Enabling Robust Production of Biorenewable Fuels and Chemicals from Biomass

Transcription

1 Enabling Robust Production of Biorenewable Fuels and Chemicals from Biomass Laura R. Jarboe, Assistant Professor Chemical and Biological Engineering Iowa State University

2 History of Biocatalysis Biotechnology through the ages: selection for desirable traits Biotechnology Revolution enables us to not just select for desired traits, but to understand, model and manipulate biological systems. The directed improvement of production, formation or cellular properties through the modification of specific biochemical reaction(s) or the introduction of new one(s) with the use of recombinant DNA technology. Metabolic Engineering Science , Stephanopoulos/Vallino, Bailey

3 Tolerance to Inhibitory Compounds biomass-derived sugars biorenewable fuel or chemical furfural 5-HMF acetate phenols Both ends of biocatalyst metabolism are affected by inhibitory compounds ethanol butanol carboxylic acids limonene Strategies for dealing with inhibition: - Selectively remove inhibitor (may increase cost) - Increase tolerance of biocatalyst to the inhibitory compound 3/25

4 Omics Analysis Generalized Strategy inhibitor-sensitive biocatalyst Ideas from literature mechanism(s) of inhibition Will discuss 3 examples for this reverse strategy: furfural, carboxylic engineering acids, pyrolytic sugars Metabolic Evolution for Tolerance Rational Engineering 4/25 inhibitor-resistant biocatalyst

5 How to Extract Sugars from Biomass? Challenge: high enzyme cost, long residence time Furfural toxicity limits biological utilization of biomass hydrolysate. How to make the bacterial biocatalyst more robust? enzymes acid, steam, pressure Challenge: inhibitors (i.e. acetic acid and furfural) fermentable sugars Work performed in Ingram Lab, University of Florida Miller, Jarboe et al AEM :613 Miller, Jarboe et al AEM :4315 Turner et al J Industr Microbiol Biotechnol 2010 biorenewables

6 Transcriptome Analysis of Furfural Challenge SF ArgR ArcA stalled translation TyrR furfural altered redox ratio biosynthesis intermediate accumulation PurR? 3 NADPH D-cys cys/met depletion? RutR furfuryl alcohol SO 3 2- H 2 S cys met SAM homoserine MetJ CysB O-acetyl-L-serine Increased transcription factor activity or metabolite abundance Decreased transcription factor activity or metabolite abundance Hypothesis: furfural is inhibitory because its reduction depletes NADPH pools, limiting H 2 S biosynthesis supplementation with D-cys, S 2 O 2-3, L-cys, cystathionine or met or use of glucose as carbon source instead of xylose improve tolerance

7 Rationally Improving Furfural Toxicity How can we rationally increase furfural tolerance? - Supplement with a metabolite that supplies reduced S (costly) - Grow on glucose instead of xylose (outside of our overall goal) - Engineer the biocatalyst for increased NADPH availability + 1 g L -1 furfural Interpretation of transcriptome data is based on known genes and pathways. Can we learn even more by reverse engineering an evolved strain? 7/25

8 Furfural Strategy inhibitor-sensitive biocatalyst Transcriptome Analysis mechanism(s) of inhibition reverse engineering (transcriptome) Metabolic Evolution for Tolerance 8/25 Rational Engineering inhibitor-resistant biocatalyst

9 Metabolic Evolution Fresh media Spent media Stressful condition, cells grow poorly A random mutation confers increased stress tolerance The mutated cell grows faster, its progeny dominate the population Another random mutation confers even more tolerance 9/25

10 Cell Mass (g L -1 ) Ethanol (g L -1 ) Reverse Engineering a Furfural-Tolerant Mutant 10 Growth Ethanol evolved mutant control Time (h) evolved mutant What is the basis of tolerance? Would like to apply to other biocatalysts so that they can be rationally engineered for furfural tolerance instead of relying on evolution Time (h) control AM1 minimal media + 9% xylose + 1 g L -1 furfural 1mM betaine, ph 6.5, 37C 150rpm 54 serial transfers, g L -1 furfural

