• KSBB Journal
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• 제19권5호
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• pp.358-362
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• 2004

Characteristics of cell growth and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] synthesis was investigated through flask and batch cultures of Alcaligenes latus and Comamonas acidovorans. The specific growth rate of C. acidovorans increased with yeast extract concentration and decreased with 1,4-butanediol concentration. Optimum glucose concentration for growth of C. acidovorans was 20 g/L. In one-step flask cultures of C. acidovorans, final dry cell weight and PHA content decreased with the ratio of 1,4-butanediol to glucose, while the 4HB fraction in copolymers gradually increased to 100 $mol\%$ with an initial 1,4-butanediol concentration of 20 g/L as single carbon source. The specific growth rate of A. latus decreased with v-butyrolactone concentration and optimum sucrose concentration for growth was 10 g/L. In batch cultures of A. latus, 4HB fraction increased with initial v-butyrolactone concentration. P(3HB-co-4HB) with 19 $mol\%$ 4HB was obtained when the initial ratio of v-butyloractone (g/L) to sucrose (g/L) was 10 : 10.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.333-336
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• 2001

An isolated phbC gene from Alcaligenes latus was reintroduced into the parent A. latus through the transformation process, and the effect of the amplified phbC gene on the biosynthesis of P(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] and P(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] in the transformant A. latus was investigated. The biosynthesis rate and content of the above copolymers increased up to 1.3-fold after enforcing its own phbC gene, and the molar fractions of 3HV and 4HB in P(3HB-3HV) and P(3HB-4HB) also changed remarkably from 35.0 to 48.0% and from 34.0 to 56.0%, respectively, showing a critical role of PHB synthase which catalyzes the polymerizing reactions between eiher 3HV or 4HB from precursor compounds and 3HB.

• Journal of Microbiology and Biotechnology
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• 제9권5호
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• pp.554-561
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• 1999

A novel estimation method was investigated for determining the concentrations of residual biomass, poly-3-hydroxybutyrate (PHB), and main nutrients including carbon and nitrogen sources, phosphate, and mineral ions from the supplied amount of ammonia solution used for a pH-control solution and nitrogen source in a PHB fermentation. The estimation equations for a batch culture and a fed-batch culture were derived from the relationship between the growth rate of residual biomass and the feed rate of the pH-control solution, and then were applied to the batch culture and the fed-batch cultures of Alcaligenes latus. This method was successfully applied to estimate the concentrations of residual biomass, PHB, and nutrients.

• Journal of Microbiology and Biotechnology
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• 제6권2호
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• pp.120-127
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• 1996

The characteristics of cell growth and poly-$\beta$-hydroxybutyrate biosynthesis of Alcaligenes latus ATCC 29713 were investigated. The PHB accumulation pattern of A. latus followed a growth-associated type where the cell growth and PHB accumulation were carried out simultaneously. Various intermediate compounds such as metabolites involved in the TCA cycle, amino acids, and saturated and unsaturated fatty acids were added to examine their effect on cell growth and PHB accumulation. Citrate, tyrosine, and palmitic acid showed the most significant increase both on cell growth and PHB accumulation. Maximum PHB concentrations were noticeably increased about 1.4 to 1.6 times higher than that of control, corresponding to 5.54, 6.45, and 6.45 g/l for citrate, tyrosine, and palmitic acid, respectively. The stimulatory effects of the supplemented metabolites were analyzed in terms of the increment of enzyme activities related to sugar catabolism and PHB biosynthesis.

• Journal of Microbiology and Biotechnology
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• 제6권6호
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• pp.425-431
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• 1996

The enzymatic characteristics of Alcaligenes latus were investigated by measuring the variations of various enzyme activities related to biosynthesis and degradation of poly-${\beta}$-hydroxybutyrate (PHB) during cultivation. All PHB biosynthetic enzymes, ${\beta}$-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase, were activated gradually at the PHB accumulation stage, and the PHB synthase showed the highest value among three enzymes. This indicates that the rate of PHB biosynthesis is mainly controlled by either ${\beta}$-ketothiolase or acetoacetyl-CoA reductase rather than PHB synthase. The enzymatic activities related to the degradation of PHB were also measured, and the degradation of PHB was controlled by the activity of PHB depolymerase. The effect of supplements of metabolic regulators, citrate and tyrosine, was also investigated, and the activity of glucose-6-phosphate dehydrogenase was increased by metabolic regulators, especially by tyrosine. The activities of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase were also activated by citrate and tyrosine, while the activity of PHB depolymerase was depressed. The increased rate and yield of PHB biosynthesis by metabolic regulators may be due to the increment of acetyl-CoA concentration either by the repression of the TCA cycle by citrate through product inhibition or by the activation of sucrose metabolism by the supplemented tyrosine.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.722-725
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• 1999

