on September 5, 2018 by guest

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1998, p Vol. 64, No /98/$ Copyright 1998, American Society for Microbiology. All Rights Reserved. Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli JONG-IL CHOI, 1,2 SANG YUP LEE, 1,2 * AND KYUBOEM HAN 3 Department of Chemical Engineering 1 and BioProcess Engineering Research Center, 2 Korea Advanced Institute of Science and Technology, Kusong-dong, Yusong-gu, Taejon , and Biotech Research Institute II, LG Chemicals, Ltd., Science Town, Taejon , 3 Korea Received 23 July 1998/Accepted 30 September 1998 Polyhydroxyalkanoates (PHAs) are microbial polyesters that can be used as completely biodegradable polymers, but the high production cost prevents their use in a wide range of applications. Recombinant Escherichia coli strains harboring the Ralstonia eutropha PHA biosynthesis genes have been reported to have several advantages as PHA producers compared with wild-type PHA-producing bacteria. However, the PHA productivity (amount of PHA produced per unit volume per unit time) obtained with these recombinant E. coli strains has been lower than that obtained with the wild-type bacterium Alcaligenes latus. To endow the potentially superior PHA biosynthetic machinery to E. coli, we cloned the PHA biosynthesis genes from A. latus. The three PHA biosynthesis genes formed an operon with the order PHA synthase, -ketothiolase, and reductase genes and were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains harboring the A. latus PHA biosynthesis genes accumulated poly(3-hydroxybutyrate) (PHB), a model PHA product, more efficiently than those harboring the R. eutropha genes. With a ph-stat fed-batch culture of recombinant E. coli harboring a stable plasmid containing the A. latus PHA biosynthesis genes, final cell and PHB concentrations of and g/liter, respectively, were obtained, resulting in a high productivity of 4.63 g of PHB/liter/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness. Recently, problems concerning the global environment have created much interest in the development of biodegradable polymers. Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates that are synthesized and intracellularly accumulated as an energy and/or carbon storage material by numerous microorganisms (1, 5, 15, 28). PHAs are considered to be good candidates for biodegradable plastics and elastomers since they possess material properties similar to those of synthetic polymers currently in use and are completely biodegradable after disposal (9). A major problem in the commercialization of PHAs in a wide range of applications is their high production cost (3, 4). Much effort has been devoted to lowering the production cost by developing more efficient fermentation and recovery processes (15, 16, 28). Poly(3-hydroxybutyrate) (PHB) is the best known member of the PHAs and has been studied most often as a model product in the development of fermentation strategies. To understand the mechanisms of PHA biosynthesis, studies on the metabolic pathways for PHA biosynthesis and molecular analyses of PHA biosynthesis genes in various bacteria have been conducted. In Ralstonia eutropha (formerly known as Alcaligenes eutrophus), acetyl coenzyme A is converted to PHB in three enzymatic steps (1, 35). A biosynthetic -ketothiolase catalyzes the formation of a carbonocarbon bond by biological Claisen condensation of two acetyl coenzyme A moieties. An NADPH-dependent acetoacetyl coenzyme A (acetoacetyl- * Corresponding author. Mailing address: Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Kusong-dong, Yusong-gu, Taejon , Korea. Phone: Fax: leesy@sorak.kaist.ac.kr CoA) reductase catalyzes the stereoselective reduction of acetoacetyl-coa to D-( )-3-hydroxybutyryl coenzyme A. The third reaction of this pathway is catalyzed by a PHA synthase, which links the D-( )-3-hydroxybutyryl coenzyme A to the growing chain of PHB by an ester bond. After the first cloning of the PHA biosynthesis genes from R. eutropha (26, 32, 34), more than 30 different PHA biosynthesis genes were cloned from various bacteria (15). Cloning of various PHA biosynthesis genes not only has provided detailed information on the structure and organization of the PHA biosynthesis genes but