11 Decreased Furfural Reduction Rate in Evolved Strain Cell Mass (g L -1 ) in vivo Furfural Reduction -1 ) ( m mol min -1 mg dcw L-cys OALS 10/25 control DyqhD DdkgA DyqhD, control +yqhd DdkgA ace parent furfural 3 NADPH H 2 S SO 3 2- evolved mutant furfuryl alcohol YqhD, DkgA SO Parent +1 g L -1 furfural control DdkgA DyqhD, DdkgA DyqhD The evolved mutant found its own way to increase availability of NADPH: silencing of the furfural reductase YqhD Have since found that mutation of YqhC is the basis of yqhd silencing Subsequent rational engineering described in Wang et al AEM 2011

12 Furfural Conclusions Strategies for increasing furfural tolerance: - Mitigate cys depletion by supplementation ($$) - Increase NADPH availability - Use glucose as carbon source (outside our project goal) - Use transhydrogenase to convert NADH to NADPH (effective) - Silence NADPH-dependent aldehyde reductase (effective) furfuryl furfural alcohol YqhD 3 NADPH cys/met depletion SO 3 2- H 2 S cys met Take-away lesson: Interpretation based on existing biocatalyst knowledge is effective, but we still have much to learn about even our most well-characterized biocatalysts 11/25

13 Bio-mass Derived Sugars engineered biocatalysts Commodity Chemicals ethanol insulin butanol lactic acid 1,3-propanediol succinate lycopene amorphadiene 12/25 engineered biocatalysts (E. coli, yeast) chemical intermediates catalysis catalysis industrial chemicals One-Use Carbon transportation fuels

14 Specific growth rate (hr -1 ) 0.8 Glucose Short-Chain Carboxylic Acids R COOH R a-olefins 13/ C6 C8 C10 Problem: Carboxylic acidproducing biocatalysts must be able to tolerate carboxylic acids at high titer. But these compounds inhibit biocatalyst growth Carboxylic acid concentration (mm)

15 Flux Analysis Ideas from literature Omics Analysis mechanism(s) of inhibition inhibitor-sensitive biocatalyst reverse engineering Metabolic Evolution for Tolerance Rational Engineering 14/25 inhibitor-resistant biocatalyst

16 Growth Rate (h -1 ) Transcriptome Analysis /25 10mM ~10% growth inhibition Concentration C8 (mm) gene name function Fold Change p-value yagu inner membrane protein that contributes to acid resistance ffs 4.5S RNA signal recognition particle (SRP) ybas glutaminase dps stationary phase nucleoid complex that sequesters iron ompx overexpression increases sigmae activity; outer membrane protein ybjc predicted inner membrane protein ompf porin; allows passage of solutes bhsa involved in stress resistance and biofilm formation ycgz predicted protein ymga involved in biofilm formation rpsv 30S rrna protein subunit gadc part of glutamate-dependent acid resistance system (AR2) gadb part of glutamate-dependent acid resistance system (AR2) mara regulates genes involved in resistance (antibiotics, oxidative stress, solvents, heavy metals) flxa Qin prophage, predicted protein cfa cyclopropane fatty acid synthase yeed conserved protein yeee putative permease ddg palmitoleoyl acytltransferase; used to incorporate palmitoleate into lipid A instead of laurate ygdi putative lipoprotein mscs mechanosensitive channel; induced by osmotic stress ygiw conserved protein yhbw conserved protein yhcn conserved protein ompr regulator component of two-component system; responds to EnvZ; EnvZ senses changes in yhid predicted Mg-ATPase, may be involved in acid resistance hdeb acid stress chaperone hdea acid stress chaperone hded acid resistance membrane protein gade activator of glutamate-dependent acid resistance gadw regulates glutamate dependent acid resistance system (GAD) gadx regulates glutamate-dependent acid resistance system (GAD) gada glutamate decarboxylase, part of glutamate-dependent acid resistance system osmy hyperosmitcally inducible periplasmic protein micf anti-sense RNA, inhibits ompf translation

