to MCL repeating units. It has been demonstrated in previous studies that SCL-MCL PHA copolymers that are comprised of 8-95% SCL and 5-2% MCL repeating units have material properties that are very similar to common petroleum-based plastics. To date, we have: (i) engineered a strain of E. coli to produce the highest levels of MCL PHAs from glucose reported and (ii) engineered the first strain of E. coli to produce both SCL and MCL PHA polymers from fatty acids with defined and controllable repeating unit compositions. The objective of this particular application is to further develop these strains so that they can produce SCL- MCL PHA and PHA-co-Polylactic acid (PLA) copolymers with desirable material properties from the renewable sugar feedstocks, glucose, and xylose (specific aim 1) and to produce new PHA-co-PLA copolymers with specific repeating unit compositions from lactic acid and fatty acids (specific aim 2). Although these studies are related, the steps to achieve the goals of these aims are independent from one another and can be accomplished in parallel. Successful completion of these studies will lead to novel polymer production platforms with the potential to overcome the lack of diversity and control over repeating unit compositions of existing bacterial PHA producing systems. Moreover, the proposed studies and research training activities are expected to have a broad impact on society, ranging from improving upon the science of biodegradable and biobased polymer synthesis to the development of new biomaterials for research, industrial, and biomedical applications. The engineered E. coli strains will be key platforms that investigators can apply towards producing new PHA polymers. Broader Impacts of the proposed work. The research associated with this proposal is intrinsically interdisciplinary in nature and will be performed in a diverse training environment. The PI currently mentors a large group of students from several fields including bioprocess engineering, biochemistry, chemistry, and biotechnology. In addition to providing interdisciplinary training and mentoring for graduate and undergraduate students at SUNY-ESF, we will collaborate with the SUNY Onondaga Community College Collegiate Science and Technology and Entry Program (CSTEP) and Louis Stokes Alliance for Minority Participation Program (LSAMP) to provide summer research experiences for underrepresented minority students at the community college level. Additionally, Dr. Nomura will provide seminars as a faculty mentor to CSTEP and LSAMP students with the objective of increasing the number of college graduates from groups that are traditionally underrepresented in science and engineering.

5 TABLE OF CONTENTS For font size and page formatting specifications, see PAPPG section II.B.2. Total No. of Pages Page No.* (Optional)* Cover Sheet for Proposal to the National Science Foundation Project Summary (not to exceed 1 page) 1 Table of Contents 1 Project Description (Including Results from Prior 15 NSF Support) (not to exceed 15 pages) (Exceed only if allowed by a specific program announcement/solicitation or if approved in advance by the appropriate NSF Assistant Director or designee) References Cited 8 Biographical Sketches (Not to exceed 2 pages each) 2 Budget 5 (Plus up to 3 pages of budget justification) Current and Pending Support 2 Facilities, Equipment and Other Resources 1 Special Information/Supplementary Documents 3 (Data Management Plan, Mentoring Plan and Other Supplementary Documents) Appendix (List below. ) (Include only if allowed by a specific program announcement/ solicitation or if approved in advance by the appropriate NSF Assistant Director or designee) Appendix Items: *Proposers may select any numbering mechanism for the proposal. The entire proposal however, must be paginated. Complete both columns only if the proposal is numbered consecutively.

6 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics A. Specific Aims To reduce dependency on non-renewable sources such as fossil fuels for energy, materials production, and transportation, alternative methods that employ renewable resources must be explored. Ubiquitous in modern society, plastics remain the prime example for which the major chemical precursors are derived from non-renewable petroleum sources. In 28, 331 million barrels of petroleum were used for plastic production in the United States, corresponding to 4.6% of total petroleum use (124). In addition to using a non-renewable precursor, only 7.1% of the total plastics produced were recovered via recycling (125), and as a result, most of these plastics made their way into landfills or directly into the environment. Most petroleum-based plastics are not biodegradable and therefore present serious problems once they enter the environment. This has been highlighted by research on large garbage patches in the Pacific gyre, where plastic garbage accumulates in the ocean (95). One way to address the numerous issues resulting from the use of petroleum-based plastics is to use biodegradable plastics produced from renewable resources. The biological synthesis of the biodegradable polyhydroxyalkanoate (PHA) polymers has potential to address both the issues of sustainability and biodegradability. PHAs are stereospecific, bacterially produced polymers that, unlike petroleum-based plastics, can be completely degraded to CO 2 and H 2 O upon disposal. These polymers have a number of uses: as bulk-commodity plastics, in marine environments, and in a variety of high-value medical applications (6, 11, 143). There are two key factors that limit commercialization of PHA polymers: (i) the inability to control the repeating unit composition of PHA polymers to produce materials with desired properties and (ii) limitations in carbon feedstock utilization to produce the required monomers to make polymers with those desired properties. Improved production of PHA copolymers is the primary target of this proposal. The physical properties of PHAs are based on the number of carbon atoms in the individual monomer units incorporated by bacterial enzymes into the polymer chains. Short-chain-length (SCL) PHAs consist of monomers of 3-5 carbons in length, while medium-chain-length (MCL) PHAs consist of monomers of 6-14 carbons in length. Polymers composed of SCL subunits have thermoplastic properties, while polymers composed of MCL subunits have elastomeric properties. It was shown previously that SCL-MCL PHA copolymers comprised of 8-95% SCL repeating units and 5-2% MCL repeating units have material properties similar to polyethylene and polypropylene (1, 75). SCL PHA homopolymers such as poly-3- hydroxybutyrate (PHB) generally display high crystallinity that results in low impact strength and melting temperatures close to the thermal decomposition temperature, whereas SCL-MCL PHA copolymers have lower crystallinity and thus exhibit improved toughness and ductility similar to polyethylene, and melting temperature ranges that are well below the thermal decomposition temperature (74). Despite the physical advantages of SCL-MCL PHAs, the limited availability of these materials has restricted research to explore applications of this class of PHAs. Our studies on metabolic pathways and enzymes for the production of this class of polymers will open new doors in PHA research by increasing the production of SCL-MCL and other novel PHAs. This proposal aims to improve PHA biopolymer production using proven metabolic engineering techniques to diversify the supply of monomers for PHA production and increase overall production of the polymers. We recently engineered two strains of Escherichia coli that point towards the success of our proposed work. The first strain is capable of producing the highest amount of MCL PHA from glucose to date (132). We chose to target this production because (i) previous studies produced extremely limited amounts of MCL PHA from glucose (26, 55, 8, 81), and (ii) the MCL PHA repeating unit is essential for disrupting the crystalline lattice of SCL PHA for improved material properties (1). The second strain is capable of producing PHA polymers with specifically defined repeating unit compositions from fatty acids (116, 117). We chose to target this production because (i) previous studies produced polymers with limited control over the PHA polymer composition (63, 91, 127, 128), and (ii) this system allows for the use of chemically modifiable fatty acids to expand the suite of accessible physical and chemical properties of the PHA polymers beyond what can be produced from sugars (117). In this proposal we will further enhance these E. coli strains to improve production of PHA copolymers with superior material properties from renewable substrates. To achieve this goal, we propose the following specific aims: Aim 1: Produce PHA and PHA-co-polylactic acid (PLA) copolymers from sugars in E. coli. Under this aim, we will extend the synthetic PHA production pathway in E. coli beyond SCL or MCL PHA to include specific copolymers of SCL-MCL PHA and PHA-co-PLA to produce novel, biobased polymers with improved material properties. In addition, we will explore control of the molecular weight of the polymers through the engineering of the ribosomal binding sites (RBSs) and order of gene expression in synthetic operons for genes encoding proteins involved in PHA production. Finally, we will engineer the Christopher T. Nomura (SUNY-ESF) NSF 1

7 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics CRP protein in E. coli in order to alleviate carbon catabolite repression (CCR) and simultaneously consume xylose and glucose for PHA production. Aim 2: Improve production of PHA polymers with defined repeating unit compositions from fatty acids in E. coli. In this aim, we will produce PHA-co-PLA copolymers with defined repeating unit composition from fatty acids and lactic acid by co-expressing the propionyl-coa transferase (PCT) enzyme in our recently engineered E. coli LSBJ strain (115, 117). This strain will be further engineered to improve yields of PHA by deletion of genes (arca and ompr) encoding transcriptional repressors of the oxidation pathway in E. coli to increase substrate availability for bioplastic production. B. Background and Significance B1. Biobased, biodegradable thermoplastics. Finding sustainable biobased and biodegradable replacements for petroleum-based thermoplastics is a global challenge due to diminishing fossil resources, increasing atmospheric CO 2 (23), and increased plastic pollution in the environment (95) Although there are a number of biobased materials available such as polysaccharides (starches, cellulose, chitosan, etc.) available, there are only two known biobased, biodegradable thermoplastics: polylactic acid (PLA) and polyhydroxyalkanoates (PHAs). PLA polymers are biobased materials with excellent biocompatibility, transparency, and compostability with bulk-commodity and biomedical uses (1, 23, 24, 16, 122). Despite the promising uses of PLA, there are notable drawbacks to this biomaterial: (i) PLA has traditionally been made through a complex multi-step process that includes fermentative production of lactic acid followed by chemical synthesis. The latter step utilizes either polycondensation reactions, which result in low molecular weight polymers with poor mechanical properties, or ring opening polymerization, which employ toxic metal catalysts (16). (ii) PLA polymers in and of themselves have relatively poor impact and heat resistance properties, prompting research to either use in vitro copolymerization (35) or blend PLA with other polymers in order to improve the material properties (76, 99). In contrast, PHAs are another major class of biobased, polymers that are produced intracellularly by microbes via the polymerization of hydroxyacyl-coa substrates which can be derived from renewable carbon feedstocks (65, 19). PHA polymers have a wider array of physical and chemical properties as compared to PLA, dictated by their repeating unit composition and molecular weights. PHA copolymers comprised of SCL and MCL repeating units have favorable material properties (1) and recently copolymers of PHA and PLA have been made with interesting material properties (112). Although PHAs have great potential as replacements for petroleum-based plastics, the main concern facing their adaptation is the control over the repeating unit composition and thus material properties of the polymer. This proposal aims to advance our knowledge of biodegradable polymer production through the use of innovative and proven metabolic engineering techniques to improve PHA and PHA-co-PLA copolymer production in E. coli. B2. Engineering E. coli for the production of PHA and PHA-co-PLA copolymers from sugars. Production of PHAs in E. coli has two distinct advantages over production in native producers: (i) native PHA producing microorganisms have PHA depolymerases, which catalyze the cleavage of the ester bonds of the polymer. This allows these bacteria to degrade PHAs for energy when there is no longer an excess of carbon and reduces the overall yield of polymer production (7, 11), and (ii) native PHAproducing organisms have transcriptional regulatory systems in place to inhibit PHA synthesis (47). E. coli has neither depolymerases nor regulatory systems for PHA production. In addition, the genome sequence for E. coli is known (11), and strains have been commonly used for high-level SCL PHA production (4, 59). The production pathway of the SCL PHA polyhydroxybutyrate (PHB) has been fully characterized and consists of three enzymes: a beta-ketothiolase (PhaA) that condenses two molecules of acetyl-coa to Figure 1. SCL PHA substrate biosynthetic pathway. Biosynthesis acetoacetyl-coa, an NADPH-dependent of (R)-3-hydroxybutyrl-CoA is catalyzed in a two-step reaction. Two reductase (PhaB) that reduces acetoacetyl- molecules of acetyl-coa are condensed to acetoacetyl-coa by the - CoA to 3-hydroxybutyryl-CoA, (Fig. 1), ketothiolase, PhaA. Acetoacetyl-CoA is subsequently reduced by the reductase PhaB to the precursor for PHB polymerization by the PHA and a PHA synthase (PhaC) is used to synthase (PhaC). polymerize 3-hydroxybutyryl-CoA into polyhydroxybutyrate (PHB). This pathway has been extensively utilized for production of PHB in native Christopher T. Nomura (SUNY-ESF) NSF 2

