无外源脯氨酸发酵产反式-4-羟脯氨酸重组大肠杆菌的构建及发酵优化

为实现无外源脯氨酸的条件下直接从葡萄糖转化生成反式-4-羟脯氨酸,本实验采用定点突变和基因共表达的方法构建重组大肠杆菌:首先对L-脯氨酸生物合成途径中的关键酶谷氨酸激酶进行定点突变E143A、K145A,以增强L-脯氨酸生物合成能力;然后引入脯氨酸4-羟化酶基因,通过两个基因的共表达可以实现从葡萄糖到反式-4-羟脯氨酸的连续转化,而不再需要添加外源L-脯氨酸。得到的L-脯氨酸生物合成能力增强的菌株在摇瓶阶段L-脯氨酸的产量可达到1.4 g/L;pro BA2与hyp双基因共表达菌株的反式-4-羟脯氨酸产量为98.9 mg/L,比原始菌株提高了一倍,经发酵优化后得到培养基为:葡萄糖10 g/L,胰蛋白胨15 g/L,硫酸亚铁3 mmol/L,硫酸镁1 g/L,磷酸氢二钾3 g/L,氯化钙0.015 g/L,在这个培养条件下反式-4-羟脯氨酸的产量为220.0 mg/L,比优化前提高1.2倍。      

代谢工程改造大肠杆菌合成L-异亮氨酸的研究

以L-苏氨酸生产菌Escherichia coli THRD为出发菌株,利用基因重组技术替换ilvLXGMEDA启动子并过表达解除L-异亮氨酸反馈抑制的ilvA和ilvIH,以期获得L-异亮氨酸生产菌.将ilvLXGMEDA启动子替换为强启动子Ptrc并敲除ilvLXGM后获得ILE01菌株,于该菌株中分别过表达解除L-异亮氨酸反馈抑制的ilvIH及共表达解除L-异亮氨酸反馈抑制的ilvA和ilvIH,获得菌株ILE02和ILE03,其L-异亮氨酸产量分别达到1.75 g/L和2.19g/L.针对ILE03 α-酮丁酸积累量过高的问题,通过改变操纵子中ilvA和ilvIH的顺序调节其转录水平,获得菌株ILE04,其L-异亮氨酸产量达2.85 g/L.利用ILE04于5L发酵罐中进行发酵实验,L-异亮氨酸产量、发酵强度及转化率分别为5.23 g/L、0.17 g/(L·h)及4.6％.     

大肠杆菌合成琥珀酸的代谢工程研究进展

琥珀酸是一种重要的平台化合物,广泛地应用于食品、医药和化工等领域,被美国能源部列为12种最具潜力的可大宗生产的 化学品之首。 大肠杆菌（Escherichia coli）是目前生产琥珀酸的主要菌株,利用生物法发酵生产琥珀酸因具有原料可再生利用、绿色环 保等优势而成为当今研究的热点。该文从代谢调控的角度出发分析总结了大肠杆菌在发酵产琥珀酸中所应用到的方法策略,如消除 竞争途径、过表达关键酶、提供还原能量、扰动磷酸戊糖途径等,着重突出了代谢控制发酵产琥珀酸方面的研究进展,并对今后的发 展提供了新思路。     

重组大肠杆菌产普鲁兰酶发酵条件优化

在7L发酵罐规模,利用自诱导培养方式,对重组大肠杆菌产普鲁兰酶发酵条件进行优化研究,主要考察了发酵过程中控制溶解氧浓度、p H和采用补料策略对菌体生长和普鲁兰酶表达的影响。结果表明,最佳发酵培养条件:溶氧浓度维持在20%~30%;p H保持在7.60;第9 h时,以0.8 g/(L·h)的速度流加甘油;在20 h,以0.2 g/(L·h)的速度流加乳糖。此条件下,重组菌的细胞浓度OD600 nm从12.58上升到42.25,提高了3.36倍;普鲁兰酶酶活力从356 U/ml上升到1 200 U/ml,提高了3.37倍。     

Proteome analysis of a temperature-inducible recombinant Escherichia coli for poly-beta-hydroxybutyrate production

