产聚羟基脂肪酸酯细菌的筛选与鉴定

【目的】聚羟基脂肪酸酯(polyhydroxyalkanoates,PHAs)是一种生物可降解的天然高分子聚酯,本研究的目的是从广东省某啤酒厂废弃的活性污泥中分离筛选PHAs产生菌。【方法】首先,从活性污泥中分离PHAs产生菌。分离方法分3步:(1)富集培养PHAs产生菌;(2)通过苏丹黑B染色法进行初筛;(3)挑选PHAs产量较高的菌株,然后对细胞内提取产物进行分析,最后通过生理生化试验和16SrRNA基因序列分析法对该菌株进行鉴定。【结果】从广东省某啤酒厂的活性污泥样品中筛选获得PHAs产生菌HG-B-1,被鉴定为嗜麦芽寡养单胞菌(Stenotrophomonas maltophlia)。细胞染色分析、胞内提取物的红外光谱分析表明HG-B-1胞内贮藏物为PHAs。该菌株在以蔗糖为碳源、牛肉膏为氮源的发酵培养基中,37℃振荡培养24h,PHAs产量可达细胞干重的23.4%。【结论】本文从广东省某啤酒厂的活性污泥中筛选得到PHAs产生菌,获得了一株新型的PHAs产生菌,为进一步研究和开发新型的PHAs产生菌提供了菌源和基础资料。     

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。     

细菌聚羟基脂肪酸酯检测方法研究进展

细菌聚羟基脂肪酸酯(polyhydroxyalkanoates,PHAs）是存在于许多细菌细胞内的聚合物,是一种新型的生物材料,在生态研究中可作为营养指标。回顾有关PHAs的研究方法的同时介绍用FT-IR技术从细胞水平快速定性和定量分析细菌PHAs。     

利用废弃物发酵法生产聚羟基烷酸PHAs

聚羟基烷酸(PHAs)是一种可降解聚合物,与石化塑料相比它具有生物降解性及生物相容性等优点,在不久的将来必然有广阔的应用前景。生产PHAs的主要方法是发酵法,在过去的几十年里传统的深层发酵法生产PHAs的工艺已经得到深入的研究,近些年固态发酵法生产PHAs也吸引了越来越多研究者的关注。     

由细菌产生的新型生物高分子材料——具新型结构的聚羟基脂肪酸酯PHAs简介

许多微生物能产生聚－β－羟基丁酸酯（PHB）,与淀粉一样作为细胞内的能量和碳源储存物。另外在一定条件下,细菌还可积累具不同结构的单体的共聚松。由荧光假单胞菌Pseudomonas积累的PHAs的结构单元可以是多种多样的。碳链长度可在6－14之间,可由各种饱和的和不饱和的3－羟基脂肪酸组成的。P．oleovorans以正烷烃、正烷基酯或正烷醇为基质,得到的PHAs是以结构单元长度与基质相同的3－羟基脂肪酯为主要组成部分的无视共聚物。含有高达九个碳原子测链的PHAs已可由P.oleovorans利用正烷烃和脂肪酸来合成。P.oleovorans利用1－烯烃或烯酸可合成倒链具不饱和健的聚－β－羟基烷烯酯。通过改变基质中正坡泛与1－烯径的比例,PHAs的不饱和度可由0增加到50％。当以1－辛烯和1－癸烯为碳源时,所得的聚酯主要由β－羟基脂肪酯组成,而链端则是不饱和的β－羟基烷烯酯,侧链可由丙基变化到庚基。最近的研究表明,在生物合成过程中还可在PHAs的侧基上引入部分含Br、Cl、CN的苯基的官能团,或在PHAs合成过程中,可进行β－氧化形成含3－羟酰基的中间产物和再次对脂肪酸进行生物合成。P．oleovorans的这种可将官能团引入聚合物分子链中的特征．为聚合物进行剪裁提供了可能性。     