8 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics and recombinant sources. PHB can be synthesized to approximately 8% of the cell dry weight in recombinant E. coli (88, 15, 126). However, use of PHB as a thermoplastic is limited since it is a relatively brittle material, becoming less brittle dependent on increasing molecular weight (8, 46, 58). In order to synthesize MCL PHAs in recombinant E. coli, the substrate, (R)-3- hydroxyacyl-coa, must be produced within the cell. If feedstocks (i.e. sugars) that are unrelated to the final structure of PHA are to be used to generate PHA polymers, intermediates must go through the fatty acid biosynthesis pathway (Fig. 2). Fatty acid biosynthesis is dissociated in E. coli and begins with the dedicated step of converting acetyl-coa to malonyl-coa by the acetyl-coa carboxylase enzyme (19, 36). This intermediate then undergoes a transacylation reaction to replace the CoA moiety with an acyl carrier protein (ACP) and proceeds through a number of metabolic conversions until it reaches a 3-ketoacylreductase, FabG, which converts 3-keto-acyl-ACP to (R)-3-hydroxyacyl-ACP. The critical link to PHA synthesis lies in the conversion of this acyl-acp moiety to an acyl-coa moiety. The PHA synthase (PhaC) does not recognize Figure 2. Metabolic pathways for producing MCL PHA from unrelated carbon the ACP moiety attached to sources such as sugars in recombinant E. coli. Unrelated carbon sources such as the (R)-3-hydroxyacyl sugars are first converted to acetyl-coa followed by a condensation reaction by acetylsubstrate. The ACP moiety CoA carboxylase to produce malonyl-coa. A. Dissociated fatty acid biosynthesis in E. must be replaced by CoA on coli. B. TesA dependent pathway for production of MCL PHA in E. coli must also go through the -oxidation pathway and leads to inefficient production of polymer (55). C. the substrate in order to be PhaG-AlkK dependent pathway for production of MCL PHA in E. coli has shown the polymerized by the PHA highest level of production to date of MCL PHA polymers from glucose (132). D. FabH synthase. There have been dependent pathway for production of PHA polymers from glucose relies on specific point few studies investigating the mutations to the native FabH protein and spurious transacylase activity that occurs during link between fatty acid its overexpression in E. coli (FabH*). Although polymers with desired repeating unit metabolism and PHA compositions can be made, yields achieved are low (7, 8, 82). biosynthesis. A study by Klinke, et al. (55) demonstrated that recombinant expression of a modified thioesterase (TesA) in the presence of a PHA synthase led to low levels of MCL PHA accumulation. Another study by Rehm, et al. (92) demonstrated that PhaG could be used to mediate production of MCL PHA in the presence of triclosan, a fatty acid biosynthesis inhibitor. Our lab has taken two metabolic engineering strategies to produce MCL PHA precursors from sugars: (i) overexpression of FabH proteins with specific point Christopher T. Nomura (SUNY-ESF) NSF 3

9 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics mutations and the FabG protein, which leads to the production of SCL-MCL PHA copolymers with desired repeating unit compositions at low yields (7, 77, 79-82); and (ii) co-expression of phag and alkk, which has resulted in for the highest level of MCL PHA production from glucose in E. coli to date (132). The first study to demonstrate that E. coli could be engineered to produce polylactic acid (PLA) polymers in vivo was performed by Taguchi et al. (112), and the metabolic route to this synthesis is shown in Fig. 3. This study has been the catalyst for a tremendous amount of research in the field and has resulted in a number of publications on PLA polymer production in E. coli (5, 67, 69, 86, 13, 112, Figure 3. Synthetic pathway for polylactic acid (PLA) production in E. coli. Pyruvate is converted to D-lactate by lactate dehydrogenase (LdhA). Lactate is then converted to lactyl-coa by propionyl-coa transferase (PCT). Lactyl-CoA is a substrate for the engineered PHA synthase enzyme [PhaC1(STQK)] for polymer production. 113, ). These previous studies have focused on PLA and PLA-co-SCL PHA production. Although there have been two studies to date that have produced PLA-co-MCL PHA copolymers (67, 12), the yields of PLA-co-PHA polymers have been low and the resulting polymer has a repeating unit composition comprised of random MCL PHA repeating units of different sizes with PLA. B3. Engineering E. coli to produce PHA polymers from fatty acids. Pathways for the production of PHA polymers from fatty acids are shown in Fig. 4. It has been shown by our group (115, 117) and others (27, 87, 119) that E. coli can be engineered to produce PHA polymers from fatty acids. There are two main routes to convert oxidation intermediates to substrates for PHA production: The first is the conversion of enoyl-coa to (R)-3-hydroxyacyl-CoA substrates via (R)-specific-enoyl-CoA hydratases such as PhaJ (27, 98, ), MaoC, PaaG, PaaF, and YdbU (85). The other route is via conversion of 3-ketoacyl- CoA to the (R)-3-hydroxyacyl-CoA substrates for polymerization using 3- ketoacyl reductases such as FabG (81, 93, 111) or RhlG (13). The major shortcoming of these studies is that oxidation of fatty acids leads to an uncontrolled repeating unit composition within the PHA polymers produced. Therefore, there is little control over the physical Figure 4. Synthetic pathways for the production of PHA polymers from fatty acids in E. coli. A. Fatty acids are activated for degradation by the acyl-coa ligase, FadD where they enter the -oxidation pathway. B. 3- Ketoacyl reductases such as FabG and RhlG can be overexpressed to convert 3-ketoacyl-CoA to (R)-3-hydroxyacyl-CoA. C. (R)-specific enoyl- CoA hydratases can be overexpressed to convert enoyl-coa to (R)-3- hydroxyacyl-coa. D. PhaC polymerizes the precursors generated by the two pathways into PHA. properties of the PHA polymers produced. Also, to date, there have only been two studies looking at the production of PLA-co-MCL PHA polymers (67, 12), but it is expected that this class of polymers will possess desirable material properties in a similar way that SCL-MCL PHA copolymers do (1). These shortcomings have led to the impetus for our proposed study to engineer E. coli strains capable of producing a series of PHA and PHAco-PLA copolymers with specifically defined repeating unit compositions in order to produce biopolymers with relevant material properties, such as improved ductility and toughness and clarity. C. Preliminary Studies C1. Discovery of a new enzymatic link for producing higher levels of MCL PHA production from sugar in E. coli. Although previous studies had established the PhaG enzyme as an important link to the production of MCL PHAs from sugars in native PHA producing microorganisms (26, 39, 4), cloning and Christopher T. Nomura (SUNY-ESF) NSF 4

10 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics expression of the phag gene with a PHA synthase produced extremely low amounts of PHA in E. coli and relied on the addition of the fatty acid biosynthesis inhibitor triclosan for production (92). Thus, one of our early goals was to develop a system by which high levels of MCL PHAs could be produced from sugars in E. coli. We hypothesized that native MCL PHA producing bacteria such as pseudomonads must have other proteins involved in MCL PHA monomer supply in addition to the PhaG enzyme. We further hypothesized that the PhaG protein was not acting as an acyl-coa:acp transacylase as proposed in previous studies (26, 39, 4) but rather was acting as an ACP-specific thioesterase to produce (R)-3- hydroxy acids (Fig. 2C), thus requiring an acyl-coa ligase to produce the (R)-3-hydroxyacyl-CoA substrate required for polymerization by the PhaC enzyme. Towards the goal of identifying candidates for this enzyme activity, we systematically monitored expression of genes putatively encoding acyl-coa ligase enzymes in MCL PHA producer Pseudomonas putida by quantitative PCR (qpcr). Based on its high level of induction during PHA production and homology to acyl-coa ligases, the open reading frame PP763 (alkk) was identified as Table 1: Production of MCL PHAs from sugars in E. coli potentially Relevant genes %PHA a Composition (mol%) b Reference important in the 3HB 3HHx 3HO 3HD 3HDD production of tesa, phac2 2.3% ND ND (55) phag, phac1 2-3% ND ND ND 1 ND (92) MCL PHA fabh(f87t), 1.8% ND ND ND (8) polymers from phac Ac glucose in P. fabh(f87t), fabg, 4.5% ND (79) putida (132). phac1(stqk) PP763 and phag phag, alkk, 11.6% ND (132) phac1(stqk) were cloned into a PHA as a percentage of total cell dry weight; b mol% of PHA polymer; tesa: thioesterase A gene the ptrc99a from E. coli without a leader sequence; phac2: PHA synthase from Pseudomonas oleovorans; phag: vector and co- gene from P. putida; phac1: PHA synthase gene from P. putida; fabh(f87t): 3-ketoacyl-ACP transformed with synthase III gene from E. coli with F87T mutation; phac Ac: PHA synthase gene from Aeromonas a plasmid caviae; fabg: 3-ketoacyl-ACP reductase from E. coli: phac1(stqk): PHA synthase gene from Pseudomonas sp with S325T and Q481K mutations; alkk: 3-hydroxyacyl-CoA ligase from P. harboring an putida. ND: not detected. engineered PHA synthase [known as PhaC1(STQK)] with broad substrate specificity towards both SCL and MCL 3- hydroxyacyl-coa (114) and lactyl-coa (112) substrates into E. coli. This has resulted in the highest levels of MCL PHA produced from glucose in E. coli to date among all methods (Table 1). These results demonstrate our ability to successfully engineer a pathway for the production of MCL PHA monomers from sugar in E. coli. C2. The molecular weight of PHA can be controlled by relative expression levels of PHA biosynthetic enzymes. Aside from repeating unit composition, another critical determinant of PHA material properties is the molecular weight of the polymer. The weight-average molecular weight (M w ) of the SCL PHA polymer, poly-3-hydroxybutyrate (PHB) produced by native PHA producing bacteria is usually in the range of to Da (11). However, expression of the SCL PHA producing pathway (Fig. 1) in E. coli has led to the production of PHB with ultrahigh-molecular-weight with M w exceeding Da (2, 3, 16, 51, 57, 58). The higher molecular weight gives PHB higher mechanical strength, and therefore we engineered E. coli to effectively produce ultrahigh-molecular-weight PHB through rearrangement of gene order relative to a promoter for the phacab operon to control expression levels of the various enzymes involved in SCL PHA production (38). We accomplished this by using ordered gene assembly in Bacillus subtilis (OGAB) (118) to arrange the pha gene order in a single step (Fig. 5). These constructs had all possible iterations of gene order for the phacab genes and were used to transform E. coli to examine the effect of gene order and expression level on M w of PHB Figure 5. Plasmids (pgets19-pha-series) used to examine the effect of gene rearrangement on M w of PHA. phaa, phab, phac genes are derived from R. eutropha. Plasmids were constructed by OGAB method with various pha gene orders (#1~6 represent individual plasmid gene orders, for example #1 represents phaabc). Christopher T. Nomura (SUNY-ESF) NSF 5

11 SusChEM: Engineering E. coli for improved production of biodegradable plastics polyhydroxyalkanoate (PHA)-based polymers produced. It was anticipated that the gene position relative to the promoter would result in different levels of gene expression and thus activities of the respective enzymes. This, in turn, would affect the yield and M w of PHB polymers produced. Polymers were isolated from each of the strains and the molecular weight distributions of the isolated polymers were examined. These results demonstrated that gene order within synthetic operons relative to the promoter had a dramatic effect on the molecular weights of the polymer produced. Placement of either of the genes encoding monomer-supplying enzymes near the promoter resulted in the production of PHB polymers with the highest molecular weights; whereas, placement of the PHA synthase near the promoter resulted in the production of polymers with lower molecular weights (Fig. 6). We determined that the expression levels of the individual enzymes were dictated by the order relative to the promoter via immunoblotting and enzymatic activity assay for each of the PHA monomer-supplying enzymes and the PHA synthase (Fig. 7). Results indicate the importance of the position of the gene relative to the promoter for the expression, production, and activity of each respective enzyme. The closer the gene is to the promoter, the higher the expression level and greater the activity of the protein produced. This provides a simple but powerful method where relative enzyme activities in a metabolic pathway can be controlled simply through placement of genes in synthetic operons relative to a common promoter. In practical terms for this proposal, these results clearly demonstrate our ability to control the molecular weights of PHA polymers by varying the expression levels of genes encoding monomer-supplying enzymes relative to the PHA synthase gene. C3. An engineered strain of E. coli to produce PHA copolymers from fatty acids with defined repeating unit compositions. Fatty acids are another important renewable carbon feedstock that can be used for PHA production (27, 81, 85, 87, 93, 98, 111, ). However, a shortcoming of previous studies is that the PHA polymers produced consist of a random, uncontrolled mixture of repeating units. This arises because -oxidation degrades each fatty acid by two carbons after a complete cycle, thus the intermediates that are incorporated into the PHA polymers consist of various degradation products from these cycles that have been intercepted and converted into PHA. Since control of repeating unit composition is critical to produce polymers with desirable material properties, we sought to engineer E. coli to produce PHA polymers with defined repeating unit compositions from fatty acids. The parental strain used for this study was E. coli LS5218, which carries two significant mutations that make it ideal for producing PHA polymers from fatty acids: (i) this strain carries a fadr Figure 6. Molecular weight distributions of PHB synthesized in recombinant E. coli harboring the pget19 pha-series. PHB was purified from the cells at 12 h (dashed lines) or 72 h (solid lines). Determination of molecular weight was carried out in triplicate, and typical distributions for each polymer are shown. Figure 7. Immunoblot analysis and enzyme activities of PHA synthase (PhaC) and the monomer-supplying enzymes (PhaA and PhaB) in recombinant E. coli strains harboring the pget19 pha-series. Crude extracts (1 g for PhaA and PhaB, 5 g for PhaC) were prepared for each engineered strain and used for immunoblot analysis. Enzyme assays were carried out in triplicate with results shown as averages with standard deviations. Christopher T. Nomura (SUNY-ESF) NSF 6