A recombinant Escherichia coli strain harboring the lambdap(R)-p(L) promoter and heterologous poly-beta-hydroxybutyrate (PHB) biosynthesis genes was shown to accumulate PHB when the incubation temperature was changed from 34 degrees C to temperatures higher than 37 degrees C. In the present research, total gene expression patterns of the recombinant E. coli before and after induction were investigated by two-dimensional gel electrophoresis. Proteins encoded by serS, sucC, trpA, and alaS were found to be expressed before induction of phb genes at a culture temperature of 34 degrees C. On the other hand, proteins encoded by metG, rplI, and carA were found to be expressed after induction achieved by increasing the temperature to 40 degrees C. In the case of plasmid-free cells, all the selected genes have been shown to be expressed except metG, and ibpA and ibpB among the heat-shock proteins. The heat-shock proteins were found to be upregulated upon induction of phb genes, which may be due to the stress caused by the accumulation of PHB granules as well as by the temperature upshift. The changes in the expression of some of the metabolic pathway-related proteins before and after induction were interpreted in relation to the consumption of NADPH and acetyl-CoA for PHB synthesis.     

Effect of modifying metabolic network on poly-3-hydroxybutyrate biosynthesis in recombinant Escherichia coli

The regulatory mechanism for poly-3-hydroxybutyrate (PHB) biosynthesis in recombinant Escherichia coli is markedly different from that of Ralstonia eutropha (formerly, Alcaligenes eutrophus) since the former efficiently synthesizes PHB during growth without any nutrient limitation. To analyze how the central metabolic pathways should be balanced with pathways necessary for cell growth and PHB formation, a stoichiometric model was developed to predict the theoretical maximum PHB production capacity for different metabolic variants. Flux analysis results illustrated the importance of the availability of acetyl-CoA and NADPH for achieving the maximum yield of PHB. In order to examine whether the increased availability of the above substances can enhance PHB synthesis in recombinant E. coli, both genetic and environmental perturbations were attempted. Several E. coli K12 derivatives, namely, HMS174, TA3516 (pta-/ack-), and DF11 (pgi-), were transformed with a plasmid which contains the native phb operon. The fermentation characteristics of these recombinant strains were studied and compared. In this study we examined the effects of intracellular acetyl-CoA accumulation, which may promote PHB synthesis in vivo, by perturbations induced from attenuation of acetate kinase and phosphotransacetylase (TA3516, blocked in the acetate pathway) and by cultivation of E. coli HMS174 on gluconate; it can convert gluconate to acetyl-CoA at a higher rate. The effects of intracellular accumulation of NADPH were investigated by introducing a perturbation induced from attenuation of phosphoglucose isomerase, which redirects the carbon flow to the pentose-phosphate (PP) pathway. Results from the analyses of these perturbations indicate that intracellular buildup of acetyl-CoA may not be able to promote PHB synthesis in vivo. On the other hand, since the biosynthesis of PHB in the pgi- mutant strain can utilize the NADPH overproduced through the PP pathway, the growth of the pgi- mutant on glucose was recovered, indicating that the overproduction of NADPH might be able to enhance PHB synthesis.     

Enhanced production of human epidermal growth factor by a recombinant Escherichia coli integrated with in situ exchange of acetic acid by macroporous ion-exchange resin

A macroporous ion-exchange resin was adopted for the in situ exchange of acetic acid during human epidermal growth factor (hEGF) production by recombinant Escherichia coli JM101. The results of this study suggest that in situ exchange by macroporous resin can improve E. coli growth, decrease acetate accumulation and enhance hEGF production.     

Enhancement of retinal production by supplementing the surfactant Span 80 using metabolically engineered Escherichia coli

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.     

高温微好氧发酵策略强化重组大肠杆菌D-乳酸合成途径

D-乳酸作为一种重要的手性中间体和聚乳酸合成的原料,其生产已越来越受到人们的重视。Escherichia coli CICIM B0013-070是一株能同型发酵合成D-乳酸的基因重组菌。比较该菌株不同温度、不同供氧强度下的生长情况及发酵产酸性能。结果显示,采用微好氧发酵,在37、40℃温度下菌体量仍在增长;当发酵温度高于42℃后,菌体生长受到一定抑制,菌体量保持稳定,而乳酸的比合成速率和乳酸得率均有所提高。另外,对比微好氧发酵和限氧发酵的乳酸比合成速率发现,前者为后者的1.5～2倍;且微好氧条件下,葡萄糖到乳酸的得率随温度的提高逐渐增加,较高温度下接近甚至超过了限氧条件相同发酵温度下的得率。上述结果表明,在非严格限氧的条件下,可以通过发酵温度的提高,实现发酵阶段菌体生长和D-乳酸合成的代谢流微调,维持D-乳酸高生产强度的同时提高D-乳酸得率。     

Effect of post-induction substrate oscillation on recombinant alkaline phosphatase production expressed in Escherichia coli

