重组大肠杆菌合成聚（3-巯基丙酸）和聚（3-羟基丁酸-3-巯基丙酸）的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株。前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚（羟基丁酸戊酸）等多种生物聚酯［Liu and Steinbüchel, Appl. Environ. Microbiol. 66:739743］。利用该重组大肠杆菌,通过生物催化作用合成了3巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物。实验首先研究了3巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚（3-巯基丙酸）可达6.7%(占细胞干重),合成聚（3-羟基丁酸—3-巯基丙酸）（分子中3-巯基丙酸:3-羟基丁酸=3:1）可达24.3%。实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的。还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究。聚（3-巯基丙酸）或聚（3-羟基丁酸-3-巯基丙酸）等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值。     

重组肺炎克雷伯氏菌转化甘油为聚3-羟基丙酸

聚3-羟基丙酸[Poly(3-hydroxypropionate),P3HP]是一种生物可降解及生物相容的新型聚羟基脂肪酸酯。目前已鉴定的生物均不能天然合成P3HP。采用PCR克隆鼠伤寒沙门氏菌的丙醛脱氢酶(Pdu P)基因及罗尔斯通氏菌的聚羟基脂肪酸酯合成酶(Pha C)基因,构建共表达载体,转化肺炎克雷伯氏菌后获得两株重组菌。以甘油为唯一碳源进行摇瓶发酵,pdu P和pha C共用tac启动子的工程菌K.p(p ET-tac-pdu P-pha C)产生0.054 g/L的P3HP,而pdu P和pha C各自独用tac启动子的工程菌K.p(p ET-tac-pdu P-tac-pha C)产生0.091 g/L的P3HP。     

重组大肠杆菌转化甘油合成聚3-羟基丙酸-co-乳酸

聚羟基脂肪酸酯作为性质优良的生物塑料,引起了广泛的关注。由于聚羟基脂肪酸合成酶PhaC特异性较强,难以通过生物合成方法获得含乳酸单体聚合物。为了实现乳酸的聚合,PhaC的筛选至关重要。以甘油为底物,通过引入Klebsiella pneumoniae的甘油脱水酶DhaB123及其激活因子GdrAB以及Salmonella typhimurium LT2的丙醛脱氢酶基因PduP,获得3-羟基丙酰辅酶A;通过引入Megasphaera elsdenii DSM 20460的丙酰辅酶A转移酶PCT,获得乳酰辅酶A;并对3种不同聚羟基脂肪酸合成酶的作用进行考察。在Pseudomonas putida的原始酶PhaC1或者PhaC2的作用下,不能实现乳酸的聚合;而在双位点突变(Ser325Thr和Gln481Lys)的PhaC1(STQK)存在条件下,重组菌可以利用甘油合成聚3-羟基丙酸-co-乳酸。经过对溶氧、有机氮源等发酵条件的优化,聚3-羟基丙酸-co-乳酸的产量可以达到0.22g/L,占细胞干重的3.2%,是含乳酸单体聚合物生物合成研究的一次有益尝试。     

生物合成聚3-羟基丙酸酯的研究进展

聚3-羟基丙酸酯(P3HP)作为聚羟基脂肪酸酯家族(PHAs)中的新型热塑性塑料,具有生物降解性和生物相容性等优点。目前,未见野生微生物可以合成P3HP的报道,生产途径主要为化学法和生物法。其中,通过化学法或添加3-HP单体及其结构类似物作为前体的P3HP合成效率低、成本高且不具环保性;而通过构建和改造工程菌的生物代谢途径,能够利用廉价、可再生的碳源,已经逐渐成为研究热点。文中综述了国内外P3HP生物合成研究进展,并对甘油途径、丙二酸单酰辅酶A(Malonyl-Co A)途径和β-丙氨酸途径等合成方法进行了优缺点分析,为生物合成P3HP的深入研究奠定理论基础。     

