Role of xylose transporters in xylitol production from engineered Escherichia coli

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.     

Doubling the catabolic reducing power (NADH) output of Escherichia coli fermentation for production of reduced products

Homofermentative production of reduced products requires additional reducing power output (NADH) from glucose catabolism. Anaerobic expression of the pyruvate dehydrogenase complex (PDH, encoded by aceEF‐lpd, a normal aerobic operon) is able to provide the additional NADH required for production of reduced products in Escherichia coli fermentation. The multiple promoters (pflBp(1–7)) of pyruvate formate lyase (pflB) were evaluated for anaerobic expression of the aceEF‐lpd operon. Four chromosomal constructs, pflBp(1–7)‐aceEF‐lpd, pflBp(1–6)‐aceEF‐lpd, pflBp(6,7)‐aceEF‐lpd, and pflBp6‐aceEF‐lpd efficiently expressed the PDH complex in anaerobically grown cells. Doubling the reducing power output was achieved when glucose was oxidized to acetyl‐CoA through glycolysis and pyruvate oxidation by the anaerobically expressed PDH complex (glucose →2 acetyl‐CoA + 4 NADH). This additional reducing power output can be used for production of reduced products in anaerobic E. coli fermentation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

重组大肠杆菌利用木糖发酵产高纯度L-乳酸

对重组大肠杆菌JH16利用木糖产高纯度的三一乳酸进行研究。通过无氧管驯化EscherwhiacdiJH12菌株得到E．coliJH16,驯化后的菌株茵体浓度提高了31％,乙酸积累减少了43％;在摇瓶中考察不同Mg2＋浓度对EcoliJHl6产三一乳酸的影响,确定最适Mg2＋质量浓度为0．25g/L;EcoEJH16以60g/L木糖为C源,在7L全自动发酵罐中添加0．25g／LMg2＋,乳酸积累量提高了18％,达38．18g/L,乳酸纯度高达95％;E．coliJH16在30g／L木糖和30g／L葡萄糖混合C源中,优先利用葡萄糖,当葡萄糖质量浓度低于1．56g/L后,菌体开始利用木糖进行乳酸发酵,最终得到39g／L乳酸。     

Analysis of NADPH supply during xylitol production by engineered Escherichia coli

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.     

Heterologous expression of D-xylulokinase from Pichia stipitis enables high levels of xylitol production by engineered Escherichia coli growing on xylose

Deletion of the Escherichia coli xylulokinase gene (xylB) is essential for achieving high xylitol titers from xylitol-producing E. coli strains growing on glucose in the presence of xylose. Our study suggests that this is due to XylB-catalyzed toxic synthesis of xylitol-phosphate. This activity prohibits the use of xylose as the sole carbon source during xylitol production by E. coli. To overcome this limitation we turned to the yeast Pichia stipitis, which naturally produces xylitol, as a source of xylulokinase (Xyl3). We examined the effects of plasmid-based expression of Xyl3 versus XylB on growth and xylitol production by engineered E. coli strains. Xylulokinase activity assays show similar levels of functional expression of both enzymes (determined as activity on xylulose), and reveal significantly more activity on xylitol by XylB compared to Xyl3. (31)P NMR confirms the production of xylitol-phosphate from in vitro reactions with XylB. Lastly, the replacement of xylB with XYL3 results in drastically enhanced xylitol titers from E. coli strains co-expressing xylose reductase during growth on xylose.     

Improved NADPH supply for xylitol production by engineered Escherichia coli with glycolytic mutations

Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

Bioaccumulation of mercury from wastewater by genetically engineered Escherichia coli

Genetically engineered E. coli, which express both a Hg2+ transport system and metallothionein, were tested for their ability to remove mercury from wastewater. The wastewater contained more than ten different ions, including 2.58 mg/l mercury, and its pH was 9.6. Mercury uptake was faster from the wastewater than from distilled water, probably because of the higher ionic strength, as the high pH had little effect on mercury accumulation. EDTA also stimulated mercury uptake rather than inhibiting it. A hollow-fiber bioreactor was used to retain induced cells for continuous mercury uptake. The cells removed more than 99% of the mercury in the wastewater and the final amount of mercury accumulated was 26.8 mg/g cell dry weight, while none of the other ions were removed from the water. These results indicated that the induced cells had a high affinity and specificity for mercury.     

Biosynthesis of L-5-methyltetrahydrofolate by genetically engineered Escherichia coli

L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 μg g⁻¹ of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.     