12 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics mutation for enhanced expression of genes encoding enzymes involved in -oxidation (94), and (ii) this strain also harbors an atoc(con) mutation which results in the constitutive expression of the Ato short-chain-fatty acid uptake enzymes in E. coli (48, 89, 94). We further engineered this strain so that we could control the production of enoyl-coa by deleting the fadb and fadj genes [E. coli LSBJ] (117). This results in a strain where any fatty acid fed in is pooled as its enoyl- CoA intermediate with no loss of carbons from the substrate. For example, a 4-carbon fatty acid (butyrate) fed in results in a butenoyl-coa intermediate, and an 8-carbon fatty acid (octanoate) fed in results in pooling of an octenoyl- CoA intermediate. Specific fatty acid feeding regiments coupled with expression of the (R)-specific enoyl- CoA hydratase (PhaJ4) and PhaC1(STQK) engineered synthase results in production of PHA polymers with defined repeating unit compositions (Fig. 8). We performed a series of experiments with both individual fatty acids to produce PHA homopolymers (117) and ratios of different sized fatty acids to produce PHA copolymers with defined compositions (115). Previous studies have demonstrated that SCL-MCL PHA copolymers with 8-95% SCL repeating units and 5-2% MCL repeating units have material properties similar to the petroleum-based plastics polyethylene and polypropylene (1). Thus, we used the E. coli LSBJ strain to produce SCL-MCL PHA copolymers with defined repeating unit ratios by feeding different ratios of butyrate and octanoate. Results from this study are shown in Fig. 9, which describes the relationship between the ratios of fatty acids fed to the bacteria and the ratios of monomers in the polymer. The results from this study were used to establish a trend line by which we could predictably produce SCL-MCL PHA copolymers with specific repeating unit compositions based on the ratio Figure 8. Engineering E. coli to produce PHA polymers with defined repeating unit compositions. Several modifications in this strain result in the production of PHA polymers with defined repeating unit compositions: (i) fadr mutation results in constitutive expression of the fad genes, (ii) atoc(con) results in constitutive expression of the ato genes, (iii) deletion of the fadb and fadj genes results in the pooling of enoyl-coa intermediates of defined carbon lengths from fed fatty acids, (iv) expression of phaj results in the conversion of specific enoyl-coa substrates to (R)-3-hydroxyacyl-CoA substrates, and (v) expression of phac1(stqk) results in polymerization of the (R)-3-hydroxyacyl- CoA substrates into PHA polymers dependent on the ratio of fatty acids fed to the strain. Figure 9. The mol ratio of repeating units in poly[(r)-3-hydroxyoctanoateco-(r)-3-hydroxybutyrate] polymer samples in relation to starting fatty acid substrate ratios. The fatty acid substrates were used by E. coli LSBJ. All values were determined by 1 H NMR and represent average integrations of three separate peaks in the 1 H NMR spectra. These integrations were compared to the integration of a peak unique to the 8-carbon repeating unit. Error bars represent standard deviations about those averages. Christopher T. Nomura (SUNY-ESF) NSF 7

13 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics of fatty acids fed to E. coli LSBJ. These results demonstrate our ability to precisely control the repeating unit composition of PHA polymers from fatty acids using an engineered strain of E. coli. Furthermore, our efforts point towards a successful outcome in applying this system for the production of PHA-co-PLA copolymers with defined repeating unit compositions from fatty acids and lactic acid. D. Research Design and Methods D1. Aim 1: Produce PHA and PHApolylactic acid (PLA) copolymers from sugars in E. coli. Overview. To date, we have successfully created a synthetic pathway in E. coli for producing MCL PHA from an inexpensive carbon substrate (glucose). However until now, we have only produced MCL PHA polymers via this pathway. Therefore, under this aim we seek to extend our polymerization system to produce SCL-MCL PHA, MCL PHA-co- PLA, and SCL-MCL PHA-co-PLA copolymers. We also seek to engineer E. coli to simultaneously utilize xylose and glucose for PHA production. A summary of the desired outcomes for Aim 1 is shown in Fig. 1. Experimental Design (i) Engineering E. coli to produce SCL-MCL PHA and PHA-co-PLA copolymers from glucose. Figure 1. Engineering E. coli for the production of PHA copolymers from sugars. Simultaneous uptake of multiple sugars for the production of PHA and PHA-PLA copolymers requires a number of modifications in E. coli. Key enzymes for metabolic pathways are in bold. A. Carbon catabolite repression (CCR) results from the uptake of a preferred carbon source (glucose) by E. coli and results in exclusion of other carbon sources (lactose and glycerol). E1: enzyme I, HPr: histidine protein; EIIA: enzyme II subunit A; EIIB: enzyme II subunit B; EIIC: enzyme II subunit C; GlpK: glycerol kinase; LacY: Lactose permease. B. Engineering of the catabolite repression protein (CRP) will lead to an alleviation of CCR for the uptake of two lignocellulosic-derived sugars (glucose and xylose) and for activation of genes under the lac promoter in the absence of the lac repressor protein for expression plasmids. AC: adenylate cylase; CRP: catabolite repression protein; CRP*: camp insensitive catabolite repression protein; XylFGH: high affinity xylose transporter. C. Pathway for the production of PLA. LdhA: D-lactate dehydrogenase; PCT: propionyl-coa transferase; PhaC: PHA synthase. D. Pathway for the production of PHB. PhaA: -ketothiolase; PhaB: reductase. E. Pathway for the production of MCL PHA. AccC: acetyl-coa carboxylase; PhaG: (R)-3-hydroxyacyl-ACP thioesterase; AlkK: (R)-3-hydroxyacyl-CoA ligase. We have already demonstrated that we can engineer a strain of E. coli to produce the highest levels of MCL PHA polymers from glucose (132), as shown in Table 1, using the engineered pathway in Fig 2C. However, the polymers produced in this previous study consisted solely of a mixture of MCL PHA repeating units, limiting the overall utility of the plastic produced. Previous studies have shown that SCL-MCL PHA copolymers have desirable material properties (1, 75); so we propose to engineer E. coli to produce large amounts of SCL-MCL PHA copolymers. In order accomplish our goal, we will co-express the alkk and phag genes from the ptrcgk vector (132) with the pbbrstqkab plasmid harboring the phac1(stqk), phaa, and phab genes under transcriptional control from a constitutive Ralstonia eutropha promoter. The phac1(stqk)phaab synthetic operon in pbbrstqkab is also co-linear to the lac promoter for the plasmid, allowing for higher levels of expression through IPTG induction. The pbbrstqkab vector was derived from the broad-host range plasmid, pbbr-1mcs2 (56), and we have successfully cotransformed and co-expressed other genes from plasmids derived from pbbr-1 MCS2 and ptrc99a vectors for the production of PHA (82). The co-expression of ptrcgk and pbbrstqkab will result in Christopher T. Nomura (SUNY-ESF) NSF 8

14 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics simultaneous expression of the pathways depicted in Fig. 1D and E for the production of SCL-MCL PHA. We will grow transformed cells under uninduced (no IPTG added) and under several stages of induction (IPTG and glucose at the same time, IPTG and glucose addition delayed for 1 h and 3 h), in order to examine gene expression effect on polymer accumulation, repeating unit composition, and molecular weights. To determine the amount and composition of PHA polymer produced by this system, we will grow the cells in 1 ml of Lennox broth media and add IPTG and glucose as described above in 5-ml baffled flasks at 3 C, 2 rpm, for 24, 48, and 72 h, at which time, the cells will be harvested by centrifugation. Cells will be frozen in liquid nitrogen and lyophilized for h dependent on the total volume of cells recovered. Once lyophilized, the total cell dry weights will be determined and 15 mg of cells will be subjected to methanolysis and GC analysis in order to determine the PHA content and composition as performed previously in our lab (115, 117, 132). Fermentations will be employed to scale up the experiments if necessary in order to extract and purify polymers for further analysis by NMR for structural characterization and repeating unit composition determination; differential scanning calorimetry (DSC) for thermal analysis and determination of the glass transition temperature (Tg,) crystallization temperature (Tc), and melting temperature (Tm) of the polymers; thermogravimetric analysis (TGA) to determine the thermal degradation profile of the polymers; and tensile strength will be performed as previously reported (115, 117, 132). A similar engineering strategy will be employed to produce and characterize MCL PHA-co-PLA and SCL-MCL PHA-co-PLA copolymers produced from sugars in E. coli. We have received the following plasmids from our collaborator Dr. Seiichi Taguchi at Hokkaido University and leader in the production of PHA-PLA copolymers: (i) ptv118npctc1stqk plasmid harboring the propionyl-coa transferase (PCT) gene (1) and phac1(stqk) genes and (ii) ptv118npctc1stqkab harboring the gene encoding PCT, phac1(stqk), phaa, and phab genes. These plasmids are puc118 derivatives with ampicillin resistance cartridges. Since the ptrcgk plasmid also has an ampicillin resistance cartridge, we will transfer the alkk and phag genes to the pbbr1- MCS2 plasmid, which has a kanamycin resistance cartridge, in order to co-transform and express these genes with either ptv118npctc1stqk (resulting in pathways depicted in Fig. 1C and E to produce MCL PHA-co-PLA) or ptv118npctc1stqkab (resulting in pathways depicted in Fig. 1C, D, and E to produce SCL- MCL PHA-co-PLA). We will grow the cells as described for the SCL-MCL PHA production experiments. However, because we will be relying on native levels of lactate dehydrogenase in E. coli to generate the lactate precursor, we will grow the cells in non-baffled flasks or capped media bottles to produce a microaerobic environment to assure expression of the ldha gene (12, 49). The proposed lactyl-coa producing pathway for PHA-co-PLA polymer production in E. coli will be further enhanced by additional chromosomal modifications to our E. coli strain to direct carbon flux towards PHA and PLA precursors. It has been shown previously that a number of gene knockouts can help with lactic acid production in E. coli (49, 13, 14). We will Figure 11. Engineering E. coli for lactate production from glucose. Key gene deletions for maximizing carbon flux from sugar metabolism towards PHA and PLA polymer production are indicated in bold. Pps: phosphoenolpyruvate synthase; Dld: FAD-binding D-lactate dehydrogenase; PflAB: pyruvate formate lyase; PoxB: pyruvate oxidase; AckA: acetate kinase; Pta: phosphotransacetyase; AdhE: alcohol dehydrogenase. inactivate the chromosomal copies of genes (pps, pfla, dld, poxb, acka, pta, and adhe) encoding enzymes that would limit carbon flux to the production of PHA and PLA intermediates in E. coli (outlined in Fig. 11) using the -red system developed by Datsenko and Wanner (18). We have used the -red system to successfully generate gene deletions in E. coli (117, 132), and we are confident that the proposed gene deletions will lead to increased flux for PHA and PLA bioplastic production. Additionally, the arca deletion proposed in Aim 2 will positively influence the production of lactate in E. coli since ArcA is a negative regulator of ldha (64). The deletion of the arca gene in E. coli has resulted in marked increases in lactate production under anaerobic growth conditions (73) and this gene Christopher T. Nomura (SUNY-ESF) NSF 9

