Improved polyhydroxybutyrate (PHB) production in transgenic tobacco by enhancing translation efficiency of bacterial PHB biosynthetic genes

Polyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves.     

Polyhydroxyalkanoate synthase from Bacillus sp. INT005 is composed of PhaC and PhaR

A polyhydroxyalkanoate (PHA) biosynthesis gene locus from Bacillus sp. INT005 strain, which had been isolated from a gas field, was cloned and analyzed at the molecular level. We found that a 3.8-kbp DraI-digested fragment of genomic DNA of Bacillus sp. INT005 conferred PHA-producing ability to Escherichia coli, which was PHA-negative. The DNA fragment contained three genes, phaR, -B and -C. The activity of 3-ketoacyl-CoA reductase with NADPH was detected in the lysate from recombinant E. coli carrying the phaB gene. Although PHA synthase activity could be detected in the extract from E. coli carrying phaR, -B and -C genes, no such activity could be detected in that from E. coli carrying only the phaC gene. However, the mixture of the crude extracts of E. coli expressing phaR or phaC revealed very high PHA synthase activity. Furthermore, when His-tagged PhaC was purified by Ni-affinity chromatography from the mixture of crude extracts containing His-tagged PhaC or native PhaR, the eluate contained His-tagged PhaC and native PhaR. On the other hand, PhaR did not bind to the column directly. This purified PhaC with PhaR had 160-fold higher specific activity of PHA synthase than that without PhaR. In addition, the kinetics of the purified PhaC with PhaR revealed a lag phase that preceded the linear phase. It has been known that class III PHA synthase is composed of two different subunits, PhaC and PhaE, and phaC and phaE genes are directly linked in the genomes. Furthermore, the PHA synthase has no lag phase. We hence concluded that the PHA synthase of Bacillus sp. INT005 consists of PhaC and PhaR, and has characteristics different from class III PHA synthase.     

Enzyme-catalyzed poly(3-hydroxybutyrate) synthesis from acetate with CoA recycling and NADPH regeneration in Vitro

We established a novel enzyme-catalyzed poly(3-hydroxybutyrate) [P(3HB)] synthesis system capable of recycling CoA on the basis of the P(3HB) biosynthetic pathway in Ralstonia eutropha. The system includes purified beta-ketothiolase (PhaA), NADPH-dependent acetoacetyl-CoA reductase (PhaB), PHA synthase (PhaC), acetyl-CoA synthetase (Acs) and glucose dehydrogenase (GDH). In this system, acetyl-CoA was synthesized from acetate and CoA by Acs and ATP, and then two molecules of acetyl-CoA were condensed by PhaA to synthesize acetoacetyl-CoA, which was converted to (R)-3-hydroxybutyryl-CoA (3HBCoA) by PhaB and NADPH. The 3HBCoA was polymerized by PhaC and converted to P(3HB). In this system, the CoA molecules that were released during the condensation and polymerization reactions catalyzed by PhaA and PhaC, respectively, were reused successfully for the synthesis of acetyl-CoA. In addition, NADPH, which was consumed in the reduction of acetoacetyl-CoA, was regenerated by the action of GDH. In this system, the yield of P(3HB) synthesized from acetate as the substrate was 5.6 mg in a 5-ml reaction mixture, and the weight-average molecular weight and polydispersity were 6.64 x 10(6) and 1.36, respectively. Furthermore, CoA was reused at least 26 times, and NADPH was also regenerated at least 26 times during 24 h of reaction.     