also has allowed the creation of genetically engineered microorganisms or even plants for more efficient production of these biodegradable polymers and for the production of novel PHAs (16, 28). One of the major factors affecting the overall production cost is productivity, defined as the amount of PHB produced per unit volume per unit time. R. eutropha and Alcaligenes latus have been used most often for the production of PHB, since PHB could be produced to a high concentration with high productivity (29, 39). Recombinant Escherichia coli strains harboring the R. eutropha PHA biosynthesis genes have also been used for the production of PHB (19, 22). A PHB concentration of as high as 157 g/liter could be obtained with a ph-stat fed-batch culture (40). Recombinant E. coli has been considered a strong candidate as a PHB producer due to several advantages over wild-type PHB producers, such as a wide range of utilizable carbon sources, PHB accumulation to a high content (up to 90% of cell dry weight), fragility of cells allowing easy recovery of PHB, and no degradation of PHB during fermentation due to the lack of intracellular depolymerases (6, 17). However, the highest productivity obtained with recombinant E. coli was 3.4 g of PHB/liter/h (40), considerably lower than that obtained with A. latus (4.94 g of PHB/liter/h) (39). Downloaded from on September 5, 2018 by guest

2 4898 CHOI ET AL. APPL. ENVIRON. MICROBIOL. Since A. latus is able to produce PHB with the highest productivity reported to date, it is reasonable to assume that this bacterium possesses more efficient PHA biosynthesis enzymes. It was therefore thought that recombinant E. coli harboring the A. latus PHA biosynthesis genes might produce PHB with a higher productivity without a loss of the advantages mentioned earlier. In this study, we report the cloning and molecular analysis of the A. latus PHA biosynthesis genes in E. coli. We also report the development of several different recombinant E. coli strains harboring these genes, the characteristics of these strains with regard to growth and PHB production, and the results obtained from high-cell-density fed-batch cultures. There was a 36% improvement in PHB productivity (from 3.4 to 4.63 g/liter/h) by the newly developed recombinant E. coli strains, a result which will make it possible to economically produce this biodegradable polymer. MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. A. latus was cultivated in nutrient broth medium (Difco Laboratories, Detroit, Mich.) at 30 C. E. coli was routinely grown in Luria-Bertani (LB) medium at 37 C. LB medium supplemented with 20 g of glucose per liter was used as a PHB accumulation medium. When required, ampicillin (50 mg/liter) was added to the medium. DNA manipulation and library construction. Total genomic DNA of A. latus was isolated by the procedure described by Marmur (25). A plasmid library of A. latus total DNA was constructed by inserting A. latus genomic DNA fragments partially digested with Sau3AI into BamHI-digested puc19, followed by transformation into E. coli XL1-Blue. Plasmid DNA isolation, agarose gel electrophoresis, and transformation of E. coli were carried out as described by Sambrook et al. (30). Restriction enzymes and modifying enzymes were purchased from New England Biolabs, Beverly, Mass., and were used as recommended by the manufacturer. DNA sequence analysis. DNA sequencing was carried out by the site-sequencing method with an ABI model 377 automated DNA sequencer (The Perkin- Elmer Corp.). Computer analysis of the resulting nucleotide sequence was performed with the DNASIS DNA and protein sequence analysis program (Hitachi Software Engineering Co., Yokohama, Japan). Culture conditions for the production of PHB. For flask and fed-batch cultures of recombinant E. coli, chemically defined R (14) or MR (40) medium was used. Separately sterilized glucose and thiamine were used as supplements at final concentrations of 20 g/liter and 10 mg/liter, respectively. Fed-batch cultures were incubated at 30 C in a 6.6-liter jar fermentor (Bioflo 3000; New Brunswick Scientific Co., Edison, N.J.) containing 1.2 liters of MR medium. The culture ph was kept at 6.9 by the addition of 28% (vol/vol) ammonia water. Antifoam 289 (0.02% [vol/vol]; Sigma Chemical Co., St. Louis, Mo.) was added at the onset of cultivation. The feeding solution used for the fed-batch culture contained, per liter, the following: glucose, 700 g; MgSO 4 7H 2 O, 15 g; and thiamine, 250 mg. TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristics Reference or source Strains A. latus Wild type DSM 1123 E. coli XL1-Blue supe44 hsdr17 reca1 enda1 gyra96 thi rela1 lacf [proab laci q lacz M15 Tn10 (Tc r )] Stratagene a Plasmids puc19 Ap r lacz New England Biolabs puc18 Ap r lacz New England Biolabs psyl104 pgem-7zf( ) b derivative; parb c Ap r ; carries the R. eutropha phac-phaa-phab operon 22 psyl105 pbluescript KS( ) a derivative; parb Ap r ; carries the R. eutropha phac-phaa-phab operon 20 psyl107 pbluescript KS( ) derivative; parb Ap r ; carries the R. eutropha phac-phaa-phab operon; ftsz d 14 pjc1 puc19 derivative; Ap r ; carries the A. latus phac-phaa-phab operon This study pjc2 pgem-7zf( ) derivative; parb Ap r ; carries the A. latus phac-phaa-phab operon This study pjc3 pjc1 derivative; Ap r ; carries the A. latus phac-phaa-phab operon This study pjc4 pjc2 derivative; parb Ap r ; carries the A. latus phac-phaa-phab operon This study a Stratagene Cloning Systems, La Jolla, Calif. b Promega, Madison, Wis. c parb (hok/sok) locus of plasmid R1. d E. coli ftsz gene. The ph-stat feeding strategy was used for fed-batch cultures. When the ph rose to a value greater than its set point (6.9) by 0.1, an appropriate volume of feeding solution was automatically added to increase the glucose concentration in the culture medium to 20 g/liter. The feeding solution volume was calculated on-line with fermentation software (AFS3.42; New Brunswick Scientific Co.). Analytical procedures. Growth was monitored by measuring the optical density at 600 nm. Cell concentration, defined as cell dry weight per liter of culture broth, was determined by weighing dry cells as described previously (40). The PHB concentration was determined by gas chromatography (HP5890; Hewlett- Packard, Wilmington, Del.) with benzoic acid as an internal standard (2). PHB content was defined as the ratio of PHB to cell dry weight and expressed as a percentage. Nucleotide sequence accession number. The nucleotide sequence data reported here will appear in the GenBank nucleotide sequence database under accession no. AF RESULTS Cloning and molecular analysis of the A. latus PHA biosynthesis genes. To clone the A. latus PHA biosynthesis genes, we used the screening strategy of conferring the ability to accumulate PHB to E. coli by an introduced recombinant plasmid. The transformed E. coli cells were replica plated on solid PHB accumulation medium. Because PHB-accumulating cells form opaque colonies, the E. coli transformants harboring all of the A. latus PHA biosynthesis genes will show a turbid colony phenotype if the genes are functionally expressed. The opaque colonies were isolated and separately cultivated in LB medium containing 20 g of glucose per liter. The presence of PHB in recombinant E. coli was confirmed by microscopic observation of intracellular inclusion bodies and by gas chromatographic analysis. One recombinant clone accumulating a large amount of PHB was isolated and characterized further. It was found to harbor a 6.3-kb A. latus genomic DNA fragment, referred to as AL63. Recombinant plasmid puc19 containing AL63 was referred to as pjc1. The entire 6,286 bp of AL63 was sequenced. The open reading frames in fragment AL63 were analyzed, and the PHA biosynthesis genes were identified by a homology search (Fig. 1). The product of ORF1 (1,608 bp), a protein composed of 536 amino acids and having a molecular mass of 59,621 Da, had high amino acid identities with PHA synthases of R. eutropha (62%) (26, 32, 34), an Alcaligenes sp. (62%) (13), and Methylobacterium extorquens (63%) (38). The Cys residue (cysteine 266) and the Ser residue (serine 207) that have been found to be conserved in all PHA synthases (7, 8, 15) were also