Metabolic pathway optimization for biosynthesis of 1,2,4-butanetriol from xylose by engineered Escherichia coli

1,2,4-Butanetriol (BT) and related derivatives have been widely used in many fields, especially in the military and in medicine. In this paper, we systematically optimized the BT biosynthetic pathway. We first investigated the activities of various NADH dependent aldehyde reductases (ALRs), which catalyze the fourth reaction in the four-step pathway for BT production from xylose in E. coli, and found that a combination of multiple endogenous enzymes catalyzed aldehyde reduction in the BT production bioprocess and that YqhD in E. coli was a main ALR for BT production. In addition, ADH2 from Saccharomyces cerevisiae can effectively catalyze 3,4-dihydroxybutanal to BT. Also, YjhG was identified as the major xylonate dehydratase and was co-overexpressed with YqhD, resulting in an improvement of BT production by 30%. Moreover, we identified and eliminated the competing branch pathway by inactivating 2-keto acid reductases (yiaE). Finally, the combination of these approaches led to BT production of 5.1 g/L. In summary, our study provides insights into the biosynthetic pathway for BT production, demonstrates an effective strategy to enhance BT production, and paves the way toward in-depth research on BT biosynthesis.     

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: γ‐glutamylcysteine synthetase (γ‐GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over‐express either γ‐GCS or GS and found that γ‐GCS over‐expression resulted in inclusion body formation and impaired cell physiology, whereas GS over‐expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over‐expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild‐type cells in size (2–5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ≈2.5 times that attained in the wild‐type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli‐based biosynthesis offers a more energy‐efficient and eco‐friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Construction and characterization of an Escherichia coli strain genetically engineered for Ni(II) bioaccumulation

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase-pea metallothionein fusion protein. The resulting strain accumulated 15 micromol of Ni(II) per g (dry weight) from a 10 microM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.     

A novel genetically engineered pathway for synthesis of poly(hydroxyalkanoic acids) in Escherichia coli

A new pathway to synthesize poly(hydroxyalkanoic acids) (PHA) was constructed by simultaneously expressing butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) genes of Clostridium acetobutylicum and the two PHA synthase genes (phaE and phaC) of Thiocapsa pfennigii in Escherichia coli. The four genes were cloned into the BamHI and EcoRI sites of pBR322, and the resulting hybrid plasmid, pBPP1, conferred activities of all three enzymes to E. coli JM109. Cells of this recombinant strain accumulated PHAs when hydroxyfatty acids were provided as carbon sources. Homopolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB), or 4-hydroxyvalerate (4HV) were obtained from each of the corresponding hydroxyfatty acids. Various copolyesters of those hydroxyfatty acids were also obtained when two of these hydroxyfatty acids were fed at equal amounts: cells fed with 3HB and 4HB accumulated a copolyester consisting of 88 mol% 3HB and 12 mol% 4HB and contributing to 68.7% of the cell dry weight. Cells fed with 3HB and 4HV accumulated a copolyester consisting of 94 mol% 3HB and 6 mol% 4HV and contributing to 64.0% of the cell dry weight. Cells fed with 3HB, 4HB, and 4HV accumulated a terpolyester consisting of 85 mol% 3HB, 13 mol% 4HB, and 2 mol% 4HV and contributing to 68.4% of the cell dry weight.     

Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli

Metabolically engineered Escherichia coli strains were constructed to effectively produce novel glycolate-containing biopolymers from glucose. First, the glyoxylate bypass pathway and glyoxylate reductase were engineered such as to generate glycolate. Second, glycolate and lactate were activated by the Megasphaera elsdenii propionyl-CoA transferase to synthesize glycolyl-CoA and lactyl-CoA, respectively. Third, β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha were introduced to synthesize 3-hydroxybutyryl-CoA from acetyl-CoA. At last, the Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61–3 was over-expressed to polymerize glycolyl-CoA, lactyl-CoA and 3-hydroxybutyryl-CoA to produce poly(glycolate-co-lactate-co-3-hydroxybutyrate). The recombinant E. coli was able to accumulate the novel terpolymer with a titer of 3.90 g/l in shake flask cultures. The structure of the resulting polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the produced terpolymer possessed decreased crystallinity and improved toughness, in comparison to poly(3-hydroxybutyrate) homopolymer. This is the first study reporting efficient microbial production of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose.     

大肠杆菌利用葡萄糖和木糖合成乙醇酸、乳酸和3-羟基丁酸共聚酯

以大肠杆菌为宿主,构建了以葡萄糖和木糖为底物获得乙醇酸、乳酸和3-羟基丁酸共聚酯的生物合成途径,包括过表达塔格糖-3-差向异构酶、核酮糖激酶、醛缩酶、醛脱氢酶、丙酰辅酶A转移酶、β-酮硫解酶、乙酰乙酰辅酶A还原酶和聚合酶等。在此基础上,表达聚羟基脂肪酸酯颗粒结合蛋白,提高了聚合物的合成,重组菌的细胞干重达到3.73g/L,含有38.72wt%的共聚酯。采用混菌共培养策略,实现以葡萄糖和木糖混合物为底物合成共聚酯,摇瓶实验中细胞干重达到4.01g/L,含有21.54wt%的聚合物。文中提供了一种以葡萄糖和木糖混合物为碳源合成聚合物的方法,为下一步纤维素水解物的有效利用提供了参考。     