生物法合成3-羟基丙酸的研究进展

从3-羟基丙酸的性质出发,介绍了生物法合成3-羟基丙酸以及它在生物体内的五种代谢途径,此外还简要介绍了3-羟基丙酸在合成生物聚酯、抗植物病虫害上的一些应用。     

产3-羟基丙酸重组菌的构建及其转化甘油的研究

将连接编码甘油脱水酶的基因重组质粒pEtac-dhaB和连接编码乙醛脱氢酶编码基因aldh的重组质粒pUCtac共转化大肠杆菌,得到产3-羟基丙酸重组大肠杆菌JM109(pUCtac-aldh,pEtac-dhaB),并对影响该重组菌发酵的营养因子进行研究.试验结果表明:该重组菌转化甘油合成3-羟基丙酸的适宜培养基组成为甘油40 g/L、酵母膏6 g/L、维生素B12 0.02 g/L以及KH2PO4 7.5 g/L; 3-羟基丙酸产量和转化率分别达到4.92 g/L和12.3 %.     

聚羟基烷酸酯 (PHA) 改性研究进展

本文简述了生物制造聚羟基烷酸酯(PHA),包括聚3-羟基丁酸酯(PHB)、聚(3-羟基丁酸酯-3-羟基戊酸酯)(PHBV)、聚(3-羟基丁酸酯-4-羟基丁酸酯)(P3/4HB)、聚(3-羟基丁酸酯-3-羟基己酸酯)(PHBH)的产业化现状,综述了针对PHA材料热稳定性差、加工窗口较窄等缺点而进行的一些改性研究。选用适当方法对PHA进行改性,可使其性能得到优化,应用领域得到拓展。     

以甘油为底物利用重组大肠杆菌生产聚3-羟基丙酸

【目的】解决前期研究中所构建的以甘油为底物合成聚3-羟基丙酸(P3HP)的代谢途径中存在两个主要的问题——细胞内还原力不平衡和质粒丢失,以提高P3HP的产量。【方法】克隆来源于肺炎克雷伯氏菌的1,3-丙二醇(1,3-PDO)氧化还原酶基因,构建P3HP和1,3-PDO联产的菌株,解决细胞内还原力不平衡的问题。利用自杀性载体系统介导的同源重组技术,将甘油脱水酶及其激活因子的基因整合到大肠杆菌基因组中,提高质粒的稳定性。同时,对发酵条件进行优化。【结果】菌种改造和发酵条件优化显著提高了P3HP产量,在摇瓶条件下到达2.7 g/L,比以前的报道提高2倍,并可同时得到2.4 g/L 1,3-PDO。【结论】该重组大肠杆菌合成P3HP的产量得到提高,具有较好的工业化生产前景。     

生物合成3-羟基丙酸的代谢工程研究进展

3-羟基丙酸(3-HP)作为一种重要的平台化合物,以此为底物能够合成多种具有商业潜质的生物制品。野生菌合成3-HP产量较低,严重限制3-HP的大规模应用与生产,通过改造合成代谢通路的相关基因,构建以廉价底物为碳源的工程菌株,实现降低生产成本提高产量的目的。文中将对近年来国内外通过代谢工程合成3-羟基丙酸的研究进展进行概述,并对甘油途径、丙二酸单酰辅酶A途径、β-丙氨酸等途径合成3-HP的优缺点进行总结分析,对3-HP未来发展前景进行展望。     