3 VOL. 64, 1998 A. LATUS PHA SYNTHESIS GENE CLONING AND APPLICATION 4899 FIG. 1. Organization of A. latus PHA biosynthesis genes. (A) Restriction map of the AL63 fragment. (B) Organization of phac Al, phaa Al, phab Al, and ORF4. aa, amino acids. found in the amino acid sequence of the ORF1 product (Table 2). Therefore, ORF1 was concluded to represent a structural gene for the A. latus PHA synthase (referred to as phac Al ). ORF2 (1,029 bp), located immediately downstream of pha- C Al, encoded a protein composed of 343 amino acids and having a molecular mass of 35,406 Da. This putative gene product had high sequence identities with -ketothiolases of R. eutropha (67%) (26, 32, 34), Paracoccus denitrificans (57%) (37), and Thiocystis violacea (60%) (23). Therefore, ORF2 was concluded to represent a structural gene for the A. latus -ketothiolase (referred to as phaa Al ). The putative gene product translated from ORF3 (735 bp) had high sequence identities with acetoacetyl-coa reductases of R. eutropha (75%) (26, 32, 34) and Chromatium vinosum (64%) (24). Therefore, ORF3 was concluded to represent a structural gene for the A. latus acetoacetyl-coa reductase (referred to as phab Al ). As shown in Fig. 1B, these three genes form an operon in the TABLE 2. Partial alignment and identity of the deduced amino acid sequence of PHA synthase from A. latus with those from other organisms Microorganism or gene (reference) Amino acid sequence a amino acid % Identity of total sequence Alcaligenes latus ---DLQPDNSLIRYTV----VNALGFCVGGTI Acinetobacter sp. (31) ---DLREQNSLVNWLR----ANCIGYCIGGTL Aeromonas caviae (7) ---DMRPQNSLVAWLV----VHGIGYCIGGTA Ralstonia eutropha (26, 32, 34) ---DLQPESSLVRHVV----INVLGFCVGGTI Alcaligenes sp. (13) ---DLQPENSLIPYAV----INALGFCMGGTI Chromatium vinosum (24) ---DIQEDRSTIKGLL----VNLLGICQGGAF Methylobacterium extorquens (38) ---DLNPQKSLIGWMV----VAAAGYCVGGTL Paracoccus denitrificans (37) ---DLKPONSLIKWIV----LNAVGYCIAGTT Pseudomonas aeruginosa phac1 (36) ---DLSPDKSLARFCL----LNLLGACSGGIT Pseudomonas aeruginosa phac2 (36) ---DLSPEKSFVQYAL----VNLAGACAGGLT Pseudomonas oleovorans phac1 (10) ---DLSPEKSLARYCL----LNMLGACSGGIT Pseudomonas oleovorans phac2 (10) ---DLSSTNSFVQYML----PNLMGACAGGLT Rhodococcus ruber (27) ---DLAPGRSLAEWAV----IEVLSICLGGAM Rhodobacter sphaeroides (11) ---DLKPQNSLLKWLV----INAVGYCIAGTT Thiocystis violacea (23) ---DIQEDRSTIKGLL----VNILGICQGGAF a Dashes indicate gaps. Boldface S indicates conserved Ser; boldface C indicates conserved Cys (active site).

4 4900 CHOI ET AL. APPL. ENVIRON. MICROBIOL. order phac Al -phaa Al -phab Al, coding for PHA synthase, -ketothiolase, and reductase, respectively. The putative ribosome binding sites were identified in the spaces between phac Al and phaa Al and between phaa Al and phab Al. A putative E. coli 70 -dependent promoter-like sequence was found in the region upstream of the phac Al gene. An inverted-repeat structure, which may serve as a transcription termination signal, was found in the region downstream of phab Al and had a structural free energy of 25.1 kcal/mol. ORF4 (636 bp), which would code for a protein with a calculated molecular mass of 24,121 Da, was located upstream of phac Al. The consensus sequence of the 70 -dependent promoter was found in the region upstream of ORF4. An inverted repeat was identified in the region downstream of ORF4 (nucleotides 1030 to 1053) and had a structural free energy of 23.9 kcal/mol. Construction of recombinant plasmids harboring the A. latus PHA biosynthesis genes. Since pjc1 had a segregational instability problem during fed-batch culturing in the absence of antibiotic pressure (data not shown), a stable plasmid, pjc2, was constructed by cloning the 6.3-kb EcoRI-HindIII fragment containing the A. latus PHA biosynthesis genes into a stable high-copy-number plasmid, psyl104 (22), digested with the same restriction enzymes; thus, the R. eutropha PHA biosynthesis genes were replaced (Fig. 2). Plasmid pjc3 was constructed by removing a fragment of about 1 kb of unnecessary DNA upstream of the promoter region of the A. latus PHA biosynthesis genes in pjc1. The fragment removed from pjc1 included the structural gene of ORF4 and its putative promoter and inverted-repeated regions. Plasmid pjc4, a stable version of pjc3, was constructed by cloning the 5.3-kb EcoRI-HindIII fragment containing the A. latus PHA biosynthesis genes into similarly digested psyl104 (Fig. 2). Plasmids pjc3 and pjc4 were stably maintained during fed-batch culturing in the absence of antibiotic pressure (data not