利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

[目的]本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470(△frdBC △ldhA △ackA △focA-pflB △pdhR::pflBp6-pflBrbs-aceEF-lpd)为起始菌株,进一步敲除其乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶(L-lactate dehydrogenase,LLDH)基因,构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌.[方法]利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01,并克隆P.acidilactici的ldhL基因,利用染色体插入技术将其整合到JH01基因组,构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12,利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量.[结果]工程菌JH12在15 L发酵罐中以6％的葡萄糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为1.46 g/(L·h),乳酸生产强度为1.14 g/(L·h),乳酸的产量达到41.13 g/L.发酵产物中未检测到琥珀酸、甲酸的生成,仅有少量乙酸生成,L-乳酸纯度达95.69％(L-乳酸在总发酵产物的比率).工程菌JH12以6％的木糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h),乳酸生产强度为0.60 g/(L·h),乳酸的产量达到34.73 g/L.发酵产物中杂酸少,乳酸的纯度高达98％.[结论]本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12,该菌株不需利用外源质粒,稳定性好,可利用五碳糖进行发酵,发酵产物中杂酸少,L-乳酸的纯度高.本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持,同时也为大肠杆菌L-乳酸的工业化生产提供了参考依据.     

High level expression of a thermostable Bacillus xylose (glucose) isomerase in Escherichia coli

Summary A thermophilic Bacillus sp. producing xylose (glucose) isomerase has been isolated. Its xy/A gene when cloned in Escherichia coli and expressed gave 37.5 and 12.8 units/ mg protein respectively for xylose and glucose isomerase activities at 85°C. A single heat treatment of the crude extract purified the enzyme further yielding the highest ever recorded activities of 150 and 49.02 units /mg protein.     

Catechol 2,3-oxygenase production by genetically engineered Escherichia coli and its application to catechol determination

Catechol 2,3-oxygenase was produced by Escherichia coli, harbouring the recombinant plasmid pBH100 which contained the pheB gene cloned from phenol-degrading Pseudomonas putida BH, and was applied for the determination of catechol in the liquor. E. coli JM103 (pBH100) and C600 (pBH100) showed, respectively, about 5 and 8.5 times higher activities than that of P. putida BH. Using the crude extract prepared from the culture broth of the recombinant, catechol between 0.1 and 3.0 g/ml could be determined quantitatively in phosphate buffer, synthetic sewage and in mixtures of phenol, benzoate and sallcylate, and also in sodium pyruvate solution. In addition to catechol, 3-methylcatechol, 4-methylcatechol and 4-chlorocatechol could be determined. Oxygenase activity of the crude extract was maintained completely during the 100-day storage at –20°C after being freeze-dried with 10% acelone.M. Fujita, M. Ike, Y. Kawagoshi and N. Shinohara are with the Department of Environmental Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565, Japan. T. Kamiya is with the Central Research Laboratory of Mitsubishi Electric Co., Amagasaki, Hyogo 661, Japan.     

Production of S-lactoylglutathione by glycerol-adapted Saccharomyces cerevisiae and genetically engineered Escherichia coli cells

Summary The enzymatic production of S-lactoylglutathione was studied by applying glyoxalase I to glycerol-grown cells of Saccharomyces cerevisiae and Escherichia coli cells dosed with Pseudomonas putida glyoxalase I gene. The glyoxalase I in S. cerevisiae cells was markedly induced when the cells were grown on glycerol. The activity of the enzyme in glycerol-grown cells was more than 20-fold higher compared with that of the glucose-grown cells. By using extracts of glycerol-grown yeast cells, about 5 mmol/1 (2 g/l) of S-lactoylglutathione was produced from 10 mM methylglyoxal and 50 mM glutathione within 1 h. The extracts of E. coli cells carrying a hybrid plasmid pGI423, which contains P. putida glyoxalase I gene, showed approximately 170-fold higher glyoxalase I activity than that of E. coli cells without pGI423. The extracts were used for production of S-lactoylglutathione and, under optimal conditions, about 40 mmol/l (15 g/l) of S-lactoylglutathione was produced from 50 mM methylglyoxal and 100mM glutathione within 1 h.     

Poly(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli

Several recombinant Escherichia coli strains harboring the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes were used to produce poly(3-hydroxybutyrate), PHB, from xylose. By flask culture of TG1 (pSYL107) in a defined medium containing 20?g/l xylose, PHB concentration of 1.7?g/l was obtained. Supplementation of a small amount of cotton seed hydrolysate or soybean hydrolysate could enhance PHB production by more than two fold. The PHB concentration, PHB content, and PHB yield on xylose obtained by supplementing soybean hydrolysate were 4.4?g/l, 73.9%, and 0.226?g PHB/g xylose, respectively.