15 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics inactivation will lead to higher levels of substrate for PLA production in our engineered E. coli strain. In sum, these modifications will ensure high levels of production of substrates necessary to produce PHA and PLA polymers from sugars like glucose. (ii) Engineering E. coli for higher levels of fatty acid substrates. Although we were able to produce the highest levels of MCL PHA from glucose in recombinant E. coli to date (132), these levels are still much lower than those achieved for SCL PHA polymers such as PHB can be from glucose. It is likely that the supply of MCL PHA monomer substrates for polymerization represents a rate limiting step for (i) production of polymers and (ii) determination of the overall repeating unit composition of the polymer produced. This is especially true for the production of MCL PHA monomer units. To address this, we will overexpress the accabcd genes, which encode the acetyl-coa carboxylase (Fig 1E). Acetyl-CoA carboxylase catalyzes the first committed step for fatty acid biosynthesis and overexpression of this enzyme has resulted in increased levels of malonyl-coa and fatty acid overproduction (2). Malonyl-CoA levels and flux through fatty acid biosynthesis are critical for the production of the (R)-3-hydroxyacyl-ACP precursors to produce MCL PHA polymers. Thus, we will PCR amplify the acca, accbc, and accd genes from E. coli to be used in a synthetic operon to increase malonyl-coa levels in our E. coli strain. The genes will be cloned into the ptrcgk plasmid to make the new plasmid ptrcgk-acc. Overexpression of the acc genes for substrate precursor production has been successfully employed to produce high levels of flavonoids (62) but had negligible effects on free fatty acid production (6) in E. coli. It has been previously reported that overproduction of free fatty acids in E. coli has led to membrane stress (61). However, we do not expect to observe the same stress response for fatty acid overproduction for our engineered E. coli strain producing PHA polymers since much of the stress observed in these previous studies is due to the free fatty acids themselves. Our studies demonstrated that PhaG behaves as an (R)- specific 3-hydroxyacyl-ACP thioesterase and when expressed alone in the cell, results in the production of free 3-hydroxyacids (132). However, when a PHA synthase and 3-hydroxyacyl-CoA ligase (AlkK) are co-expressed in this system, it results in the complete conversion of the free 3-hydroxyacid substrates to PHA. Thus, it is anticipated that any surplus fatty acid intermediates generated by overexpression of the acetyl- CoA carboxylase will be rapidly converted to PHA polymers via our engineered MCL PHA production pathway, thus alleviating any stress to cells caused by the free fatty acids. (iii) Gene rearrangement to control the molecular weight of PHAbased polymers. Control over the molecular weights of polymers can have dramatic effects on the physical properties of the materials. Our previous study demonstrated that gene order and thus enzyme expression levels are partially responsible for controlling the molecular weights of PHA polymers produced in E. coli. We were able to produce PHB polymers with ultra-high-molecularweights using gene rearrangement (38). It is also known that PHA synthase activity is a key factor for controlling the molecular weight of PHA (14). An outline to apply this strategy for controlling the molecular weights of PHA-co- PLA copolymers is provided in Fig. 12. In addition to producing polymers with higher molecular weights, it is also important to produce PHA polymers with lower molecular weights. Many of the PLA biomaterials used in tissue engineering applications are of much lower molecular weights (typically Da) (41, 11) than those the PHA polymers typically produced (typically Da). It is clear from our results that high levels of expression of the phac gene encoding the PHA synthase relative to monomer supplying enzymes results in the production of polymers with lower molecular weights. In order to establish a range Figure 12. Gene rearrangement to control molecular weights of MCL PHA-co-PLA polymers. Synthetic operons for MCL PHA-co- PLA polymer production will be made via Gibson assembly. Overlaying primers will be designed to rearrange all monomer-supplying genes (PCT, phag, and alkk) relative to the PHA synthase gene encoding phac1(stqk) shown here as phac1. of molecular weights of PHA polymers produced in our engineered E. coli strains, we will assemble expression constructs with the PHA synthase and various monomer supplying genes relative to a series Christopher T. Nomura (SUNY-ESF) NSF 1

16 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics of promoters with variable strengths (lac, ara, trc, R. eutropha native pha promoter) in large expression constructs using Gibson assembly (3, 31). Briefly, Gibson assembly allows for the one-step isothermal in vitro assembly of multiple DNA fragments regardless of fragment length or end compatibility. We have shown that placement of the phac gene encoding the PHA synthase at the position nearest to the promoter results in PHA polymers with lower molecular weights while PHA polymers produced in constructs where the phac gene was furthest from the promoter had the highest molecular weight (38). Thus initial Gibson assembled constructs will be made with the phac1(stqk) gene occupying either the first or last position of the construct with genes encoding monomer-supplying enzymes shuffled into all other positions (Fig. 12). As an alternative, these can be assembled via the OGAB method we have employed for SCL PHA production (38). These constructs will be further engineered for varying levels of protein production by altering the ribosomal binding site using the RBS calculator designed and maintained by Howard Salis ( (96, 97). The RBS calculator will allow us to calculate the relative expression of each of the proteins based on designed RBS sites to alter translation from.5-fold to 1,-fold higher than the original rate of translation within a factor of 2.3 (97). We will use the RBS calculator to design primers with engineered RBSs for the PCT, phag, alkk, and phac1(stqk) by three increments (.5, 1, and 1-fold of the native RBS). This will result in controlled expression of the genes in the pathway, which should lead to differences in the molecular weights of any polymers produced based on our previous study with the SCL PHA polymer PHB (38). (iv) Engineering E. coli for simultaneous consumption of sugar substrates. The use of lignocelluosic biomass as an alternative growth substrate for biobased chemical and energy production has the potential to reduce the overall cost of producing PHA-based bioplastics. A key to using the various carbon substrates generated from biomass for the production of value-added products such as PHAs is the control of carbon catabolite repression (CCR). In E. coli, the major proteins involved in CCR are shown in Fig. 1A and B, and include the transcription activator cyclic AMP (camp) receptor protein; CRP (also known as catabolite gene-activator protein (CAP)); adenylate cyclase (AC), which generates camp; and the IIA component of the glucose-specific phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), EIIA Glc, which is also called catabolite repression resistance (Crr protein or EIIA Crr ) (32). The cyclic AMP (camp) receptor protein (CRP) has been studied in detail and is known to modulate the transcription of a large number of genes in E. coli via CCR in conjunction with camp levels (29). The prototypical example of CCR is the preferential use of glucose over other sugars in growth media. Normally, CRP must bind to camp in order to act as a transcriptional activator. A number of efforts have been made to alleviate catabolite repression through mutation of the CRP protein (14, 45, 52, 139). We will take advantage of the well-characterized CRP* mutant (53), which has three significant mutations (I112L, T127I and A144T) that lead to camp activation independence in our proposed study. A DNA fragment containing partial sequence for the crp* gene with the kanamycin resistance fragment from plasmid pkd13 and Figure 13. Strategy for engineering CRP* into E. coli for the production of PHA-based biopolymers. The mutant partial sequence of the yhfk gene will be construct contains the following: partial crp* sequence with synthesized (total size 1652 bp). The native crp point mutations for I112L, T127I, and A144T underlined and in R gene of E. coli will be replaced via -red bold, Km cartridge from pkd13, and partial sequence of the substitution using the helper plasmids pkd46 yhfk gene. This will be co-transformed with the pkd46 plasmid and pcp2 as outlined by Datsenko and to facilitate incorporation into the chromosome through homologous recombination. The antibiotic cartridge can be Wanner (18) (Fig. 13). The antibiotic marker can removed using the FLP recombinase from the pcp2 plasmid. be removed via the FLP recombinase as described previously (18). CRP engineering has been successfully utilized for xylitol production from xylose and glucose (17), and we will draw upon the wealth of transcriptomic and phenotypic data available for CRP* E. coli strains (53) to optimize the production of PHA polymers from xylose and glucose feedstocks. An interesting finding from the study by Khankal, et al. (53) is that there are elevated NADPH levels in the CRP* E. coli strains grown on glucose as compared to the wild type strain. This observation bodes well Christopher T. Nomura (SUNY-ESF) NSF 11

17 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics for our PHA production studies since the synthesis of fatty acid precursors is partially dependent on NADPH levels (Fig. 2A). We fully expect that CRP* will work to facilitate PHA production from the simultaneous consumption of glucose and xylose in our engineered E. coli. As an alternative and supportive strategy, we can also overexpress xylr. This has been demonstrated as an effective strategy for co-utilization of hemicellulosic sugars (xylose, arabinose, glucose) for enhancing ethanol production in E. coli (33) and should be an effective strategy for the production of PHA-based polymers from hemicellulosic sugars as well. D2. Aim 2: Improve production of PHA polymers with defined repeating unit compositions from fatty acids in E. coli. In this aim, we will produce PHA-co-PLA copolymers with defined repeating unit compositions by co-expressing the propionyl-coa transferase (PCT) enzyme in our recently engineered E. coli LSBJ strain (115, 117). This strain will be further engineered to improve yields of PHA by deletion of genes encoding negative transcriptional regulators (arca and ompr) of the -oxidation pathway in E. coli to increase substrate availability for PHA production. Successful completion of this aim will lead to higher levels of production of both PHA polymers and copolymers and for the first time, the production of PHA-co-PLA copolymers with defined repeating unit compositions. Experimental Design (i) Deregulating repression of -oxidation in E. coli. We are using E. coli LSBJ as the parental strain for the production of PHA polymers with defined repeating unit composition from fatty acids (Fig. 8). This strain utilizes the -oxidation pathway to generate intermediates for PHA synthesis. In order to increase flux through this pathway, it is necessary to engineer or delete negative transcriptional regulators of -oxidation and fatty acid uptake in E. coli. These have been identified in a previous study (25), and an outline of proposed modifications is shown in Fig. 14. Modifications include the deletion of genes encoding transcriptional regulators (fadr, arca, and ompr) that downregulate expression of the fad regulon and engineering of the CRP protein as outlined in Aim 1 so that transcription initiation is independent of glucose. We have previously engineered the E. coli LSBJ strain that carries an inactivated fadr, constitutively expressed atoc, and inactivated fadb and fadj genes for the production of PHA polymers with defined repeating unit compositions (115, 117). For the proposed study, we Figure 14. Engineering transcriptional regulators in E. coli for enhanced fatty acid uptake. Regulation of the E. coli fad regulon responds to several environmental stimuli including: long chain fatty acids (LCFA), glucose, oxygen, and osmotic stress. These external stimuli influence a number of regulatory proteins (in bold) including FadR, catabolite repression protein, ArcA-P, and OmpR. The fad regulon components are in italics and are either positively regulated as indicated by a black arrow or repressed as denoted by a gray. will further engineer this strain for optimized utilization of fatty acids as feedstocks for PHA polymer production by additionally inactivating the arca and ompr genes using the red system (117). A total of three new E. coli strains will be developed in this subaim: LSBJ-arcA, LSBJ-ompR, and LSBJ-arcA-ompR. PHA polymers will be produced in each strain as we have done for E. coli LSBJ, and polymer and growth yields will be calculated as previously described (115, 117). ArcA mutations have already been performed successfully for other strains of E. coli (64, 73) and it is known that ArcA has a regulatory role for the fad genes of E. coli (15). Although E. coli arca mutants have successfully employed for PHB production (71, 72), they have yet to be used for MCL PHA production. In addition, as the deletion of arca is expected to have a benefit for the production of lactate from sugars as outlined in Aim 1. Thus, this mutation can benefit the production of PHA and PHA-co- PLA polymers in both aims of our proposal. OmpR has been shown previously to repress expression of fadl, which encodes the protein responsible for uptake of long-chain fatty acids in E. coli (37). In order to remove this repression, ompr will be also be inactivated both independently and successively with arca in E. coli LSBJ via -red recombination (18). It is anticipated that the inactivation of these repressors will lead to higher levels of both fatty acid substrate uptake and PHA production via our engineered -oxidation pathway in E. coli Christopher T. Nomura (SUNY-ESF) NSF 12