Poly[(R)-3-hydroxybutyrate] formation in Escherichia coli from glucose through an enoyl-CoA hydratase-mediated pathway

In this study, a new metabolic pathway for the synthesis of poly[(R)-3-hydroxybutyrate] [P(3HB)] was constructed in a recombinant Escherichia coli strain that utilized forward and reverse reactions catalyzed by two substrate-specific enoyl-CoA hydratases, R-hydratase (PhaJ) and S-hydratase (FadB), to epimerize (S)-3HB-CoA to (R)-3HB-CoA via a crotonyl-CoA intermediate. The R-hydratase gene (phaJ(Ac)) from Aeromonas caviae was coexpressed with the PHA synthase gene (phaC(Re)) and 3-ketothiolase gene (phaA(Re)) from Ralstonia eutropha in fadR mutant E. coli strains (CAG18497 and LS5218), which had constitutive levels of the beta-oxidation multienzyme FadB(Ec). When grown on glucose as the sole carbon source, the cells accumulated P(3HB) up to an amount 6.5 wt% of the dry cell weight, whereas the control cells without phaJ(Ac) or fadR mutation accumulated significantly smaller amounts of P(3HB). These results suggest that PhaJ(Ac) and FadB(Ec) played an important role in supplying monomers for P(3HB) synthesis in the pathway. Furthermore, by using this pathway, a P(3HB)-concentration-dependent fluorescent staining screening technique was developed to rapidly identify cells that possess active R-hydratase.     

Production system for biodegradable polyester polyhydroxybutyrate by Corynebacterium glutamicum

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] production by Corynebacterium glutamicum was developed by introducing the phbCAB operon derived from Ralstonia eutropha. P(3HB) synthase activity was detected in this recombinant C. glutamicum carrying a cell surface protein gene promoter. Intracellular P(3HB) was microscopically observed as inclusion granules and its content was calculated to be 22.5% (w/w) with a number average molecular weight of 2.1x10(5) and a polydispersity of 1.63.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

可降解材料P(3HB-co-4HB)的性能分析

本文对聚(3-羟基丁酸酯-co-4-羟基丁酸酯)(P(3HB-co-4HB))的基本性能和降解性能进行了研究,分析了降解机理及降解影响因素.通过差示扫描量热仪、偏光显微镜、拉力机等表征了P(3HB-co-4HB)的结晶及力学性能.采用扫描电子显微镜,并通过热失重分析,考察了降解过程中表面形态和样品质量等的变化.结果表明:P(3HB-co-4HB)是一种环境友好、可降解的环保材料,其物理性能、球晶尺寸和球晶形态随着4HB含量的不同而改变.     

Precise control of repeating unit composition in biodegradable poly(3-hydroxyalkanoate) polymers synthesized by Escherichia coli

The composition of medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) biopolymers is normally an uncontrollable random mixture of repeating units with differing side chain lengths. Attempts to generate MCL PHA homopolymers and control repeating unit composition have been published in native PHA-producing organisms but have limited ranges for the different sizes of repeating units that can be synthesized. In this study, a new Escherichia coli-based system that exhibits control over repeating unit composition for both MCL PHAs and short-chain-length (SCL) PHAs has been developed, covering an unprecedented range of repeating units. The fadB and fadJ genes from the β-oxidation pathway were eliminated from the chromosome of E. coli LS5218. The subsequent blockage in β-oxidation caused a buildup of enoyl-CoA intermediates, which were converted to PHAs by an (R)-specific enoyl-CoA hydratase (PhaJ4) and PHA synthase [PhaC1(STQK)] expressed from a plasmid DNA construct. Fatty acid substrates were converted to PHAs with repeating units equal in the number of carbon atoms to the fatty acid substrate. The broad substrate specificities of the PhaJ4 and PhaC1(STQK) enzymes allowed for the production of homopolymers with strict control over the repeating unit composition from substrates of four to twelve carbons in length. Polymers were purified and analyzed by GC, GC-MS, and NMR for structural composition and by DSC, TGA, and GPC for thermal and physical characteristics. This study marks the development of the first single biological system to achieve consistent repeating unit control over such a broad range of repeating units in PHAs.     