shown). To examine whether the expression of the cloned genes was affected by the lac promoter in puc vectors, pjc1r and pjc3r were constructed by reverse orientation of the A. latus PHA biosynthesis genes in pjc1 and pjc3, respectively. Recombinant E. coli XL1-Blue strains harboring pjc1, pjc3, pjc1r, and pjc3r were cultivated in PHB accumulation medium and MR medium supplemented with glucose and compared for PHB production. Cell growth and PHB accumulation in strains XL1-Blue(pJC1) and XL1-Blue(pJC1R) were similar. They were also similar in the strains harboring pjc3 and pjc3r. Furthermore, induction with isopropyl- -D-thiogalactopyranoside (IPTG) at a final concentration of 1 mm did not increase PHB production in XL1-Blue(pJC1) and XL1- Blue(pJC3). These results suggested that the A. latus PHA biosynthesis enzymes were constitutively expressed in E. coli from the natural promoter. Cell growth and PHB synthesis in various recombinant E. coli strains. Recombinant E. coli strains harboring four different plasmids (pjc1, pjc2, pjc3, and pjc4) containing the A. latus PHA biosynthesis genes and recombinant E. coli strains harboring the psyl series of plasmids (psyl104, psyl105, and psyl107) containing the R. eutropha PHA biosynthesis genes were cultivated for 66 h in defined R medium supplemented with 20 g of glucose per liter and 50 mg of ampicillin per liter at 30 C (Fig. 3). XL1-Blue(pJC3) and XL1-Blue(pJC4) grew to higher cell concentrations and accumulated more PHB than the other recombinant strains. The final cell and PHB concentrations obtained with XL1-Blue(pJC4) were 7.45 and 5.30 g/liter, respectively. These concentrations are much higher than those obtained with recombinant E. coli XL1-Blue containing psyl107, the best version of the plasmids containing the R. eutropha PHA biosynthesis genes (14). Better cell growth and PHB production were achieved by removal of the unnecessary DNA fragment upstream of the promoter of the cloned A. latus PHA biosynthesis genes. Fed-batch cultures of recombinant E. coli harboring the A. latus PHA biosynthesis genes. ph-stat fed-batch culturing of recombinant E. coli strains harboring the A. latus PHA biosynthesis genes was carried out. For the recombinant strains harboring plasmids pjc3 and pjc4, ampicillin was not added during the entire cultivation. All recombinant E. coli strains harboring the A. latus PHA biosynthesis genes, XL1- Blue(pJC1), XL1-Blue(pJC2), XL1-Blue(pJC3), and XL1- Blue(pJC4), grew to cell concentrations higher than 177 g/liter. XL1-Blue harboring pjc3 or pjc4 accumulated more PHB (up to 73% cell dry weight) than XL1-Blue harboring pjc1 or pjc2 (50 to 60% cell dry weight). As an example, the time profiles of cell growth and PHB production during fed-batch culturing of XL1-Blue(pJC4) are shown in Fig. 4. The final cell concentration, PHB concentration, and PHB content obtained in 30.6 h were g/liter, g/liter, and 73%, respectively, resulting in a very high PHB productivity of 4.63 g/liter/h. DISCUSSION The use of PHA as a substitute for nondegradable petroleum-derived plastics hinges on the ability to produce it at a cost that is competitive with that incurred in the production of conventional plastics. From studies on the design and economic evaluation of the processes used for the production of PHAs (4), it was found that PHA productivity is one of the most important factors determining overall production cost. Even though there are more than 300 different microorganisms that are known to synthesize PHAs in nature, only a few bacteria can produce PHB to an extent that meets commercial interest (15, 16). The highest PHB productivity was achieved with the fed-batch culture of A. latus (39). It was reasoned that the efficient synthesis of PHB in A. latus was due to the greater catalytic power of the PHA biosynthesis enzymes in this bacterium. Previous studies showed that recombinant E. coli was superior to wild-type PHB producers in all aspects except for productivity (6, 12, 18, 21, 33). Therefore, economical production of PHB by recombinant E. coli was thought to be possible if PHB productivity could be increased by cloning the A. latus PHA biosynthesis genes. The cloning strategy was based on the previous observation that recombinant E. coli harboring