可完全生物降解生物材料项目在鲁投产

深圳市意可曼科技有限公司在山东邹城举办可完全生物降解材料项目投产庆典,这标志着意可曼生物科技有限公司拥有完全自主知识产权、通过基因工程菌种构造法进行生物发酵合成生物高分子材料聚羟基烷酸酯（PHAs）的技术正式实现批量化生产。该项目的产品品级包括注塑级PHAs、吹膜级PHAs、吹瓶级PHAs、板片级PHAs、可发性PHAs、纺丝／无纺布级PHAs、生物弹性体、3-羟基丁酸酯等,可广泛应用于农业、环境、生化、微电、能源、医用等领域。     

细菌聚羟基脂肪酸酯检测方法研究进展*

细菌聚羟基脂肪酸酯（polyhydroxyalkanoates,PHAs）是存在于许多细菌细胞内的聚合物,是一种新型的生物材料,在生态研究中可作为营养指标。回顾有关PHAs的研究方法的同时介绍用PT-IR技术从细胞水平快速定性和定量分析细菌PHAs。     

微生物发酵法生产聚羟基脂肪酸酯的研究进展

聚羟基脂肪酸酯(Polyhydroxyalkanoates,PHAs)是一类由羟基脂肪酸单体通过酯化聚合得到的高分子化合物,因具有传统石油基塑料类似的力学特征、100%生物降解性和生物相容性而被认为是最有潜力的绿色环保材料之一。受限于其高昂的生产成本,PHAs作为绿色环保材料的应用推广困难。文中分别从细胞形态调控、代谢途径构建、廉价碳源利用和开放式发酵技术开发等方面详细介绍了目前有效降低PHAs生产成本的方法。尽管大部分研究成果还局限于实验室阶段,但是研究方法和方向为实现低成本PHAs的工业化生产提供了理论指导。     

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。     

废弃活性污泥中产聚羟基脂肪酸酯菌株Bacillus sp. PB-3的发酵条件优化

通过尼罗红染色法结合荧光显微镜镜检,从废弃活性污泥中分离得到1株高产聚羟基脂肪酸酯(PHAs)的菌株Bacillus sp.PB-3,经气相色谱法鉴定该菌株胞内产物为聚β-羟基丁酸酯(PHB)。对培养基成分及发酵条件优化后,获得最佳培养方案:12 g/L的葡萄糖为C源,2 g/L的牛肉膏为N源,初始pH 7.5,培养基装液量80 mL,转速为200 r/min,37℃培养48 h,PHB质量分数可达菌体干质量的32.09%,比优化前提高30%。     

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polylactic acid (PLA) and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] employing metabolically engineered Escherichia coli strains by the introduction of evolved Clostridium propionicum propionyl-CoA transferase (Pct _Cp ) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Using this in vivo PLA biosynthesis system, we presently report the biosynthesis of PHAs containing 2-hydroxybutyrate (2HB) monomer by direct fermentation of a metabolically engineered E. coli strain. The recombinant E. coli ldhA mutant XLdh strain expressing PhaC1 _Ps6-19 and Pct _Cp was developed and cultured in a chemically defined medium containing 20 g/L of glucose and varying concentrations of 2HB and 3HB. PHAs consisting of 2HB, 3HB, and a small fraction of lactate were synthesized. Their monomer compositions were dependent on the concentrations of 2HB and 3HB added to the culture medium. Even though the ldhA gene was completely deleted in the chromosome of E. coli, up to 6 mol% of lactate was found to be incorporated into the polymer depending on the culture condition. In order to synthesize PHAs containing 2HB monomer without feeding 2HB into the culture medium, a heterologous metabolic pathway for the generation of 2HB from glucose was constructed via the citramalate pathway, in which 2-ketobutyrate is synthesized directly from pyruvate and acetyl-CoA. Introduction of the Lactococcus lactis subsp. lactis Il1403 2HB dehydrogenase gene (panE) into E. coli allowed in vivo conversion of 2-ketobutyrate to 2HB. The metabolically engineered E. coli XLdh strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene successfully produced PHAs consisting of 2HB, 3HB, and a small fraction of lactate by varying the 3HB concentration in the culture medium. As the 3HB concentration in the medium increased the 3HB monomer fraction in the polymer, the polymer content increased. When Ralstonia eutropha phaAB genes were additionally expressed in this recombinant E. coli XLdh strain, P(2HB-co-3HB-co-LA) having small amounts of 2HB and LA monomers could also be produced from glucose as a sole carbon source. The metabolic engineering strategy reported here should be useful for the production of PHAs containing 2HB monomer.     

Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6–19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.     

Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter

Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (ΔpflA, Δpta, ΔackA, ΔpoxB, Δdld, and a dual mutant; ΔpflA?+?Δdld) and their parent strain, BW25113, were grown on 20 g l^?1 xylose for P(LA-co-3HB) production. The single deletions of ΔpflA, Δpta, and Δdld increased the LA fraction (58–66 mol%) compared to BW25113 (56 mol%). In particular, the ΔpflA?+?Δdld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in ΔpflA, Δpta, and Δdld mutants, and BW25113. The ΔpflA?+?gatC strain achieved a productivity of 8.3 g l^?1, which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l^?1 xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l^?1. On the other hand, the ΔpflA?+?Δdld strain grown on 30 g l^?1 xylose synthesized 6.4 g l^?1 P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000–114,000.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Economic considerations in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by bacterial fermentation

The process for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB/V)] by bacterial fermentation and its recovery was analysed. The effects of various factors such as P(3HB/V) content, P(3HB/V) productivity, P(3HB/V) yield and 3-hydroxyvalerate (3HV) fraction in P(3HB/V) on the production cost of P(3HB/V) were examined. The increase in the 3HV yield on a carbon source did not significantly decrease the production cost when the 3HV fraction was 10 mol%, because the cost of the carbon substrate for 3HV was relatively small in terms of the total cost. However, at a 3HV fraction of 30 mol%, the 3HV yield on a carbon source had a significant effect on the total P(3HB/V) production cost. The production cost of P(3HB/V) increased linearly with the increase in the 3HV fraction in P(3HB/V). Received: 8 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Recent advances in polyhydroxyalkanoate production by bacterial fermentation: mini-review.

Poly(3-hydroxybutyrate) [P(3HB)] and other polyhydroxyalkanoates (PHAs) have been drawing much attention as biodegradable substitutes for conventional nondegradable plastics. For the economical production of P(3HB), various bacterial strains, either wild-type or recombinant, and new fermentation strategies were developed for the production of P(3HB) with high concentration and productivity. To reduce the cost of carbon substrate, several processes for P(3HB) production from cheap carbon sources were also developed. P(3HB) can now be produced to a content of 80% of cell dry weight with the productivity greater than 4 g/l per h. Fermentation strategy was also developed for the efficient production of medium chain length PHA by high cell density culture. With all these advances, P(3HB) and PHAs can be produced by bacterial fermentation at a cost (ca. $2/kg) similar to that of other biodegradable polymers under development.     

Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli

In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB)s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB)s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

Xylose-based hydrolysate from eucalyptus extract as feedstock for poly(lactate-co-3-hydroxybutyrate) production in engineered Escherichia coli

Woody extract-derived hemicellulosic hydrolysate, which was obtained from dissolving pulp manufacturing, was utilized as feedstock for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] in engineered Escherichia coli. The hydrolysate was composed of mainly xylose and galactose, and contained impurities mainly acetate, which was found to inhibit the polymer synthesis rather than the cell growth. Thus, acetate and other impurities were removed through active charcoal and ion-exchange columns. Using the purified hydrolysate, P(LA-co-3HB) was successfully produced (cell dry weight 8.6 g/L, polymer concentration 5.4 g/L, LA fraction 5.5 mol%, polymer content 62.4%), the amount of which was comparable to that obtained using reagent grade xylose and galactose. Therefore, the hydrolysate from woody extract is considered as an abundant, inexpensive and efficient feedstock applicable to consolidated process for P(LA-co-3HB) production, when the removal of acetic acid was satisfactorily accomplished.