谷氨酸棒杆菌3-脱氧-α-阿拉伯庚酮糖酸-7磷酸合成酶基因的克隆与过量表达

本实验对谷氨酸棒杆菌(Corynebacterium glutamicum) AS10111及其亚硝基胍诱变获得的丙氨酸和酪氨酸双营养缺陷型、抗3- 甲基色氨酸突变菌株JLC1 中3- 脱氧- α- 阿拉伯庚酮糖酸-7 磷酸合成酶(DS)基因进行克隆和序列分析,将突变体来源DS 基因构建pZ8-1-DSM 重组质粒,在谷氨酸棒杆菌突变株JLC1 中进行表达,重组菌株JLC1(pZ8-1-DSM)DS 酶活力分别是宿主菌和野生型菌株DS 酶活力的5.9 和7.3 倍,色氨酸产量达到14.90g/L,较宿主菌提高了65%。这表明通过在谷氨酸棒杆菌中过量表达3- 脱氧- α- 阿拉伯庚酮糖酸-7 磷酸合成酶基因可以有效提高该酶活力和色氨酸的产量。     

Mutations to the active site of 3-ketoacyl-ACP synthase III (FabH) increase polyhydroxyalkanoate biosynthesis in transgenic Escherichia coli

Polyhydroxyalkanoate (PHA) production has been enhanced with engineered 3-ketoacyl-ACP synthase III (FabH) enzymes that accept diverse fatty acyl-ACP substrates and convert them to fatty acyl-CoA substrates for polymerization by PHA synthase enzymes resulting in the production of diverse polymers. Two mutations in the monomer supplying enzyme FabH, His244Ala and the Asn274Ala, were investigated to assess the impact of these mutations on PHA monomer production. PHA production increased more than six-fold with the mutation His244Ala in the FabH enzyme. Engineering of the FabH enzyme for improved PHA monomer supply led to a more productive system for PHA copolymer production.     

利用CRISPRi技术构建乙醛酸生物合成Corynebacterium glutamicum工程菌

以谷氨酸棒状杆菌(Corynebacterium glutamicumATCC 13032)为出发菌株,敲除其支流代谢关键酶乳酸脱氢酶合成基因lldh,建立规律间隔成簇短回文重复序列干扰(clustered regularly interspaced short palindromic repeats interfer...     

Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity

A rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaC(Ac)), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB(-)4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA.     

Up-cycling of PET (polyethylene terephthalate) to the biodegradable plastic PHA (polyhydroxyalkanoate)

The conversion of the petrochemical polymer polyethylene terephthalate (PET) to a biodegradable plastic polyhydroxyal-kanoate (PHA) is described here. PET was pyrolised at 450 degrees C resulting in the production of a solid, liquid, and gaseous fraction. The liquid and gaseous fractions were burnt for energy recovery, whereas the solid fraction terephthalic acid (TA) was used as the feedstock for bacterial production of PHA. Strains previously reported to grow on TA were unable to accumulate PHA. We therefore isolated bacteria from soil exposed to PET granules at a PET bottle processing plant From the 32 strains isolated, three strains capable of accumulation of medium chain length PHA (mclPHA) from TA as a sole source of carbon and energy were selected for further study. These isolates were identified using 16S rDNA techniques as P. putida (GO16), P. putida (GO19), and P. frederiksbergensis (GO23). P. putida GO16 and GO19 accumulate PHA composed predominantly of a 3-hydroxydecanoic acid monomer while P. frederiksbergensis GO23 accumulates 3-hydroxydecanoic acid as the predominant monomer with increased amounts of 3-hydroxydodecanoic acid and 3-hydroxydodecenoic acid compared to the other two strains. PHA was detected in all three strains when nitrogen depleted below detectable levels in the growth medium. Strains GO16 and GO19 accumulate PHA at a maximal rate of approximately 8.4 mg PHA/l/h for 12 h before the rate of PHA accumulation decreased dramatically. Strain GO23 accumulates PHA at a lower maximal rate of 4.4 mg PHA/l/h but there was no slow down in the rate of PHA accumulation over time. Each of the PHA polymers is a thermoplastic with the onset of thermal degradation occurring around 308 degrees C with the complete degradation occurring by 370 degrees C. The molecular weight ranged from 74 to 123 kDa. X-ray diffraction indicated crystallinity of the order of 18-31%. Thermal analysis shows a low glass transition (-53 degrees C) with a broad melting endotherm between 0 and 45 degrees C.     