the R. eutropha PHA biosynthesis genes could synthesize and intracellularly accumulate PHB when grown in a suitable medium, such as LB medium containing glucose (26, 32, 34). A 6.3-kb A. latus genomic DNA fragment (AL63) coding for the PHA biosynthesis enzymes was cloned in E. coli by screening the transformants that appeared as opaque colonies. Nucleotide sequence analysis of the cloned AL63 fragment indicated that the phac Al, phaa Al, and phab Al genes were clustered in a single operon (phacab), as in R. eutropha (26, 32, 34). There was one more open reading frame (ORF4) in front of the promoter region of the PHA biosynthesis operon. Its product had high amino acid sequence identity (58%) with glutathione S-transferase, but its actual function is not known. Since ORF4 did not seem to affect PHB synthesis, two derivatives, pjc3 and pjc4, were constructed by deleting this region. The use of antibiotics in a large-scale fermentation is not desirable. Since pjc1 and pjc3 showed instability problems during PHB production in the absence of antibiotic pressure, stable derivatives pjc2 and pjc4 were constructed by trans-

5 VOL. 64, 1998 A. LATUS PHA SYNTHESIS GENE CLONING AND APPLICATION 4901 FIG. 2. Construction of pjc1, pjc2, pjc3, and pjc4. Abbreviations: stb, parb locus of plasmid R1; ORI, origin of replication. Bold lines and arrows represent the DNA fragment from A. latus. ferring the A. latus PHA biosynthesis genes into the psyl104 backbone, which contains the parb locus of plasmid R1 for plasmid stabilization (22). The growth of recombinant E. coli strains harboring four different plasmids containing the A. latus PHA biosynthesis genes and PHB production were compared with those of recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes. PHB production was higher for recombinant E. coli XL1-Blue(pJC3) and XL1-Blue(pJC4)

6 4902 CHOI ET AL. APPL. ENVIRON. MICROBIOL. FIG. 3. Growth of recombinant E. coli strains harboring various plasmids and PHB production after cultivation at 30 C for 66 h in defined R medium containing 20 g of glucose per liter (L). than for recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes. A better comparison could be made with plasmids pjc4 and psyl104, since they share the same plasmid backbone and have the PHA biosynthesis genes cloned in the same orientation. The only difference is the source of the PHA biosynthesis genes (A. latus for pjc4 and R. eutropha for psyl104). When XL1-Blue(pJC4) and XL1- Blue(pSYL104) were cultured under the same conditions, XL1-Blue(pJC4) had higher cell and PHB concentrations than XL1-Blue(pSYL104) (Fig. 3). To determine if the new recombinant E. coli strains harboring the A. latus PHA biosynthesis genes could produce PHB more efficiently, fed-batch culturing was carried out. XL1- Blue(pJC1) and XL1-Blue(pJC2) grew to high concentrations (higher than 177 g of cell dry weight/liter), but the PHB contents were below 60%. On the other hand, fed-batch cultures of XL1-Blue(pJC3) and XL1-Blue(pJC4) showed high concentrations of cells and PHB (higher than 140 g of PHB/liter) with high productivity. In particular, PHB concentration and PHB productivity obtained with XL1-Blue(pJC4) were as high as g/liter and 4.63 g of PHB/liter/h, respectively. Recently, metabolic engineering approaches were taken to develop several recombinant bacteria and transgenic plants (16, 28) for more efficient production and recovery of PHB. Recombinant E. coli strains harboring the R. eutropha PHA biosynthesis genes have been one of the most successful examples in this aspect, except for PHB productivity. This study provided a strategy of enhancing PHB productivity by developing recombinant E. coli strains harboring the more efficient PHA biosynthesis machinery of A. latus. The advantages of the use of recombinant E. coli plus the ability to produce PHB with high productivity should make it possible to produce PHB with a high level of economic competitiveness. ACKNOWLEDGMENTS We thank S. H. Choo and H. S. Yoon for help in DNA sequencing. This work was supported by the Ministry of Science and Technology and by LG Chemicals, Ltd. FIG. 4. Time profile of cell concentration, PHB concentration, and PHB content during fed-batch culturing of XL1-Blue(pJC4) in chemically defined medium. L, liter. REFERENCES 1. Anderson, A. J., and E. A. Dawes Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54: Braunegg, G., B. Sonnleitner, and R. M. Lafferty A rapid gas chromatographic method for the determination of poly- -hydroxybutyric acid in microbial biomass. Eur. J. Appl. Microbiol. Biotechnol. 