Microorganisms are exposed to fast changes in microenvironment in large scale bioreactors. Because of their fast response to the changes, overall performance of biological system in small scale differs from large scale. Hence the variations in the environment that microorganisms are living in are mimicked in small scale. For this purpose one way is to feed substrate into the bioreactor in an oscillatory profile. In this work two different types of triangular oscillatory feeding profiles were applied as the post induction feeding strategy in intracellular recombinant alkaline phosphatase production expressed in Escherichia coli to find out if this biological system behaves in inhomogeneous environment differently. On line and offline measurements provide evaluation of product quality and quantity. Then the results of the experiments were compared with those of the control run at which constant feeding rate was executed. The results showed that oscillatory feeding at which cells were not starved led to higher yield of protein per substrate (0.027 C-mol/C-mol) and higher activity per protein (0.79 U/mg) when compared to a constant feeding rate (0.011 C-mol/C-mol and 0.11 U/mg).     

重组大肠杆菌全细胞合成D-苯基乳酸

苯基乳酸(phenyllactic acid,PLA)是一种重要的有机酸,广泛应用于食品、医药、化妆品等领域。通过构建D-乳酸脱氢酶重组大肠杆菌BL21(DE3)/p ET28a-ldh D利用全细胞催化合成D-苯基乳酸。对重组大肠杆菌的诱导及转化条件进行了优化,最终确定了最佳的诱导条件:OD600=0. 8时,添加IPTG至终浓度为0. 2 mmol/L,在25℃下诱导4 h;最佳的转化条件:pH 7. 0磷酸缓冲液,13 g/L苯丙酮酸钠,5 g/L葡萄糖,30 g/L菌体(干重),37℃,200 r/min,转化2 h。在最优的条件下,苯基乳酸的产物浓度达到5. 2 g/L,产率为40%。该文还探索了苯丙酮酸钠与葡萄糖分批添加对苯基乳酸合成的影响,并初步得到了最佳工艺,经3 h转化,苯基乳酸产物质量浓度和产率分别提高到7. 38 g/L和52. 7%,显示了较好的应用前景,也为后续酶的改造奠定了基础。     

过量表达葡萄糖脱氢酶对E.coli羟基脂肪酸合成能力的影响

在工程大肠杆菌生产羟基脂肪酸的基础上,为解决构建的以葡萄糖为底物合成羟基脂肪酸(HFAs)代谢途径中存在的细胞内还原力(NADPH)不平衡的问题,克隆了来源于巨大芽孢杆菌葡萄糖脱氢酶(GDH)基因,构建了胞内HFAs和NADPH联产的工程菌株;实验结果表明该重组大肠杆菌合成HFAs的产量得到显著提高,摇瓶条件下HFAs产量达到173.9 mg/L。具有较好的工业化开发前景。      

Construction and selection of the novel recombinant Escherichia coli strain for poly(beta-hydroxybutyrate) production

Heterogeneous cloning of Vitreoscilla hemoglobin gene (vgb), lytic genes of phage lambda with S amber mutation (S(-)RRz) and PHB biosynthetic genes (phbCAB) in the same host strain E. coli JM105 was carried out for production of poly(beta-hydroxybutyrate) (PHB). A superior novel strain, VG1 (pTU14), was constructed and selected, which contained the vgb gene in the chromosomal DNA and the plasmid pTU14 containing S(-)RRz and phbCAB genes. When cultured in 100 ml of LBG medium in a 300-ml flask, all of the exogenous genes in VG1 (pTU14) were expressed. The cell concentration of VG1 (pTU14) grown by batch culture in a flask reached 10.2 gl(-1); PHB concentration, PHB content and PHB yield, which is the ratio of the PHB accumulation to the glucose consumption, were 8.54 gl(-1), 84% and 0.43 gg(-1), respectively. When cultured by batch-feeding of glucose in a 300-ml shaking flask, the cell concentration and PHB content reached 26 gl(-1) and over 96%, respectively.     