17 Intracellular ph From omics analysis: C8 stress involves acid stress Control +20mM C8 C8 HCl +20mM +20mM C8 ph=7 HCl +20mM HCl ph=7 +2% Ethanol Even when the external media is maintained at ph 7.0, the presence of short-chain carboxylic acids can result in a drop in cytoplasmic ph HA H + A- HA HA H + A- media: ph can be controlled inside cell: ph cannot be controlled, ph is critical Engineering Approach: - Utilize native acid resistance systems - Express proton-buffering peptides - Pump out the carboxylic acids Royce, Liu et al, in preparation

18 Membrane polarization % Mg2+ released by chloroform E. coli carboxylic acid stress/production involves membrane damage Carboxylic Acids Produced (g/l) Membrane fluidity Octanoic acid (mm) Membrane leakage Royce, Liu et al, in preparation Octanoic acid (mm) total carboxylic acids Mg leakage Time (h) The presence of carboxylic acids impacts membrane fluidity and integrity, with stronger impact than ethanol or heat shock. Similar effects were seen during carboxylic acid production. 0 % Mg 2+ released relative to CHCl 3

19 10 control 10 control OD C8 OD C8 parent strain evolved mutant Time (h) Time (h) Goal: Reverse engineer fatty acid tolerance, learn new strategies for dealing with fatty acids Genome sequence data analysis in progress 19/25 Royce, in preparation

20 Biorenewable Chemicals from Biomass BIOCATALYSIS Brown Gold sugarcane bagasse Lake Okeechobee, Florida

21 How to Extract Sugars from Biomass? Challenge: complex, unstable mixture, low sugar content, inhibitors Benefit: fast, cheap, applicable to any biomass type thermochemical processing (pyrolysis) acid, steam, pressure Challenge: high enzyme cost, long residence time enzymes Challenge: inhibitors (i.e. acetic acid and furfural) fermentable sugars Hybrid processing: Thermochemical processing of biomass, Biological utilization of thermochemical products. biorenewables

22 Ethanol, wt% Sugar, wt% Engineering Pyrolytic-Sugar Utilizing Biocatalysts Sugar utilization levoglucosan glucose Existing biocatalysts can easily be engineered for utilization of levoglucosan as carbon/energy source with same redox, ATP demand as glucose time (hr) Ethanol production levoglucosan glucose time (hr) 22/25 LB + pure sugars, 37C, ph 6.5 Layton et al Bioresource Tech 2011

23 Pyrolytic Sugar Strategy inhibitor-sensitive biocatalyst Transcriptome Project outcome: A list of modifications to implement in existing bacterial Analysis biocatalysts to enable pyrolytic sugar utilization. mechanism(s) of inhibition reverse engineering Metabolic Evolution for Tolerance Rational Engineering 23/25 inhibitor-resistant biocatalyst

24 Inhibitor Tolerance Conclusions - Product toxicity or contaminants in dirty sugars limit production of biorenewable fuels and chemicals - Finding the mechanism of toxicity enables rational engineering for inhibitor tolerance - Reverse engineering of evolved strains can reveal (a) the mechanism of inhibition (b) useful mutations and (c) increase characterization of existing workhorse strains 24/25

25 Acknowledgements PI: Lonnie Ingram Researcers: Elliot Miller, Brelan Moritz, Christy Baggett Collaborators: K.T. Shanmugam, Priti Pharkya, David Nunn EEC Graduate Students: Liam Royce, Ping Liu Researchers: Matt Stebbins, Brittany Rover, Emily Rickenbach, Ben Hanson, Jennifer Au Collaborators: Jackie Shanks, Julie Dickerson, Ramon Gonzalez, Kai-Yu San Researchers: Zhanyou Chi, Tao Jin, D. Layton, M. Deaton, S. Steffen, J. Kuyper, B. Sorensen, A. Rossinger Collaborators: Zhiyou Wen, Robert C. Brown, D.W. Choi Funding: NSF Energy for Sustainability, Iowa Energy Center, ISU Bioeconomy Institute, ISU Plant Sciences Institute CBET Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.