Filamentation-suppressed recombinant Escherichia coli strain harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes and the E. coli ftsZ gene was constructed and cultivated for the production of poly(3-hydroxybutyrate) [P(3HB)] with high concentration and high content. By the pH-stat fed-batch culture of this recombinant E. coli strain XL1-Blue(pJC5), the final cell concentration and P(3HB) concentration obtained in 44.25h were 172.2g cell dry weight/l and 141.9g P(3HB)/l, respectively, resulting in productivity of 3.21g P(3HB)/l-h. More importantly, the P(3HB) content obtained was 82.4 wt %, which was significantly higher than that obtained with the recombinant E. coli harboring only the PHA biosynthesis genes.

• KSBB Journal
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• 제14권4호
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• pp.422-428
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• 1999

Alcaligenes latus 유래의 phbC 유전자를 A. eutrophus에 재도입시킨 형질전환균주를 이용하여 높은 4HB 몰분율을 갖는 P(3HB-4HB)의 고농도 생산을 시도하였다. 형질전환균주는 총균체량, P(3HB-4HB) 농도, 그리고 축적률에서 모균주에 비해 다소 증가한 반면 P(3HB-4HB)내의 4HB 몰분율은 23.5 mol%로 모균주의 12.3 mol%에 비해 현저히 증가하였다. 이는 phbC유전자의 증폭으로 인해 해당과정에서 생성된 3HB와 전구물질인 ${\gamma}$-butyrolacton에서 전환된 4HB의 중합반응이 촉진되기 때문으로 사료된다. 또한 ${\gamma}$-butyrolacton의 농도 $Mg^{2-}$ 이온, 그리고 citrate 첨가량이 P(3HB-4HB)의 농도, 축적률, 그리고 4HB 몰분율에 미치는 영향을 검토하였다. P(3HB-4HB)내의 4HB 몰분율을 증대시키기 위하여 일반적으로 사용되는 2단계 배양법을 변형시켜 ${\gamma}$-butyrolacton과 citrate의 첨가시기를 늦춘 2단계 배양법을 활용하여 P(3HB-4HB)내의 4HB 몰분율을 61.0 mol%로 증가시킬 수 있었다. 또한 ${\gamma}$-butyrolacton의 첨가량을 조절하여 P(3HB-4HB)내의 4HB 몰분율이 92.0 mol%에 이르는 homopolymeric P(4HB)를 생산할 수 있었으며, 그 구조를 $^1$H-NMR을 통해 확인하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.731-734
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• 2001

An efficient fermentation strategy for the high level production of ultra-high-molecular weight poly(3-hdyroxybutyrate) (PHB) was developed. Although the cell and PHA concentrations obtained by flask cultures at different initial pH (6.0 or 6.9) were almost same level, the molecular mass of PHB produced were quite different along with the initial pH. When a recombinant Escherichia coli XL1-Blue harboring pJC2 containing the Alcaligenes latus PHB biosynthesis genes was cultivated in flask culture (pH 6.0), the PHB having a very high molecular weight of 22 MDa could be produced while only below 1 MDa at initial pH 6.9. The ultra-high-molecular weight PHB could be synthesized to high concentration of 89.8 g/L resulting in the PHB productivity of 2.07 g/L-h by simple fed-batch culture. In this study, we report that PHB having various molecular mass can be produced by employing metabolically engineered E. coli strains harboring the plasmids of different copy numbers containing the A. latus phbCAB genes.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.321-324
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• 2001

R. eutropha 의 PHA 생합성 유전자를 포함하는 플라스미드 pSYLl07을 가진 재조합 대장균 GCSC6576 과 A. latus PHA 생합성 유전자를 포함하는 플라스미드 pJC4 한 가진 fadR atoC 돌연변이주 재조합 대장균 LS5218 의 유청으로부터 P(3HB- co -3HV) 합성을 비교하였다. 재조합 대장균 LS5218의 pH-stat 유가식 배양결괴 39 시간에 균체농도 31.8 g/L. P(3HB-co-3HV) 농도 10.6 g/L. P(3HB-co-3HV) 함량 33.4 wt%. 3HV 함량 6.26 mol%를 얻을 수 있었다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.196-200
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• 2004

The supra molecular weight poly(〔R〕-3-hydroxybutyrate) (PH B), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineered Escherichia coli strain and its fermentation for high level production of supra molecular weight PHB. Recombinant E. coli strains, harboring plasm ids of different copy numbers containing the Alcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinant E. coli XL1-Blue, harboring a medium-copy-number pJC2 containing the A. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced in Ralstonia eutropha or recombinant E. coli.