6: Byrom, D Polymer synthesis by microorganisms: technology and economics. Trends Biotechnol. 5: Choi, J., and S. Y. Lee Process analysis and economic evaluation for poly(3-hydroxybutyrate) production by fermentation. Bioprocess. Eng. 17: Doi, Y Microbial polyesters. VCH, New York, N.Y. 6. Fidler, S., and D. Dennis Polyhydroxyalkanoate production in recombinant Escherichia coli. FEMS Microbiol. Rev. 103:

7 VOL. 64, 1998 A. LATUS PHA SYNTHESIS GENE CLONING AND APPLICATION Fukui, T., and Y. Doi Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae. J. Bacteriol. 179: Gerngross, T. U., K. D. Snell, O. P. Peoples, A. J. Sinskey, E. Csuhai, S. Masamune, and J. Stubbe Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity. Biochemistry 33: Holmes, P. A Biologically produced PHA polymers and copolymers, p In D. C. Bassett (ed.), Developments in crystalline polymers, vol. 2. Elsevier, London, England. 10. Huisman, G. W., E. Wonink, R. Meima, B. Kazemier, P. Terpstra, and B. Witholt Metabolism of poly(3-hydroxyalkanoates) by Pseudomonas oleovorans: identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J. Biol. Chem. 266: Hustede, E., A. Steinbüchel, and H. G. Schlegel Cloning of poly(3- hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus. FEMS Microbiol. Lett. 93: Kusaka, S., H. Abe, S. Y. Lee, and Y. Doi Molecular mass of poly[(r)- 3-hydroxybutyric acid] produced in a recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 47: Lee, I., S. Nam, Y. Rhee, and J. Kim Cloning and functional expression in Escherichia coli of polyhydroxyalkanoate synthase (phac) gene from Alcaligenes sp. SH-69. J. Microbiol. Biotechnol. 6: Lee, S. Y Suppression of filamentation in recombinant Escherichia coli by amplified FtsZ activity. Biotechnol. Lett. 16: Lee, S. Y Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49: Lee, S. Y Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol. 14: Lee, S. Y E. coli moves into the plastic age. Nat. Biotechnol. 15: Lee, S. Y., and H. N. Chang High cell density cultivation of Escherichia coli W using sucrose as a carbon source. Biotechnol. Lett. 15: Lee, S. Y., and H. N. Chang Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies. Can. J. Microbiol. 41(Suppl. 1): Lee, S. Y., K. M. Lee, H. N. Chang, and A. Steinbüchel Comparison of Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid), and morphological changes. Biotechnol. Bioeng. 44: Lee, S. Y., A. P. J. Middelberg, and Y. K. Lee Poly(3-hydroxybutyrate) production from whey using recombinant Escherichia coli. Biotechnol. Lett. 19: Lee, S. Y., K. S. Yim, H. N. Chang, and Y. K. Chang Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) in Escherichia coli. J. Biotechnol. 32: Liebergesell, M., F. Mayer, and A. Steinbüchel Analysis of polyhydroxyalkanoic acid-biosynthesis genes of anoxygenic phototrophic bacteria reveals synthesis of a polyester exhibiting an unusual composition. Appl. Microbiol. Biotechnol. 40: Liebergesell, M., and A. Steinbüchel Cloning and nucleotide sequences of genes relevant for biosynthesis of poly(3-hydroxybutyric acid) in Chromatium vinosum strain D. Eur. J. Biochem. 209: Marmur, J A procedure for the isolation of deoxyribonucleic acids from microorganisms. J. Mol. Biol. 3: Peoples, O. P., and A. J. Sinskey Poly- -hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbc). J. Biol. Chem. 264: Pieper, U., and A. Steinbüchel Identification, cloning and molecular characterization of the poly(3-hydroxyalkanoic acid) synthase structural gene of the gram-positive Rhodococcus ruber. FEMS Microbiol. Lett. 96: Poirier, Y., C. Nawrath, and C. Somerville Production of polyhydroxyalkanoates, a family of biodegradable plastics and elastomers, in bacteria and plants. Bio/Technology 13: Ryu, H. W., S. K. Hahn, Y. K. Chang, and H. N. Chang Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phosphate limitation. Biotechnol. Bioeng. 