• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.310-316
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• 2001

Poly-3-hydroxybutyrate (PHB) synthase catalyzed the last enzymic step to synthesize the intracellular PHB of Rhodobacter sphaeroides. No PHB was detected when the phbC coding for PhB synthase was interrupted, and its expression was regulated at the level of transcription. The cellular PHB content increased about four- to six-fold during the growth transition from the exponential to the early stationary phase under both aerobic and photoheterotrophic conditions. The PHB content during the aerobic growth seemed to be determined by the PhB synthase activity. However, the PHB synthase activity of photoheterotrophically grown cells did not correlate with the PhB content, suggesting a photoheterotrophic regulation different from the aerobic control. Thus, the PHB content of R. sphaeroides was regulated at the transcription level only under aerobic conditions.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.1031-1037
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• 2001

By a sequential action of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, two molecules of acetyl-CoA re converted into D-3-hydroxybutyryl-CoA, a substrate for PHB synthase to form poly-3-hydroxybutyryl-CoA, a substrate for PHB synthase to form poly-3-hydroxybutyrate (PHB) of rhodobacter sphaeroides. The ${\beta}$-ketothiolase gene, phbA, and acetoacetyl-CoA reductase gene, phbB, were cloned and analyzed for their expression. Enzyme activities of ${\beta}$-ketothiolase and acetoacetyl-CoA reductase showed constitutive levels during aerobic and photoheterotrophic growth of R. sphaeroides. In addition, no difference of each enzyme activity was observed between cells grown aerobically and photoheterotrophically. The constitutive level of the enzyme activities are regulated according to the growth phases along with growth conditions. Thus, phbAB expression is not determinative in regulating the PB content. On the other hand, phbA-deleted cell AZI accumulated only $10\%$ PHB of the wild-type, and an elevated dosage of phbAB in trans in R. sphaeroides resulted in a higher content of PHB, indicating that phbAB codes for the enzymes responsible for providing the main supply of subsyrate for PHB synthase. PHB formation by an alternative pathway that does not does not depend on the phbA-and phbB-coding enzymes is also proposed.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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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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• v.13 no.3
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• pp.477-481
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• 2003

The community escape response of Rhodobacter sphaeroides is exerted through the action of CerR and CerI, which code for a LuxR-type regulatory protein and acylhomoserine lactone synthase, respectively. Deletion of chromosomal DNA including cerR and cerI (mutant RI) or insertional interruption of cert (mutant AP3) resulted in two-fold increase in the cellular poly-${\beta}$-hydroxybutyrate (PHB) content In comparison with the wild-type under aerobic growth conditions. The PHB synthase (PhbC) activities of the cer mutants were doubled, and the enzyme expression was regulated at the level of phbC transcription. Thus, CerR, possibly in response to autoinducer (AI), appears to modulate the PHB content of aerobically grown cells by downregulating phbC transcription.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.215-222
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• 1997

The regulatory mechanism of the flux of acetyl-CoA in Alcaligenes eutrophus in unbalanced growth conditions was investigated using a PHB-negative mutant and transformants reintroduced PHB-biosynthesis enzymes through the transformation of cloned phbCAB genes. The PHB-negative mutant was defected absolutly in PHB synthase but partially in ${\beta}$-ketothiolase and acetoacetyl-CoA reductase, and excreted substantial amount of pyruvate to culture broth at late growth phase. The excretion was due to the inhibitory effect of acetyl-CoA on the activity of pyruvate dehydrogenase. The cloned phbC and phbCAB genes were transformed to the PHB-negative mutant strain to reintroduce PHB biosythesis enzymes. Pyruvate excretion could be decreased substantially but not completely by transformation of PHB synthase alone, while pyruvate excretion was ceased by transformation of all three PHB biosynthesis enzymes. To identify the most critical PHB biosynthesis enzyme influencing on the flux of acetyl-CoA, the effect of the variation of PHB biosynthesis enzymes on pyruvate dehydrogenase was investigated. ${\beta}$-Ketothiolase influenced the activity of pyruvate dehydrogenase more sensitively than PHB synthase. ${\beta}$-Ketothiolase, the first step enzyme of PHB biosynthesis that condense acetyl-CoA to acetoacetyl-CoA, seems to be the major enzyme determining the flux of acetyl-CoA to PHB biosynthesis or TCA cycle, and the rate of PHB biosynthesis in A. eutrophus.

• KSBB Journal
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• v.13 no.1
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• pp.96-100
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• 1998

Two promoters (trc and P$\rho$) were inserted in PHA operon derived from Alcaligenes eutrophus to obtain high chain molecules of polyhydroxybutyrate (PHB) in recombinant Escherichia coli. Newly designed PHA operon was used to control the gene expressions of hydroxybutyric CoA and PHA polymerization, separately. Plasmids containing new synthetic operon was transformed into E. coli DH5$\alpha$ and analyzed for PHB production. Without induction of the PHA biosynthetic operon, PHA synthase which has low activity might supply low concentration of initiator during the polymerization reaction, resulting very high molecular weight of polymer. An increase of PHB average molecular weight was observed with decreased IPTG (isopropyl $\beta$ -Dithiogalactosidase) concentration. When no IPTG was added to the culture of E. coli DH5$\alpha$ /$\rho$ SJS1 which contained two promoters in PHA operon, high chain polymer having an average molecular weight of $2.5{\times}10^7$ was achieved. Analysis of the enzyme activities of PHA biosynthetic enzymes suggests that PHA synthase, the enzyme responsible for polymerizing 3-hydroxybutyric CoA, controls the molecular weight of PHB produced in vivo.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.5
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• pp.281-288
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• 2002

Recombinant Escherichia coli strain harboring the ${\lambda}$pR-pL promotor and heterologus poly-${\beta}$-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression of phb genes was induced by a temperature upshift from $33^{\circ}C\;to\;38^{\circ}C$. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lac-tate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.

• Journal of Applied Biological Chemistry
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• v.42 no.1
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• pp.1-6
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• 1999

Poly-${\beta}$-hydroxybutyrate (PHB) and enzymes related PHB metabolism have been measured in nitrogen-fixing symbiosis of chickpea and cowpea plants. Bacteroids from chickpea and cowpea contained PHB to 0.8% and 43% of their dry weight, respectively, whereas the free-living cells CC 1192 and I 16 produced $285{\pm}55mg$ and $157{\pm}18mg$ of PHB g (dry weight)$^{-1}$. To further understand why chickpea bacteroids contained little PHB, the enzyme activities of PHB metabolism (3-ketothiolase, acetoacetyl-CoA reductase, PHB depolymerase, and 3-hydroxybutyrate dehydrogenase), the TCA cycle (malate dehydrogenase, citrate synthase, and isocitrate dehydrogenase), and related reactions (malic enzyme, pyruvate dehydrogenase, and glutamate:2-oxoglutarate transaminase) were compared in extracts from chickpea and cowpea bacteroids and the respective free-living bacteria. Significant differences were observed between chickpea and cowpea bacteroids and between the bacteroid and free-living forms of CC 1192, with respect to the capacity for some of these reactions. It is indicated that a greater potential for oxidizing malate to oxaloacetate in chickpea bacteroids could be a factor that favors the utilization of acetyl-CoA in TCA cycle rather than for PHB synthesis.