Effects of medium composition on production of 5-aminolevulinic acid by recombinant Escherichia coli

The recombinant Escherichia coli BL21(DE3) harboring hemA from Agrobacterium radiobacter, which was engineered in our previous work, was used for the extracellular production of 5-aminolevulinic acid (ALA). The effects of various physiological factors, such as the concentrations of precursors (glycine, succinic acid and glucose) and the inhibitor 5-aminolevulinate dehydratase (levulinic acid), on the ALA accumulation in the fermentation broth were investigated in both shake flasks and a jar fermentor. Among these precursors, glycine exhibited the strongest ability to inhibit cell growth, while glucose mainly inhibited ALA formation. The optimum initial concentrations of glycine, succinic acid and glucose were found to be 2.0, 10.0 and 2.0 g/l, respectively. Levulinic acid (LA; 30 mM) was fed to the fermentation broth at the end of the exponential cell growth phase (about 8 h), and the intracellular activity of ALA dehydratase was efficaciously suppressed. Repeating the optimum composition of the medium in a stirred tank fermenter resulted in 1.49 g/l ALA. Furthermore, the fed batch of the precursors and inhibitor further increased ALA production up to 3.01 g/l.     

High cell density cultivation of recombinant Escherichia coli for production of rat procarboxypeptidase B

The recombinant Escherichia coli harboring pPT/proCPB (procarboxypeptidase B) gene was constructed for the overexpression of rat proCPB gene. The proCPB expression was controlled by the rrnB P2 promoter fused with lac operator. In the fed batch fermentations, the expression of proCPB was accelerated by the temperature shift from 30 to 37°C without lac operon inducers such as isopropyl-1-thio-β-d-galactoside (IPTG) and lactose. Fermentation strategies including the 3-step increase was optimized to harvest high titers of cell growth and ProCPB. The ideal results of optical density 80 and 66.7% of ProCPB content were obtained through the optimized 3-step shift fed-batch fermentation from 30 to 37oC for 6 hr. After refolding and activation of ProCPB to CPB by trypsin treatment, the CPB activity of 39,375 U/L with specific activity 135 U/mg was obtained in the culture broth. In the conversion reaction by ProCPB, preproinsulin was successfully transformed into insulin.     

pH及溶氧对重组大肠杆菌发酵生产5-氨基乙酰丙酸的影响

以重组大肠杆菌DALA为实验菌株,研究了该菌株在机械搅拌通风发酵罐发酵过程中pH以及溶解氧对5-氨基乙酰丙酸（ALA）积累的影响。结果发现,发酵前期（0～27 h）pH保持为6.5;稳定期后期（28～48 h）,pH为6.0时有利于5-ALA的积累。其次,通过控制转速与通气量调节发酵液中的溶氧,发现发酵前期转速为500 r/min,通气量为2 vvm;稳定期后期,转速降低至250 r/min,通气量减少为1 vvm,有利于重组菌DALA发酵生产5-ALA,在此条件下发酵5-ALA的产量可达到3.46 g/L。     

基因工程大肠杆菌生产重组别藻蓝蛋白(holo-SA-apcA)发酵条件优化

利用Design-Expert 8.0软件,对大肠杆菌产重组别藻蓝蛋白(holo-SA-apc A)的发酵条件进行优化。首先运用Plackett-Burman两水平设计,对影响重组蛋白表达量的8个因素进行评价,选择有显著影响的3个因素,即p H值、诱导温度、接种量。在此基础上,再用最陡爬坡实验快速逼近以上3个因素的最大响应区域,最后经过Box-Behnken设计和响应面分析,通过求解回归方程得到最优条件为:p H 8.0、诱导温度18℃、接种量4%。在此条件下重复发酵实验3次,重组蛋白表达量达52.3 mg/L,与预测值基本一致。     

Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine

Beta-(pyrazol-1-yl)-L-alanine (beta-PA), a model nonproteinaceous amino acid, was specifically synthesized by two methods using recombinant Escherichia coli cells that express cysteine synthase, comprising serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS-A) and related enzymes from E. coli. In the first method (method A), recombinant cells that express wild-type SAT, OASS-A, acetate kinase (AK), and phosphotransacetylase (PTA) showed the highest beta-PA production. beta-PA was produced at 140 mM from 200 mM L-serine and 200 mM pyrazole under optimum conditions. Using the cells expressing SATDeltaC20 (truncated SAT), OASS-A, AK, and PTA, beta-PA was produced at a level of only 80 mM, whereas O-acetyl-serine (OAS) was found to be secreted into the broth. Under optimum conditions, OAS accumulated at levels of around 105 mM from 300 mM L-serine. Thus, in the second method (method B), the secreted OAS was used as the substrate for the syntheses of beta-PA and beta-(triazol-1-yl)-L-alanine (beta-TA). The OAS that accumulated in the broth was efficiently converted to beta-PA and beta-TA at levels of around 90 mM from 105 mM OAS using free OASS-A. In both methods A and B, the addition of glucose was essential for the efficient production of beta-PA and OAS, respectively.