Optimization of L-lactic acid feeding for the production of poly-D-3-hydroxybutyric acid by Alcaligenes eutrophus in fed-batch culture

We investigated optimization of the feeding of L-lactic acid for the production of poly-D-3-hydroxybutyric acid [P(3HB)] by Alcaligenes eutrophus in a fed-batch culture system. An acidic substrate solution was fed automatically so as to maintain the pH of the culture liquid at 7.0. Feeding of a substrate solution containing 45% (w/v) L-lactic acid, 6.2% (w/v) sodium L-lactate, 5.8% (w/v) ammonia water and 1.8% (w/v) potassium phosphate [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10], allowed the L-lactate concentration in the culture liquid to be maintained at approximately 2 g/l and the cell concentration reached 27.4 g/l after 15 h of cultivation. To promote P(3HB) production, a two-stage fed-batch culture consisting of a culture for cell growth and one for P(3HB) accumulation was carried out. When the substrate solution, whose C N molar ratio was 23, was fed during the P(3HB) accumulation phase, the cell concentration and the P(3HB) content in the cells reached 103 g/l and 57.6% (w/w), respectively, in 51.5 h.     

转谷氨酰胺酶酶原在大肠杆菌中的重组优化表达

茂原链霉菌(Streptomyces mobaraensis)转谷氨酰胺酶酶原(pro-transglutaminase,pro-TG)在重组大肠杆菌中的表达量低,限制了其在食品、化妆品、纺织行业中的应用。通过对转谷氨酰胺酶酶原的基因序列进行密码子优化,降低其GC含量,提高了它在大肠杆菌中的表达水平,结果表明经优化后转谷氨酰胺酶原的表达量达到了优化前的4.4倍。为提高转谷氨酰胺酶的比活力,通过基于融合PCR的定点突变技术将转谷氨酰胺酶第二位的丝氨酸突变为脯氨酸,突变后转谷氨酰胺酶的比活力达到了突变前的1.26倍。上述研究结果表明,密码子优化及定点突变技术可以应用于优化转谷氨酰胺酶酶原在大肠杆菌中的表达。     

Cloning, sequence analysis, and expression in Escherichia coli of gene encoding N-Benzyl-3-pyrrolidinol dehydrogenase from Geotrichum capitatum

The gene encoding N-benzyl-3-pyrrolidinol dehydrogenase (DDBJ/EMBL/GenBank accession no. AB294179), a useful biocatalyst for producing (S)-N-benzyl-3-pyrrolidinol, was cloned from the genomic DNA of Geotrichum capitatum JCM 3908. The gene contained an open reading frame consisting of 1023 nucleotides corresponding to 340 amino acid residues. The subunit molecular weight was calculated to be 39,000. The predicted amino acid sequence did not have significant similarity to those of N-benzyl-3-pyrrolidinone reductases reported previously. From 30 mM N-benzyl-3-pyrrolidinone, (S)-N-benzyl-3-pyrrolidinol was obtained with a yield >99.9% and an enantiomeric excess >99.9% in 1-h and 2-h reactions without NADH addition by the resting cells of Escherichia coli HB 101 strains harboring the expression plasmids pSG-POBS and pSF-POBS that possess the glucose dehydrogenase gene and formate dehydrogenase gene as an NADH-reproducing system, respectively, besides the N-benzyl-3-pyrrolidinol dehydrogenase gene. N-Benzyl-3-pyrrolidinol dehydrogenase activity (0.56 U/mg) was observed in E. coli (pSG-POBS), which was 17-fold the specific activity observed in G. capitatum JCM 3908.     