18 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics LSBJ. Greater flux and uptake of the feedstocks of this pathway will increase overall yields and decrease incubation times to produce PHA polymers from fatty acids. (ii) Production of MCL PHA-co-PLA polymers with defined repeating unit compositions. Our previous studies have demonstrated that we can produce PHA homopolymers (117) and PHA copolymers (115) with defined repeating unit composition strain by supplying specific ratios of fatty acid feedstocks to the E. coli LSBJ strain (Figs. 8. and 9). This represents the first system by which the repeating unit composition of PHA polymers can be controlled. We will now extend this control to PHA-co-PLA copolymers by the co-expression of the PCT gene with phaj4 and phac1(stqk) in E. coli LSBJ and co-feeds of specific concentrations of lactic acid and fatty acids (Fig. 15). The PCT gene will be PCR amplified from the ptv118n plasmid (112), sublconed into the pbbr-c1j4sii vector (117) harboring the phaj4 and phac1(stqk) genes, and transformed into E. coli LSBJ. The transformed strain will be grown in the presence of octanoate and lactic acid at various feed ratios (1:, 9:1, 6:4, 5:5, 4:6, 1:9, and :1, octanoate:lactate) to produce MCL PHA-co-PLA copolymers with defined repeating unit compositions. In addition, we will perform growth and polymer production experiments using undecylenic acid and lactic acid at similar ratios. Undecylenic acid is an 11- carbon fatty acid with a terminal alkene, allowing the insertion of a chemically reactive handle into our polymers. PHA polymers containing Figure 15. Engineering E. coli for precise control of MCL PHA and PLA repeating unit composition. E. coli LSBJ has repeating units with terminal alkenes have been key mutations [atoc(con), fadr, fadb, fadj] for the acquisition chemically modified to expand the number of and conversion of small organic acids like lactic acid and fatty applications for drug delivery (17, 18) and acids. With the co-expression of the (R)-specific enoyl-coa shape-memory polymers (44). It is anticipated hydratase, PhaJ, PHA synthase, PhaC, and propionyl-coa that we will be able to control the ratio of PHA to transferase, PCT, the strain can easily be engineered for the production of PHA-co-PLA polymers. The ratio of PHA to PLA PLA in the copolymers produced in a manner incorporated into the copolymer will be dependent on the ratio similar to what we have established for PHA of feedstocks (fatty acids: lactic acid) available to the strain. copolymers (115). This will be the first study to By feeding different ratios of lactic acid and fatty acid to the produce PHA-co-PLA copolymers with defined cells and determining the polymer composition, copolymers compositions, allowing for unprecedented with desired ratios of PHA to PLA repeating units can be made in a manner similar to SCL-MCL PHA copolymers depicted in control over the material properties of next Figures 8 and 9. The system shown here is for MCL PHA-cogeneration of biobased, biodegradable plastic PLA polymer production but can be readily adapted for the materials. production of SCL PHA-co-PLA polymers with desired repeating unit compositions. D3. Timeline. To complete these studies as outlined below, two full-time graduate research assistants (Ms. Lucia Salamanca-Cardona and Ms. Xian Wang) will be require. Tasks for the two aims have minimal overlap and thus can be pursued in parallel. Task Year 1 Year 2 Year 3 Aim 1 (Salamanca-Cardona) (i) Engineering E. coli to produce PHA polymers from glucose (ii) Engineering E. coli for higher levels of MCL monomer production (iii) Gene rearrangement for molecular weight control of PHA (iv) PHAs from multiple sugar sources Aim 2 (Wang) (i) Deregulation of -oxidation in E. coli (ii) PHA-PLA copolymers with defined repeating unit composition Christopher T. Nomura (SUNY-ESF) NSF 13

19 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics E. Broader Impacts E1. Broader Scientific Impact. The success of this project will have broad societal impacts since biodegradable plastics produced from renewable carbon feedstocks can provide sustainable replacements for petroleum-based non-biodegradable plastics. There have been numerous life-cycle analyses that demonstrate that biobased plastics such as PHA polymers have a much smaller environmental footprint than petroleum-based plastics (5, 34, 54, 9). Because PHA-based plastics are biodegradable in a number of environments, including compost (42, 66), soil (123), and marine environments (43), they will alleviate the impact of non-biodegradable plastics accumulating in the environment, such as those seen in the Pacific garbage gyre. The metabolically engineered strains to be developed in the proposed study will increase precursors availability and yields for PHA-based polymers. Importantly, for the first time, controlled composition and thus material properties of PHA-based polymers will be achieved by this study, extending the applications of these polymers beyond bulk-commodity uses and into specialty plastics with specific material properties. In addition, many of the substrates (e.g., acetyl-coa, malonyl- CoA, fatty acids) for PHA polymer production are important precursors for other value-added products, such as biofuels (21, 22, 28, 6) and fine chemicals (21, 22, 28, 62). Thus, the strains to be developed in this study have the potential to be widely used for the production of other valuable bioproducts. E2. Broader Educational Impact. The proposed research project will be performed in an intrinsically interdisciplinary environment. I currently mentor a large group of students from several fields including bioprocess engineering, biotechnology, biochemistry, and polymer chemistry majors. This variety of disciplines fosters an outstanding training environment for students to collaborate across disciplines in order to achieve the goals of this project. I have worked to expand opportunities for elementary school, high school, undergraduate, and graduate students to participate in polymer and metabolic engineering projects. My group currently consists of 5 graduate students, with 3 of these graduate students being women and two underrepresented minorities. Two of the female graduate students (Ms. Lucia Salamanca-Cardona, also an underrepresented minority, and Ms. Xian Wang), will be directly involved in all aspects of the proposed research. At least one other graduate student (Mr. Alex Levine) will also be involved in the proposed research, but will receive financial support from other funding sources. In addition, for the past three years, I have been teaching an upper-division, introductory graduate study course in Microbiology for Bioprocessing through the Paper and Bioprocess Engineering Department at SUNY-ESF and it is expected that elements of this study can be integrated into current course materials to expand the educational impact of the proposed project. (i) Previous and current broader impact activities. In my previous NSFsponsored research, I have worked with 5 th -1 th grade students from traditionally underrepresented groups drawn from the Syracuse City School District in conjunction with the ESF Summer Camps Investigating Ecology in Neighborhood and City Environments (SCIENCE) and Stewards of Syracuse (S.O.S.) programs at SUNY- ESF. For these summer programs, a series of exercises to practice the scientific method were developed and lab experiments were performed with students. One of the lab exercises is shown in Fig. 16, upper photo. In addition, I have been a research mentor for the Syracuse Biomaterials Institute (SBI) and Louis Stokes Alliance for Minority Participation (LSAMP) summer programs for the past two years. I have supervised two LSAMP students (Ms. Jennifer Quinn from Clarkson University and Ms. Ada Ozumba from Trinity University in San Antonio). This last summer (212) I mentored Ms. Ivory Patterson (Fig. 14, bottom photo), an NSF-sponsored REU student from the Historical Black College University (HBCU), Hampton University. The research conducted by Ms. Patterson is directly related to the proposed research project. Contributions from Ms. Patterson and Ms. Ozumba will result in co-authorship on a publication to be submitted soon and point to our ability to directly involve underrepresented science and engineering students in Figure 16. Our research leads to positive broader impacts in science and engineering. Top panel, Dr. Nomura lectures students from the S.O.S program at SUNY- ESF before a lab assignment. Similar lab experiments were designed for ESF SCIENCE participants. Bottom panel, Dr. Nomura supervised Ivory Patterson, for the NSF-sponsored SBI-REU program in 212. Christopher T. Nomura (SUNY-ESF) NSF 14

20 SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics meaningful and productive research projects. (ii) Proposed broader impact activities for this study. For the current proposed study, I will expand the participation and involvement for underrepresented minorities in science and engineering at the community college level through a new partnership with The State University of New York Onondaga Community College (SUNY OCC). The goal of our broader impacts for the proposed study is to introduce lab competency and increase interest and retention of underrepresented and economically disadvantaged students enrolled in science and engineering majors. To achieve this goal, we will collaborate with Drake Harrison, the director for the Collegiate Science and Technology Entry Program (C-STEP) and LSAMP programs at SUNY OCC to recruit one student to participate in a research and training program in the lab for 1 weeks each summer (see attached letter of support). The C-STEP and LSAMP programs are dedicated to increasing the number of college graduates from both traditionally underrepresented ethnic groups and individuals from economically disadvantaged families who are interested in pursuing careers in science and technology. SUNY OCC is a community college in Syracuse, NY with ~11,5 students from in and around Central New York. Dr. Nomura will give presentations on the research project and on opportunities for summer research at SUNY-ESF to C-STEP and LSAMP students at SUNY OCC each year prior to selection of a student to participate in the program. The selected student will start off learning basic lab techniques and move on to a greater contribution to the overall goals of the project. The student will have the opportunity to learn from graduate students in the laboratory and will attend weekly meetings and journal clubs to provide a meaningful experience in a research laboratory. An exit interview at the end of the experience will be used to assess areas for improvements. Follow up will be performed in collaboration with Drake Harrison regarding the student s experience and their career path after their research internship in the lab. These activities will lead to broader participation in science and engineering and increase the retention of transfer students from underrepresented backgrounds once they reach a 4-year university. F. Results from Prior NSF Support: Protein and Metabolic Engineering for the Production of Biodegradable Plastics (DMR-9785, PI: Nomura; Period: 7/1/9-6/31/12; amount: $378,). Results from the DMR-9785 funding have provided data for the current proposal. The intellectual merit component of this award focused on metabolic engineering for PHA production. Summary of results of completed work: We developed a transformation protocol for biopolymer producing bacteria. qpcr was used to identify enzymes involved in MCL PHA biopolymer synthesis from the native PHA producing microorganism Pseudomonas putida. These studies led to the discovery and development of a new pathway to produce MCL PHA polymers from unrelated carbon sources such as sugars at the highest levels reported to date. This funding also allowed us to engineer the first and only Escherichia coli strains to date capable of producing PHA polymers with specifically defined repeating unit compositions. The broader impacts component included: (i) work with the ESF SCIENCE program to design scientific experiments with biodegradable polymers targeted towards local underrepresented minority (URM) middle and high school students in order to teach the scientific method; (ii) involvement of 35 undergraduate students from SUNY-ESF, Syracuse University, Clarkson University, Trinity College, Hampton College (15 women, 6 URMs) in the laboratory working on various research projects; and (iii) training of four Ph.D. students (3 women, 1 URM), 2 M.S. students (1 URM woman), and 1 postdoc. In addition, one of the graduate students (Alex Mueller), supported by this grant received a NSF EAPSI fellowship and conducted research on biodegradable polymer production in Dr. Seiichi Taguchi s laboratory at Hokkaido University, strengthening our collaborative efforts. We have been highly productive over the lifetime of this grant and this prior NSF support has led to the publication of 15 peerreviewed articles thus far (9, 38, 68, 7, 84, , , 141, 142) and 2 patent applications (78, 83). Christopher T. Nomura (SUNY-ESF) NSF 15