55: Sambrook, J., E. F. Fritsch, and T. Maniatis Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 31. Schembri, M. A., R. C. Bayly, and J. K. Davies Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located. FEMS Microbiol. Lett. 118: Schubert, P., A. Steinbüchel, and H. G. Schlegel Cloning of the Alcaligenes eutrophus genes for synthesis of poly- -hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J. Bacteriol. 170: Sim, S. J., K. D. Snell, S. A. Hogan, J. Stubbe, C. Rha, and A. J. Sinskey PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat. Biotechnol. 15: Slater, S. C., W. H. Voige, and D. E. Dennis Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly- -hydroxybutyrate biosynthetic pathway. J. Bacteriol. 170: Steinbüchel, A Polyhydroxyalkanoic acids, p In D. Byrom (ed.), Biomaterials: novel materials from biological sources. Stockton, New York, N.Y. 36. Timm, A., and A. Steinbüchel Cloning and molecular analysis of the polyhydroxyalkanoic acid gene locus of Pseudomonas aeruginosa PAO1. Eur. J. Biochem. 209: Ueda, S., T. Yabutani, A. Maehara, and T. Yamane Molecular analysis of the poly(3-hydroxyalkanoate) synthesis gene from a methylotrophic bacterium, Paracoccus denitrificans. J. Bacteriol. 178: Valentin, H. E., and A. Steinbüchel Cloning of the Methylobacterium extorquens polyhydroxyalkanoic acid synthase structural gene. Appl. Microbiol. Biotechnol. 39: Wang, F., and S. Y. Lee Poly(3-hydroxybutyrate) production with high polymer content by fed-batch culture of Alcaligenes latus under nitrogen limitation. Appl. Environ. Microbiol. 63: 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:

8 ERRATA Cloning of the Alcaligenes latus Polyhydroxyalkanoate Biosynthesis Genes and Use of These Genes for Enhanced Production of Poly(3-hydroxybutyrate) in Escherichia coli JONG-IL CHOI, SANG YUP LEE, AND KYUBOEM HAN Department of Chemical Engineering and BioProcess Engineering Research Center, Korea Advanced Institute of Science and Technology, Kusong-dong, Yusong-gu, Taejon , and Biotech Research Institute II, LG Chemicals, Ltd., Science Town, Taejon , Korea Volume 64, no. 12, p , Page 4898, column 2, Results, line 16: 6.3-kb should read 6.4-kb. Line 19: 6,286 bp should read 6,433 bp. Page 4899, Fig. 1. Figure 1 should appear as shown below. FIG. 1. Column 1, line 4: 1,029 bp should read 1,176 bp. Line 5: 343 amino acids should read 392 amino acids. Line 6: 35,406 Da should read 40,519 Da. Page 4900, column 1, line 21: 6.3-kb should read 6.4-kb. Line 31: 5.3-kb should read 5.4-kb. Column 2, Discussion, line 25: 6.3-kb should read 6.4-kb. Page 4901, Fig. 2: pjc1 (9-kbp), pjc2 (9.7-kbp), pjc3 (8-kbp), and pjc4 (8.7-kbp) should read pjc1 (9.1-kbp), pjc2 (9.8-kbp), pjc3 (8.1-kbp), and pjc4 (8.8-kbp), respectively. 1361

9 Inactivation of Cryptosporidium parvum Oocysts by Ammonia MICHAEL B. JENKINS, DWIGHT D. BOWMAN, AND WILLIAM C. GHIORSE Section of Microbiology, Division of Biological Sciences, and Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York Volume 64, no. 2, p , p. 786, Table 1. Table 1 should read as shown below. TABLE 1. Inactivation rates of C. parvum oocysts exposed to measured concentrations of ammonia a [NH 3 ] (mol/liter) K 95% CI/h b Days to reach % inactivation c d e a Based on data from the dye permeability assay after a 24-h exposure. b It was assumed that oocyst inactivation was a first-order process. The coefficient of inactivation was determined by regressing ln (P 0 /P t ) against time (derived from the equation P t P 0 e Kt, where P 0 is the initial percentage of viable oocysts, P t is the percentage of viable oocysts at time t, in hours, and K is the coefficient of inactivation). The 95% confidence intervals (CI) were determined by multiplying the Student t value at the appropriate degree of freedom and at an level (two-sided) of by the standard deviations of K. c Calculated by the equation t ln (P 0 /P t )/K. d This concentration of NH 3 and exposure time were used in the validation experiment shown in Table 2. e A power function, y 2.523x (r ), that fit the regression of [NH 3 ] against days to reach % inactivation was used to determine the concentration of ammonia that would reduce the viability of oocysts by % in 1 day. The K value for this concentration of ammonia was then derived. Page 787, column 2, line 9: 26.5 days should read 55.1 days. 1362