弱化呼吸链水平对代谢工程大肠杆菌聚羟基丁酸乳酸酯合成的影响

聚3-羟基丁酸乳酸酯[Poly(3-hydroxybutyrate-co-lactate), P(3HB-co-LA)],属于聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHA)家族的一员,是一种具有良好生物相容性和可降解性的天然高分子生物材料。文中通过在大肠杆菌中引入来源于富养罗尔斯通氏菌Ralstonia eutropha的β-酮硫解酶、乙酰乙酰Co A还原酶、来源于丙酸梭菌Clostridium propionicum的丙酰Co A转移酶突变体以及荧光假单胞菌Pseudomonas fluorescens strain 2P24来源的PHA合成酶突变体等异源酶,成功实现了一步法利用葡萄糖合成P(3HB-co-LA),其中乳酸组分的摩尔百分比达到1.6%,聚合物含量为83.9wt%。在此基础上,通过敲除辅酶Q8合成所需的黄素异戊烯基转移酶基因(ubi X)来弱化呼吸链水平,从而增强乳酸积累,并进一步缺失乳酸脱氢酶基因(dld)以减少乳酸在发酵后期转化成丙酮酸,最终将P(3HB-co-LA)中乳酸组分的摩尔百分比提高至14.1%,而聚合物含量为81.7wt%。上述实验结果表明,采用弱化呼吸链水平策略可有效提高聚合物中乳酸组分的摩尔百分比,从而提供了一种改变生物合成聚合物中单体组分含量的新思路。     

需钠弧菌(Vibrio natriegens)合成聚羟基丁酸的研究

研究结果表明,V.natriegens可以利用葡萄糖,果糖,以及糖蜜为碳源合成聚羟基丁酸[Poly(3HB)] ,当以糖蜜为碳源时,积累的Poly(3HB)达到细胞干重的28．4％,实验结果还表明,Poly(3HB)的积累滞后于细胞生长,在培养前加入过量的碳源,不仅没有Poly(3HB)积累,还抑制细胞的生长,测定了与Poly(3HB)合成相关的PHA聚合酶,β-酮硫解酶和乙酰乙酰CoA还原酶的活性。结果表明,伴随Poly(3HB)合成,PHA聚合酶活性从无到有,β－酮硫解酶活性提高了10倍以上。进一步通过利用脂肪酸合成代谢抑制物－浅蓝菌素（cerulenin),研究了脂肪酸从头合成途径与Poly(3HB)合成途径的关系。发现浅蓝菌素能够明显降低细胞Poly(3HB)的累积。根据以上结果,推测在V.natrigens中可能存在两条代谢途径参与Poly(3HB)的合成。     

Degradation of poly(3-hydroxybutyrate) by soil streptomycetes

The ability of 64 soil streptomycetes to degrade poly(3-hydroxybutyrate) [P(3HB)] was evaluated on Pridham and Lyons mineral salts agar medium overlayered with the same medium containing 0.2% P(3HB). The streptomycete isolates were grown on this overlayered medium and the degradation was detected by the formation of clear zone surrounding the growth. Four potent degrader isolates identified as species of Streptomyces were selected. Degradation of P(3HB) by these isolates was studied for a period of 8 days. The rate of degradation increased with increase in concentration of P(3HB) in the medium while it decreased with the supplementation of readily utili- zable carbon sources like glucose, fructose and sucrose. All four isolates also degraded the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB–co–3HV)] in solid medium but to a lesser extent. However, the isolates were equally efficient in degrading P(3HB) in liquid medium.     

A New Pathway for Poly(3-hydroxybutyrate) Production in Escherichia coli and Corynebacterium glutamicum by Functional Expression of a New Acetoacetyl-coenzyme A Synthase

A biosynthetic pathway for poly(3-hydroxybutyrate) [P(3HB)] was developed in Escherichia coli and Corynebacterium glutamicum by an acetoacetyl-coenzyme A (CoA) synthase (AACS) recently isolated from terpenoid-producing Streptomyces sp. strain CL190. Expression of AACS led to significant productions of P(3HB) in E. coli (10.5 wt %) and C. glutamicum (19.7 wt %).     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli

ABSTRACT: BACKGROUND: Poly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as gamma-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB). RESULTS: Recombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies. CONCLUSIONS: Significant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.     