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27 111. Taguchi, K., Y. Aoyagi, H. Matsusaki, T. Fukui, and Y. Doi Co-expression of 3- ketoacyl-acp reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB11 strain. FEMS Microbiol Lett 176: Taguchi, S., M. Yamada, K. Matsumoto, K. Tajima, Y. Satoh, M. Munekata, K. Ohno, K. Kohda, T. Shimamura, H. Kambe, and S. Obata. 28. A microbial factory for lactate-based polyesters using a lactate-based polyesters using a lactate-polymerizing enzyme. Proc Natl Acad Sci U S A 15: Tajima, K., X. Han, Y. Satoh, A. Ishii, Y. Araki, M. Munekata, and S. Taguchi In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG452 with acquired LA-polymerizing activity. Appl Microbiol Biotechnol 94: Takase, K., S. Taguchi, and Y. Doi. 23. Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase gene. J Biochem 133: Tappel, R. C., J. M. Kucharski, J. M. Mastroianni, A. J. Stipanovic, and C. T. Nomura Biosynthesis of poly[(r)-3-hydroxyalkanoate] copolymers with controlled repeating unit compositions and physical properties.. Biomacromolecules Accepted Tappel, R. C., and C. T. Nomura Recent Advances in Polyhydroxyalkanoate Biosynthesis in Escherichia coli In K. Khemani and C. Scholz (ed.), Degradable Polymers and Materials: Principles and Practice (2nd Edition). Oxford University Press, New York Tappel, R. C., Q. Wang, and C. T. Nomura Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanoate) polymers synthesized in Escherichia coli. J Biosci Bioeng 113: Tsuge, K., K. Matsui, and M. Itaya. 23. One step assembly of multiple DNA fragments with a designed order and orientation in Bacillus subtilis plasmid. Nucleic Acids Res 31:e Tsuge, T., T. Fukui, H. Matsusaki, S. Taguchi, G. Kobayashi, A. Ishizaki, and Y. Doi. 2. Molecular cloning of two (R)-specific enoyl-coa hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS Microbiol Lett 184: Tsuge, T., T. Hisano, S. Taguchi, and Y. Doi. 23. Alteration of chain length substrate specificity of Aeromonas caviae R-enantiomer-specific enoyl-coenzyme A hydratase through site-directed mutagenesis. Appl Environ Microbiol 69: Tsuge, T., K. Taguchi, T. Seiichi, and Y. Doi. 23. Molecular characterization and properties of (R)-specific enoyl-coa hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation. Int J Biol Macromol 31: Tsuji, H. 25. Poly(lactide) stereocomplexes: formation, structure, properties, degradation, and applications. Macomol Biosci Tsuji, H., K. Suzuyoshi, Y. Tezuka, and T. Ishida. 23. Environmental degradation of biodegradable polyesters: 3. Effects of alkali treatment on biodegradation of poly(ecaprolactone) and poly[(r)-3-hydroxybutyrate) films in controled soil. J Polym Environ 11: U.S. Department of Energy, E. I. A. 29. How much oil is used to make plastic? In U. E. I. Administration (ed.) U.S. Environmental Protection Agency, O. o. R. C. a. R. 29. Municipal Solid Waste Generation, Recycling, and Disposal in the United States Detailed Tables and Figures for 28. Report Wang, F., and S. Y. Lee Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli. Appl Environ Microbiol 63: Wang, H. H., X. R. Li, Q. Liu, and G. Q. Chen Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonas putida. Appl Microbiol Biotechnol 89:

28 128. Wang, H. H., X. T. Li, and G. Q. Chen. 29. Production and characterization of homopolymer polyhydroxyheptanoate (P3HHp) by a fadba knockout mutant Pseudomonas putida KTOY6 derived from P. putida KT2442. Process Biochem 44: Wang, Q., A. P. Mueller, C. R. Leong, K. Matsumoto, K. Taguchi, and C. T. Nomura. 21. Quick and efficient method for genetic transformation of biopolymer-producing bacteria. J Chem Technol Biotechnol 85: Wang, Q., and C. T. Nomura. 21. A survey of biodegradable plastics in the U.S. BioPla 36: Wang, Q., and C. T. Nomura. 21. Monitoring differences in gene expression levels and polyhydroxyalkanoate (PHA) production in Pseudomonas putida KT244 grown on different carbon sources. J Biosci Bioeng 11: Wang, Q., R. C. Tappel, C. Zhu, and C. T. Nomura Development of a new strategy for production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) from inexpensive nonfatty acid feedstocks in recombinant Escherichia coli. Appl Environ Microbiol 78: Wang, Q., C. Zhu, T. J. Yancone, and C. T. Nomura The effect of co-substrate feeding on polyhydroxyalkanoate (PHA) homopolymer and copolymer production in recombinant Escherichia coli LS5218. J Bioprocess Eng Biorefinery 1: Yamada, M., K. Matsumoto, T. Nakai, and S. Taguchi. 29. Microbial production of lactateenriched poly[(r)-lactate-co-(r)-3-hydoxybutyrate] with novel thermal properties. Biomacromolecules 1: Yamada, M., K. Matsumoto, K. Shimizu, S. Uramoto, T. Nakai, F. Shozui, and S. Taguchi. 21. Adjustable mutations in lactate (LA)-polymerizing enzyme for the microbial production of LA-based polyesters with tailor-made monomer composition. Biomacromolecules 11: Yamada, M., K. Matsumoto, S. Uramoto, R. Motohashi, H. Abe, and S. Taguchi Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3- hydroxybutyrate)s produced by control of lactyl-coa monomer fluxes in recombinant Escherichia coli. J Biotechnol 154: Yang, T. H., Y. K. Jung, H. O. Kang, T. W. Kim, S. J. Park, and S. Y. Lee Tailormade type II Pseudomonas PHA synthases and their use for the biosynthesis of polylactic acid and its copolymer in recombinant Escherichia coli. Appl Microbiol Biotechnol 9: Yang, T. H., T. W. Kim, H. O. Kang, S. H. Lee, E. J. Lee, S. C. Lim, S. O. Oh, A. J. Song, S. J. Park, and S. Y. Lee. 21. Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase. Biotechnol Bioeng 15: Youn, H., R. L. Kerby, M. Conrad, and G. P. Roberts. 26. Study of highly constitutively active mutants suggests how camp activates camp receptor protein. J Biol Chem 281: Zhou, L., Z. R. Zuo, X. Z. Chen, D. D. Niu, K. M. Tian, B. A. Prior, W. Shen, G. Y. Shi, S. Singh, and Z. X. Wang Evaluation of Genetic Manipulation Strategies on D-Lactate Production by Escherichia coli. Curr Microbiol 62: Zhu, C., C. T. Nomura, J. A. Perrotta, A. J. Stipanovic, and J. P. Nakas. 21. Production and characterization of poly-3-hydroxybutyrate from biodiesel-glycerol by Burkholderia cepacia ATCC Biotechnol Prog 26: Zhu, C., C. T. Nomura, J. A. Perrotta, A. J. Stipanovic, and J. P. Nakas The effect of nucleating agents on physical properties of poly-3-hydroxybutyrate (PHB) and poly-3- hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV) produced by Burkholderia cepacia ATCC Polym Testing 31: Zinn, M., B. Witholt, and T. Egli. 21. Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53:5-21.

29 Biographical Sketch: Christopher T. Nomura Department of Chemistry State University of New York - Environmental Science and Forestry Syracuse, NY 1321 Phone: (315) FAX: (315) ctnomura@esf.edu A. Professional Preparation: University of California, Santa Cruz Biology with honors B.A The Pennsylvania State University Biochemistry, Microbiology, and Ph.D. 21 Molecular Biology RIKEN Institute Polymer Chemistry Postdoc B. Appointments: Associate Professor, Chemistry Department, SUNY-ESF 211-present Assistant Professor, Chemistry Department, SUNY-ESF JSPS Fellow, Polymer Chemistry Laboratory, RIKEN Institute Postdoctoral Fellow, Polymer Chemistry Laboratory, RIKEN Institute C(I). Five Publications Most Relevant to the Proposal: 1. Tappel, R.C., Kucharski, J.M., Mastroianni, J.M., Stipanovic, A.J., and C.T. Nomura (212). Biosynthesis of poly[(r)-3-hydroxyalkanoate] copolymers with controlled repeating unit compositions and physical properties. Biomacromolecules. 13(9) Hiroe, A., Tsuge, K., Nomura, C.T., Itaya, M., and T. Tsuge (212). Rearrangement of phaabc gene order leads to effective production of ultra-high-molecular-weight poly[(r)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl Environ Microbiol. 78(9) Tappel, R.C., Wang, Q., and C.T. Nomura (212). Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanoate) polymers synthesized in Escherichia coli. J Biosci Bioeng. 114(4) Wang, Q., Tappel, R.C., Zhu, C., and C.T. Nomura (212). Development of a new strategy for production of medium-chain-length polyhydroyalkanoates (MCL-PHAs) from inexpensive non-fatty acid feedstocks in recombinant Escherichia coli. Appl Environ Microbiol. 78(2) Nomura, C.T., Taguchi, K., Taguchi, S., and Y. Doi. (24). Coexpression of genetically engineered 3-ketoacyl-ACP synthase III (fabh) and polyhydroxyalkanoate synthase (phac) genes leads to shortchain-length-medium-chain-length polyhydroxyalkanoate copolymer production from glucose in Escherichia coli JM19. Appl Environ Microbiol. 7(2) C(II). Five Other Significant Publications: 6. Ashby, R.D., Solaiman, D.K..Y., Strahan, G.D., Zhu, C., Tappel, R.C., and C.T. Nomura (212). Glycerine and levulinic acid: Renewable co-substrates for the fermentative synthesis of short-chain poly(hydroxyalkanoate) biopolymers. Biores Technol Mueller, A.P. and C.T. Nomura (212). Mutations to the active site of 3-ketoacyl-ACP synthase III (FabH) increase polyhydroxyalkanoate biosynthesis in transgenic Escherichia coli. J Biosci Bioeng. 113(3) Wang, Q. and C.T. Nomura (21). Monitoring differences in gene expression levels and polyhydroxyalkanoate (PHA) production in Pseudomonas putida KT244 grown on different carbon sources. J Biosci Bioeng. 11(6) Matsumoto, K., Murata, T., Nagao, R., Nomura, C.T., Arai, S., Arai, Y., Takase, K., Nakashita, H.,

30 Taguchi, S., and H. Shimada (29). Production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymer in the plastid of Arabidopsis thaliana using an engineered 3- ketoacyl-acyl carrier protein synthase III. Biomacromolecules. 1(4), Nomura, C.T. and S. Taguchi (27). PHA synthase engineering toward superbiocatalysts for custom-made biopolymers. Appl Microbiol Biotechnol. 73(5) D. Synergistic Activities: Honors and awards: 212 ACS NERM Session Chair: Biopolymers; 211 SUNY-ESF Exemplary Researcher; 21 Co-organizer, Biodegradable and Biomass Plastics Symposium, Pacifichem, 28; Panelist and presenter World Congress on Industrial Biotechnology and Bioprocessing; NSF CBET panelist, NSF DMR panelist. Outreach and education: Faculty member, Stewards of Syracuse (SOS) and ESF Summer Camps Investigating Ecology in Neighborhoods and City Environments (ESF S.C.I.E.N.C.E.): Lectures on biodegradable plastics in the environment and liquid nitrogen demonstrations for underrepresented and at-risk middle and high school students from Syracuse City School District, Girls Inc., Boys and Girls Club, Spanish Action League, CNY Works, Catholic Charities of Onondaga County and Syracuse City Parks and Recreation (27-present). Research supervisor, Minority Health and Health Disparities International Research Training Program (MHIRT) RIKEN/UC Santa Cruz summer internship: Supervision of two female underrepresented American undergraduate students at the RIKEN Institute (24). Invited speaker, Minority Access to Research Careers (MARC), UC Santa Cruz: Genetic Engineering for Biodegradable Polymers and Getting Into and Succeeding in a Graduate Program (26). Member: Michael Szwarc Polymer Research Institute (SUNY-ESF), Center for Applied Microbiology (SUNY-ESF), Cellulose Research Institute (SUNY-ESF), Structural Biology, Biochemistry, Biophysics (SB 3 : Syracuse University, SUNY Upstate, SUNY-ESF), Syracuse Biomaterials Institute (SBI: Syracuse University, SUNY Upstate, SUNY-ESF), American Chemical Society, American Society for Microbiology. Reviewer and panelist: Appl Environ Microbiol, Appl Microbiol Biotechnol, Appl Microbiol, Appl Polym Sci, Arch Microbiol, Biochemistry, Biomacromolecules, Biores Technol, Can J Chem, FEMS Microbiol Lett, Int J Biol Macromol, J Am Oil Chem Soc, J Appl Microbiol, J Appl Polym Sci, J Bacteriol, J Biobased Mater Bioenergy, J Biomol Screen, J Biotechnol, J Chem Technol Biotechnol, J Polym Environ, Macromolecules, Mar Biotechnol, Nat Chem Biol, New Biotechnol, Polym Bulletin, Polym Deg Stabil, USDA CSREES Proposals, NSF CBET, NSF DMR. E(I). Collaborators in the last 48 months: R.D. Ashby, D.K.Y. Solaiman, G.D. Strahan (USDA). J.P. Nakas, A.J. Stipanovic (SUNY-ESF), S. Taguchi, K. Matsumoto (Hokkaido University), T. Tsuge (Tokyo Institute of Technology), C.N. Boddy (U. of Ottawa). E(II). Graduate and Postdoctoral Advisors: Ph.D. Advisor: Donald A. Bryant (The Pennsylvania State University) Postdoctoral Advisor: Yoshiharu Doi (RIKEN Institute) E(III). Research Advisor and Postgraduate-Scholar Sponsor (SUNY-ESF): Postdoctoral scholar (1): Dr. B.R. Lundgren (21-present). Graduate students (7): Ms. Q. Wang (211, currently postdoc at Tufts), Mr. R.C. Tappel (28-present), Mr. A.P. Mueller (211, currently scientist at Lanzatech), Ms. L. Salamanca-Cardona (21-present), Ms. X. Wang (21-present), Mr. A. Levine (21-present), Ms. L.C. Izquierdo (211-present). Undergraduate students: >25 supervised for independent research.