Regulating the molar fraction of 4-hydroxybutyrate in poly(3-hydroxybutyrate-4-hydroxybutyrate) biosynthesis by Ralstonia eutropha using propionate as a stimulator

The regulation of the molar fraction of 4-hydroxybutyrate (4-HB) in the poly(3-hydroxybutyrate-4-hydroxybutyrate) [P(3HB-4HB)] biosynthesis by Ralstonia eutropha (formerly Alcaligenes eutrophus) was attempted by the supplemental addition of propionate. The molar fraction of 4-HB in P(3HB-4HB) was increased significantly from 12.3 to 51.8 mol% by the addition of a small amount of propionate along with gamma-butyrolactone commonly used as a precursor for the biosynthesis of P(3HB-4HB). The mechanism of regulation by propionate was investigated by measuring the variation of enzyme activities related to the biosynthesis of P(3HB-4HB) and the level of intermediate metabolite acetyl-CoA. PHB synthase activity was induced significantly by propionate, and the acetyl-CoA concentration also increased significantly due to the additional supply of propionate. The overflowing acetyl-CoA seems to cause an inhibitory effect on the ketolysis reaction catalysing the lysis of 4-hydroxybutyryl-CoA to two molecules of acetyl-CoA; consequently, the 4-HB fraction available for polymerization increased. Accordingly, the molar fraction of 4-HB in P(3HB-4HB) biosynthesis seems to be regulated by both an increased 4-HB fraction and an activated PHB synthase due to the supplemental addition of propionate as a stimulator.     

Characterization of two 3-hydroxybutyrate dehydrogenases in poly(3-hydroxybutyrate)-degradable bacterium, Ralstonia pickettii T1

Two D-(-)-3-hydroxybutyrate (3HB) dehydrogenases, BDH1 and BDH2, were isolated and purified from a poly(3-hydroxybutyrate) (PHB)-degradable bacterium, Ralstonia pickettii T1. BDH1 activity increased in R. pickettii T1 cells grown on several organic acids as a carbon source but not on 3HB, whereas BDH2 activity markedly increased in the same cells grown on 3HB or PHB. To examine their biochemical properties, bdh1 and bdh2 were cloned and overexpressed in Escherichia coli, and their purified products were characterized. The kinetic parameters indicate that BDH1 is more suitable for converting acetoacetate to 3HB than BDH2, whereas BDH2 is more efficient for the reverse reaction than BDH1. Thus, R. pickettii T1 contains two BDHs with different biochemical properties and physiological roles: BDH1 for cell growth on organic acids other than 3HB and BDH2 for cell growth on 3HB or PHB.     

Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase

We succeeded in developing a novel method for in vitro poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3 HB-co-4 HB)] synthesis with CoA recycling using polyhydroxyalkanoate synthase and an acyl-CoA synthetase. Using this method, the monomer compositions in P(3 HB-co-4 HB)s could be controlled strictly by the ratios of the monomers in the reaction mixtures.     

Development of a novel method for feeding a mixture of -lactic acid and acetic acid in fed-batch culture of Ralstonia eutropha for poly- -3-hydroxybutyrate production

Fermentative production of poly- -3-hydroxybutyrate [P(3HB)] from a mixture of -lactic acid and acetic acid by Ralstonia eutropha was investigated. For fed-batch culture with cell density, it is necessary to control the concentration of these organic acids in the culture medium below the inhibitory level for cell growth. Therefore, a novel feeding method, termed the computer-controlled pH-stat substrate feeding method, was developed using the rate of increase of the pH (pH-increasing rate) of the culture medium as an indicator for feed control. The pH-increasing rate, which was calculated every minute by a pH meter-linked computer, represented secondary information regarding substrate consumption by cells. When the pH-increasing rate decreased to 5% of the maximum increasing rate, acidic substrate solution was fed into the fermentor until the pH was reduced to 7.00. Using this feeding strategy, the cell concentration and PHA content obtained in 42 h were 75.0 g/l and 73.1% (w/w), respectively, resulting in a high P(3HB) productivity of 1.30 g/l·h.