Degradation of microbial polyester poly(3-hydroxybutyrate) in environmental samples and in culture

Poly(3-hydroxybutyrate) [P(3HB)] test-pieces prepared from the polymer produced by Azotobacter chroococcum were degraded in natural environments like soil, water, compost and sewage sludge incubated under laboratory conditions. Degradation in terms of % weight loss of the polymer was maximum (45%) in sewage sludge after 200 days of incubation at 30°C. The P(3HB)-degrading bacterial cultures (36) isolated from degraded test-pieces showed different degrees of degradation in polymer overlayer method. The extent of P(3HB) degradation increases up to 12 days of incubation and was maximum at 30°C for majority of the cultures. For most efficient cultures the optimum concentration of P(3HB) for degradation was 0.3% (w/v). Supplementation of soluble carbon sources like glucose, fructose and arabinose reduced the degradation while it was almost unaffected with lactose. Though the cultures degraded P(3HB) significantly, they were comparatively less efficient in utilizing copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB-co-3HV)].     

Sequencing microbial copolymers of 3-hydroxybutyric and 3-mercaptoalkanoic acids by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amounts of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry analysis of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. (13)C NMR spectroscopy of intact polymer samples, with values of average block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, composition analysis by (1)H NMR of fractions obtained by size exclusion chromatography showed significant variations with molecular weight, revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The experimental NMR carbonyl dyad signal intensities were satisfactorily matched by a random sequence model when the presence of poly(3HB) was taken into account.     

In silico prediction and validation of the importance of the Entner-Doudoroff pathway in poly(3-hydroxybutyrate) production by metabolically engineered Escherichia coli

The metabolic network of Escherichia coli was constructed and was used to simulate the distribution of metabolic fluxes in wild-type E. coli and recombinant E. coli producing poly(3-hydroxybutyrate) [P(3HB)]. The flux of acetyl-CoA into the tricarboxylic acid (TCA) cycle, which competes with the P(3HB) biosynthesis pathway, decreased significantly during P(3HB) production. It was notable to find from in silico analysis that the Entner-Doudoroff (ED) pathway flux increased significantly under P(3HB)-accumulating conditions. To prove the role of ED pathway on P(3HB) production, a mutant E. coli strain, KEDA, which is defective in the activity of 2-keto-3-deoxy-6-phosphogluconate aldolase (Eda), was examined as a host strain for the production of P(3HB) by transforming it with pJC4, a plasmid containing the Alcaligenes latus P(3HB) biosynthesis operon. The P(3HB) content obtained with KEDA (pJC4) was lower than that obtained with its parent strain KS272 (pJC4). The reduced P(3HB) biosynthetic capacity of KEDA (pJC4) could be restored by the co-expression of the E. coli eda gene, which proves the important role of ED pathway on P(3HB) synthesis in recombinant E. coli as predicted by metabolic flux analysis.     

A New Beneficial Mutation in Pseudomonas sp. 61-3 Polyhydroxyalkanoate (PHA) Synthase for Enhanced Cellular Content of 3-Hydroxybutyrate-Based PHA Explored Using Its Enzyme Homolog as a Mutation Template

A newly isolated mutation (Gln508Leu) and a combination of it with previously discovered beneficial mutations in polyhydroxyalkanoate synthase 1 from Pseudomonas sp. 61-3 were found to enhance the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoate)s in recombinant Escherichia coli.     

High-cell-density cyclic fed-batch fermentation of a poly(3-hydroxybutyrate)-accumulating thermophile, Chelatococcus sp. strain MW10

Different fermentation strategies were employed for the cultivation of a new poly(3-hydroxybutyrate)-accumulating thermophilic bacterium, Chelatococcus sp. strain MW10, with the aim of achieving high-cell-density (HCD) growth and high poly(3-hydroxybutyrate) [poly(3HB)] productivity. Enhanced cultivation was achieved by a cyclic fed-batch fermentation (CFBF) technique (42-liter scale). Maximal poly(3HB) productivity was obtained during the second cycle [16.8 ± 4.2 g poly(3HB)/liter]. At the end of CFBF (265 h), an HCD of up to 115.0 ± 4.3 g cell dry weight/liter was achieved.