31 fm13rs-7 SUMMARY PROPOSAL BUDGET YEAR FOR NSF USE ONLY ORGANIZATION PROPOSAL NO. DURATION (months) SUNY College of Environmental Science and Forestry Proposed Granted PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO. Christopher Nomura NSF Funded A. SENIOR PERSONNEL: PI/PD, Co-PI s, Faculty and Other Senior Associates Funds Funds Person-months Requested By granted by NSF (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different) 1. Christopher Nomura - Principal Investigator , ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) ( 1 ) TOTAL SENIOR PERSONNEL (1-6) ,344 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( ) POST DOCTORAL SCHOLARS ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) ( 2 ) GRADUATE STUDENTS 44, 4. ( ) UNDERGRADUATE STUDENTS 5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( ) OTHER TOTAL SALARIES AND WAGES (A + B) 51,344 C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 9,575 TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 6,919 D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,.) 1 TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. INTERNATIONAL 1, F. PARTICIPANT SUPPORT COSTS 1. STIPENDS $ 5, 2. TRAVEL 3. SUBSISTENCE 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 1 ) TOTAL PARTICIPANT COSTS 5, G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2, 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 1,2 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER 21,526 TOTAL OTHER DIRECT COSTS 42,726 H. TOTAL DIRECT COSTS (A THROUGH G) 19,645 I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE) Modified Total Direct Costs (Rate: 56., Base: 83119) TOTAL INDIRECT COSTS (F&A) 46,547 J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 156,192 K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) 156,192 M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $ PI/PD NAME FOR NSF USE ONLY Christopher Nomura INDIRECT COST RATE VERIFICATION ORG. REP. NAME* Date Checked Date Of Rate Sheet Initials - ORG William nicholson 1 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

32 fm13rs-7 SUMMARY PROPOSAL BUDGET YEAR FOR NSF USE ONLY ORGANIZATION PROPOSAL NO. DURATION (months) SUNY College of Environmental Science and Forestry Proposed Granted PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO. Christopher Nomura NSF Funded A. SENIOR PERSONNEL: PI/PD, Co-PI s, Faculty and Other Senior Associates Funds Funds Person-months Requested By granted by NSF (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different) 1. Christopher Nomura - Principal Investigator , ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) ( 1 ) TOTAL SENIOR PERSONNEL (1-6) ,565 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( ) POST DOCTORAL SCHOLARS ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) ( 2 ) GRADUATE STUDENTS 45,32 4. ( ) UNDERGRADUATE STUDENTS 5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( ) OTHER TOTAL SALARIES AND WAGES (A + B) 52,885 C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 1,647 TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 63,532 D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,.) 2 TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. INTERNATIONAL 1, F. PARTICIPANT SUPPORT COSTS 1. STIPENDS $ 5, 2. TRAVEL 3. SUBSISTENCE 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 1 ) TOTAL PARTICIPANT COSTS 5, G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2, 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 1,2 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER 22,387 TOTAL OTHER DIRECT COSTS 43,587 H. TOTAL DIRECT COSTS (A THROUGH G) 113,119 I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE) Modified Total Direct Costs (Rate: 56., Base: 85731) TOTAL INDIRECT COSTS (F&A) 48,9 J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 161,128 K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) 161,128 M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $ PI/PD NAME FOR NSF USE ONLY Christopher Nomura INDIRECT COST RATE VERIFICATION ORG. REP. NAME* Date Checked Date Of Rate Sheet Initials - ORG William nicholson 2 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

33 fm13rs-7 SUMMARY PROPOSAL BUDGET YEAR FOR NSF USE ONLY ORGANIZATION PROPOSAL NO. DURATION (months) SUNY College of Environmental Science and Forestry Proposed Granted PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO. Christopher Nomura NSF Funded A. SENIOR PERSONNEL: PI/PD, Co-PI s, Faculty and Other Senior Associates Funds Funds Person-months Requested By granted by NSF (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different) 1. Christopher Nomura - Principal Investigator , ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) ( 1 ) TOTAL SENIOR PERSONNEL (1-6) ,792 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( ) POST DOCTORAL SCHOLARS ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) ( 2 ) GRADUATE STUDENTS 46,68 4. ( ) UNDERGRADUATE STUDENTS 5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( ) OTHER TOTAL SALARIES AND WAGES (A + B) 54,472 C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 11,461 TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 65,933 D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,.) 3 TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. INTERNATIONAL 1, F. PARTICIPANT SUPPORT COSTS 1. STIPENDS $ 5, 2. TRAVEL 3. SUBSISTENCE 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 1 ) TOTAL PARTICIPANT COSTS 5, G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 2, 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 1,2 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER 23,282 TOTAL OTHER DIRECT COSTS 44,482 H. TOTAL DIRECT COSTS (A THROUGH G) 116,415 I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE) Modified Total Direct Costs (Rate: 56., Base: 88133) TOTAL INDIRECT COSTS (F&A) 49,354 J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 165,769 K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) 165,769 M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $ PI/PD NAME FOR NSF USE ONLY Christopher Nomura INDIRECT COST RATE VERIFICATION ORG. REP. NAME* Date Checked Date Of Rate Sheet Initials - ORG William nicholson 3 *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

34 fm13rs-7 SUMMARY PROPOSAL BUDGET Cumulative FOR NSF USE ONLY ORGANIZATION PROPOSAL NO. DURATION (months) SUNY College of Environmental Science and Forestry Proposed Granted PRINCIPAL INVESTIGATOR / PROJECT DIRECTOR AWARD NO. Christopher Nomura NSF Funded A. SENIOR PERSONNEL: PI/PD, Co-PI s, Faculty and Other Senior Associates Funds Funds Person-months Requested By granted by NSF (List each separately with title, A.7. show number in brackets) CAL ACAD SUMR proposer (if different) 1. Christopher Nomura - Principal Investigator , ( ) OTHERS (LIST INDIVIDUALLY ON BUDGET JUSTIFICATION PAGE) ( 1 ) TOTAL SENIOR PERSONNEL (1-6) ,71 B. OTHER PERSONNEL (SHOW NUMBERS IN BRACKETS) 1. ( ) POST DOCTORAL SCHOLARS ( ) OTHER PROFESSIONALS (TECHNICIAN, PROGRAMMER, ETC.) ( 6 ) GRADUATE STUDENTS 136, 4. ( ) UNDERGRADUATE STUDENTS 5. ( ) SECRETARIAL - CLERICAL (IF CHARGED DIRECTLY) 6. ( ) OTHER TOTAL SALARIES AND WAGES (A + B) 158,71 C. FRINGE BENEFITS (IF CHARGED AS DIRECT COSTS) 31,683 TOTAL SALARIES, WAGES AND FRINGE BENEFITS (A + B + C) 19,384 D. EQUIPMENT (LIST ITEM AND DOLLAR AMOUNT FOR EACH ITEM EXCEEDING $5,.) TOTAL EQUIPMENT E. TRAVEL 1. DOMESTIC (INCL. CANADA, MEXICO AND U.S. POSSESSIONS) 2. INTERNATIONAL 3, F. PARTICIPANT SUPPORT COSTS 1. STIPENDS $ 15, 2. TRAVEL 3. SUBSISTENCE 4. OTHER TOTAL NUMBER OF PARTICIPANTS ( 3 ) TOTAL PARTICIPANT COSTS 15, G. OTHER DIRECT COSTS 1. MATERIALS AND SUPPLIES 6, 2. PUBLICATION COSTS/DOCUMENTATION/DISSEMINATION 3. CONSULTANT SERVICES 3,6 4. COMPUTER SERVICES 5. SUBAWARDS 6. OTHER 67,195 TOTAL OTHER DIRECT COSTS 13,795 H. TOTAL DIRECT COSTS (A THROUGH G) 339,179 I. INDIRECT COSTS (F&A)(SPECIFY RATE AND BASE) TOTAL INDIRECT COSTS (F&A) 143,91 J. TOTAL DIRECT AND INDIRECT COSTS (H + I) 483,89 K. RESIDUAL FUNDS L. AMOUNT OF THIS REQUEST (J) OR (J MINUS K) 483,89 M. COST SHARING PROPOSED LEVEL $ AGREED LEVEL IF DIFFERENT $ PI/PD NAME FOR NSF USE ONLY Christopher Nomura INDIRECT COST RATE VERIFICATION ORG. REP. NAME* Date Checked Date Of Rate Sheet Initials - ORG William nicholson C *ELECTRONIC SIGNATURES REQUIRED FOR REVISED BUDGET

35 Budget Justification The total amount of support requested from NSF is $483,89 over three years. The money requested from NSF will be used for the following expenses: Personnel: $19,383 over 3 years This charge includes a 5% academic year (AY) charge and 2 weeks of summer salary per year for Dr. Nomura to work on the assigned project with fringe benefit costs starting at 5.16% and increasing to 56.52% for the AY and 17% for the summer appointment. There will be two graduate students as outlined in the proposal (Ms. Lucia Salamanca-Cardona and Ms. Xian Wang) supported throughout the duration of this project. The costs of the graduate students include an indirect fringe cost starting at 14.5% in year 1 and increasing to 18.5% in year 3. Tuition: $67,195 over 3 years Funds are requested to cover the tuition of the graduate students for 3 years. The costs per student are $1,763 in year 1, $11,193 in year 2, and $11,641 in year 3. Travel: $3, over 3 years Travel money is requested to cover expenses of scientists and students to attend, participate, and present results from this study at local and national meetings. These costs will cover meeting registration, room and board, and transportation expenses. Stipend for CSTEP or LSAMP summer undergraduate student: $15, over 3 years Funding is requested to support a summer stipend for a CSTEP or LSAMP student from SUNY OCC to participate in the project as outlined in the Broader Impacts section of the proposal. This stipend will cover living expenses for an undergraduate student during each summer of the proposed work. Administrative fees for SUNY OCC CSTEP and LSAMP participation: $3,6 over 3 years Funding is requested to help defray administrative charges for managing and recruiting students for the CSTEP and LSAMP program. This will be done in collaboration with Drake Harrison at SUNY OCC in order to administrate recruiting goals as outlined in the Broader Impacts section of the proposal. Materials and supplies: $6, over 3 years Consumables, chemicals, and general lab supplies will be purchased from this budget. This charge includes supplies for general lab supplies and disposables such as custom DNA sequences for PCR reactions to manipulate DNA expression vectors for the expression of enzymes in recombinant E. coli, Gibson assembly kits from NEB, and custom gene synthesis. Additionally, reagents for genetic engineering and molecular biology, such as restriction enzymes, Qiagen RNA purification kits, Invitrogen plasmids, PrimeStar polymerase, Pfu polymerase, ligase, agarose, etc. will be required for this project. Miscellaneous lab supplies including gloves, lab coats, safety glasses, flasks, pipet tips, Petri dishes, filters, etc. will be purchased with these funds as well. Costs for DNA sequencing and analytical services from SUNY-ESF will also be paid through this funding. Indirect costs: $143,911 over 3 years SUNY-ESF will charge their federally negotiated rate of 56.% of modified total direct costs.

36 Current and Pending Support (See GPG Section II.D.8 for guidance on information to include on this form.) The following information should be provided for each investigator and other senior personnel. Failure to provide this information may delay consideration of this proposal. Other agencies (including NSF) to which this proposal has been/will be submitted. Investigator: Christopher Nomura Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Use of Synergistic Pretreatment Technologies to Produce Fermentable Sugars from Forest Biomass Source of Support: USDA-CSREES / McIntire Stennis Program Total Award Amount: $53,81 Total Award Period Covered: 8/15/1-9/3/14 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Project. Cal: Acad:.45 Sumr: Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Protein and Metabolic Engineering for the Production of Biodegradable Plastics Source of Support: National Science Foundation Total Award Amount: $378, Total Award Period Covered: 7/1/9-6/3/13 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Project. Cal: Acad:.45 Sumr: Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Biochemical Conversion of Forestry-Derived Feedstocks to Biodiesel Source of Support: USDA-CSREES / McIntire Stennis Program Total Award Amount: $53,81 Total Award Period Covered: 1/1/1-9/3/13 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Project. Cal: Acad:.9 Sumr: Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Production of Polyhydroxyalkanoates with Defined Repeating Unit Composition Source of Support: NYSERDA Total Award Amount: $75, Total Award Period Covered: 12/23/11-12/31/13 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Project. Cal: Acad:.9 Sumr: 2 weeks Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Reduction of Pharmaceutical Compounds in Waste Water Source of Support: Syracuse University Total Award Amount: $7,5 Total Award Period Covered: 4/1/12-3/31/13 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Project. Cal: Acad: Sumr: 3 weeks *If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period. NSF Form 1239 (1/99) USE ADDITIONAL SHEETS AS NECESSARY

37 Current and Pending Support (See GPG Section II.D.8 for guidance on information to include on this form.) The following information should be provided for each investigator and other senior personnel. Failure to provide this information may delay consideration of this proposal. Other agencies (including NSF) to which this proposal has been/will be submitted. Investigator: Christopher Nomura Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Use of Molecular Roadblocks to Define the Role of RpoN in Environmental Response Source of Support: National Science Foundation Total Award Amount: $8,284 Total Award Period Covered: 9/1/12 8/31/15 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Cal: Acad:.45 Sumr: 1 month Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Development of Setaria viridis as a Model for High Biomass C4 Grass Metabolic Engineering, Metabolomics, and Genome Scale Modeling Source of Support: University of North Texas Total Award Amount: $339,959 Total Award Period Covered: 11/15/12-11/14/15 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Cal: Acad:.45 Sumr: 1 month Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Characterization of a Key Regulator of Phenazines in Clinical Isolates of Pseudomonas aeruginosa Source of Support: Research Foundation of SUNY Total Award Amount: $1, Total Award Period Covered: 9/1/12-8/31/14 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Cal: Acad:.45 Sumr: Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: This Proposal: SusChEM: Engineering E. coli for Improved Production of Polyhydroxyalkanoate (PHA)-Based Biodegradable Plastics Source of Support: National Science Foundation Total Award Amount: $483,89 Total Award Period Covered: 6/1/13-5/31/16 Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Cal: Acad:.45 Sumr: 2 weeks Support: Current Pending Submission Planned in Near Future *Transfer of Support Project/Proposal Title: Source of Support: Total Award Amount: $ Total Award Period Covered: Location of Project: SUNY College of Environmental Science and Forestry, Syracuse, New York Person-Months Per Year Committed to the Cal: Acad: Sumr: *If this project has previously been funded by another agency, please list and furnish information for immediately preceding funding period. NSF Form 1239 (1/99) USE ADDITIONAL SHEETS AS NECESSARY

38 Facilities, Equipment, and Other Resources All of the equipment necessary for Dr. Nomura and his group to complete the proposed work is available. Dr. Nomura s personal lab space consists of four separate rooms in Jahn Laboratory at SUNY-ESF, including a main laboratory for general experiments (~8 sq. ft.), instrument room for bench-top fermentation and chromatography experiments (~6 sq. ft.), culture room for bacterial growth (~128 sq. ft.) and a positive air-flow clean room for RNA isolation (~175 sq. ft.). All of the equipment that Dr. Nomura needs to perform the proposed research is available at SUNY-ESF and includes: a Shimadzu 21 GC machine with AOCI2S attachment capable of high throughput analysis of polymer samples, Shimadzu LC21AHT HPLC system with UV detector and RID1A Refractive Index Detector, an additional Shimadzu LC21 GPC system with RID1A Refractive Index Detector for the measurement of polymer M w, M n, and polydispersity, an AKTA Purifier FPLC system for protein purification, BioRad IQ5 real-time quantitative PCR machine, BioRad icycle thermocycler for standard PCR reactions, BioRad GelDoc viewing system, two refrigerated microcentrifuges, three room temperature microfuges, two Sorvall Legend benchtop RT centrifuges with biocontainment rotors, two New Brunswick I26 stackable shakers, two New Brunswick BioFlo31 Fermentors with 2-L and 7-L vessels, Genysis UV/Vis scanning spectrophotometer, Nanodrop spectrophotometer for small volume nucleic acid measurements, laminar flow hood, and a Sorvall Speedvac. Vortexes, stir plates, and analytical balances are also available. There are several Windows XP and Windows 7 workstations in Dr. Nomura s laboratories for data analysis and word processing. In addition, the research group has access to shared facilities and equipment scattered throughout SUNY-ESF s Jahn Laboratory, including the following: Autoclaves, -8 C freezers, -2 C freezers, 4 C, 3 C, and 37 C incubators, Sorvall RC-5B refrigerated centrifuge, Bio-Tek Synergy HT plate reader, and walk-in cold rooms/freezers. SUNY-ESF Analytical & Technical Services (A&TS) ( will provide access to the following: light microscopy laboratory, Perkin Elmer Differential Scanning Calorimeter 4 for thermal characterization, TA Instruments 295 Hi-res Thermogravimetric analyzer for thermo-degradation analyses, Hewlett Packard Model 5989B GC/MS, Hewlett Packard Series 11 MSD LC/MS, Bruker AVANCE 3 MHz and 6 MHz NMR spectrometers, Bruker MALDI TOF/TOF Mass Spectrometer. Technical support will be provided by facilities that are in close proximity to our laboratory. Polymer characterization, including NMR and GC-MS, aspects of the proposed research project will also benefit from expertise provided by the staff of the SUNY-ESF Analytical and Technical Services (A&TS) ( In addition, Dr. Nomura is a faculty member in the Syracuse Biomaterials Institute (SBI). A full description of the facilities and programs associated with it can be found at Dr. Nomura and his group members have easy access to the facilities for SBI which include a number of key instruments for polymer characterization including: TA Instruments Q1 DSC, TA Instruments Q5 for thermogravimetric analysis, Anton Paar MCR51 rheometer, SRS QCM2 Quartz crystal microbalance, Olympus BX-51 polarizing microscope with optical microrheometer, heating/cooling stage, shear cell, spectrographic birefringence apparatus, and Mito CCD attachments, electrospinning apparatus, Pine model AFCBP1 potentiostat, Joel 56 SEM with X-ray spectrophotometer, Digital Instruments Nanoscope III AFM, Instron 135 hydraulic testing apparatus, Perkin-Elmer Spectrum One Fourier Transform Infrared Spectroometer, Perkin Elmer Pyris 1 DSC, and many other devices for polymer characterization. Electron microscopy studies can be carried out at the N.C. Brown Center for Ultrastructure Studies at SUNY-ESF ( equipped with a JEOL 2EX 8-2 KV transmission electron microscope and a JEOL 58 low vacuum scanning electron microscope equipped with an EDAX energy dispersive X-ray analyzer.

39 Data Management Plan The following is the Data Management Plan for the proposal SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics submitted to the NSF CBET division in Biotechnology, Biochemical and Biomass Engineering (BBBE). Types of data The data generated from the work described in this proposal will consist of a variety of PHA polymers, polymer characterization data, bacterial strains, plasmids, graphs, reconstructed images, photographs, movies, hand recorded observations, etc. These data will be in both raw and processed forms and will include relevant statistical analyses. These data will be collected using instruments and methods described in the proposal and will include datasets that have been generated from commonly accepted data acquisition software, with essential metadata presented as headers in the relevant electronic files, or included along with the indexed laboratory notebook narrative. In addition, data will be generated regarding CSTEP and LSAMP fellows on advancement from the community college environment into 4-year universities or into technical positions related to the science generated by the proposed project. Records of results will be labeled and stored as has hard copy, digitized images, and electronic files. In some cases, observations of bacterial physiology, viability or phenotype, polymer morphology, polymer properties, etc. will be retained in hand written or electronic notation that will be dated and labeled in dated laboratory notebooks. The data produced by these experiments will provide information about the use of PHA polymers to capture hydrophobic pollutants from water. New oil filtration materials are expected as an outcome of these studies. These results will be of interest to the environmental engineering and wastewater treatment communities and especially to those who study the filtration of water. Archiving and retention of data Original data notebooks will be retained in a secure location in the PI s laboratory with electronic data backed up on the ESF server whenever the nature of the data allows for archiving. Data will be retained at least three years beyond the award period, as required by NSF. In the event that discoveries or inventions are made in direct connection with this data, access will be granted upon request once appropriate invention disclosures and/or provisional patent filings are made. Key data relevant to the discovery will be preserved until all issues of intellectual property are resolved. Biological samples of bacterial strains, plasmids, etc. generated will be stored at 4 C and -2 C short term and for longer-term will be stored at -8 C in an ultralow freezer. Access to data and data sharing practices and policies If requested, data will be made available for sharing to qualified parties by the PI, so long as the request does not compromise intellectual property interests, interfere with publication, invade subject privacy, betray confidentiality, or precede archiving of the data. Data will be available for access and sharing in a reasonable time period, normally no longer than two years after its acquisition. Management for data and samples generated by the proposed research that comprise parts of intellectual property will be handled by the Technology Transfer office of the Research Foundation of SUNY. These properties may be transferred to individuals or universities dependent on non-disclosure and/or materials transfer agreements, or licensing agreements with the Research Foundation of SUNY and SUNY-ESF. Dissemination of data Typical routes of data dissemination will be through peer-reviewed journal articles and seminars or poster presentations at local, national, or international meetings. In addition, annual and final project reports to NSF will summarize these findings.

40 Christopher T. Nomura, Ph.D. SUNY-ESF Department of Chemistry 318 Jahn 1 Forestry Drive Syracuse, NY 1321 Dear Chris, I am writing this letter to confirm our commitment to collaborate with you on your research proposal SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA) biodegradable plastics submitted to the NSF. The Collegiate Science and Technology Entry Program (CSTEP) and Louis Stokes Alliance for Minority Participation (LSAMP) programs at Onondaga Community College share a commitment to increase the number of college graduates from ethnic groups that are traditionally underrepresented in technical-related fields and individuals who are interested in pursuing careers in science, technology, math, and engineering. We understand that if you are funded for this project that you will provide a stipend of $5 and mentoring for a CSTEP or LSAMP students for a summer research experience where the students will have the opportunity to perform research in molecular biology, microbiology, and chemistry related to your proposal. We are also looking forward to having you participate in our Research Symposium and provide seminars as a faculty mentor to CSTEP and LSAMP students in our program. Dr. Nomura agrees to provide an evaluation of the success of CSTEP/LSAMP students who participate in his summer research lab. Sincerely, Drake Harrison, Director Collegiate Science Technology Entry Program, Co-Pi ULSAMP and S-STEM Programs 4585 West Seneca Turnpike Syracuse, New York harrisod@sunyocc.edu T

41 August 31, 212 Christopher T. Nomura, Ph.D. SUNY-ESF Department of Chemistry 318 Jahn 1 Forestry Drive Syracuse, NY 1321 USA Dear Dr. Nomura, I am very happy to provide you with my strongest letter of support for your NSF proposal: SusChEM: Engineering E. coli for improved production of polyhydroxyalkanoate (PHA)-based biodegradable plastics. As part of our ongoing collaboration, I will gladly act as a collaborator and consultant to the project and provide your laboratory with the expertise and reagents required to reach your proposal goals. I am especially excited about the engineering of E. coli using a combination of our lactatepolymerizing enzyme and pathways with those providing medium-chain-length PHA monomers. This should result in the production of completely novel sets of polymers with a broad range of potential applications and represents the next frontier in producing biobased and biodegradable polymers from renewable resources. I believe that by working together, our laboratories have a great chance of reaching the goal of engineering a model bacterial system for making these polymers. I believe that your experimental approach is extremely novel and has great potential to produce high levels of PHA-based polymers with desirable material properties. I look forward to working closely with you on this project. Sincerely, Seiichi TAGUCHI, Ph. D. Professor of Molecular BioEngineering Division of Molecular Chemistry The Graduate School of Engineering Hokkaido University N14W8, Kita-ku, Sapporo , Japan staguchi@eng.hokudai.ac.jp,