基于基因转录和代谢物分析的异亮氨酸生产菌谷氨酸棒状杆菌YILW的理性改造

为获得异亮氨酸生产菌谷氨酸棒状杆菌（Corynebacterium glutamicum）YILW的理性改造策略,考察该菌株与出发菌株C. glutamicum ATCC 13032异亮氨酸合成途径中关键酶及代谢产物的差异。结果表明,C. glutamicumYILW丙酮酸羧化酶编码基因pyc的下调表达使得其胞内草酰乙酸含量降低,过表达该基因显著增加胞内草酰乙酸含量及异亮氨酸产量（分别从1.32 μmol/g（细胞干质量,下同）和5.18 g/L提高至3.32 μmol/g和5.81 g/L）,但副产物赖氨酸及胞内2-酮丁酸积累量提高。针对该问题采用强启动子替换手段过表达ilvBNC操纵子,使得其异亮氨酸产量提高至6.63 g/L。为进一步增加异亮氨酸合成,过表达输出载体编码基因brnE和brnF,其产量提高至7.31 g/L,较出发菌株C. glutamicum YILW提高41.1%,转化率提高40.0%。由此可见,在基因转录及代谢物分析结果指导下理性过表达pyc、ilvBNC操纵子及brnE和brnF能够显著提高异亮氨酸产量并降低副产物浓度。     

代谢工程改造谷氨酸棒杆菌合成四氢嘧啶

为了获得四氢嘧啶生产菌株并利用此菌株提高四氢嘧啶产量,以谷氨酸棒杆菌（Corynebacterium glutamicum）野生菌株ATCC13032为研究对象,以赖氨酸积累量为评价指标,强化了天冬氨酸-β-半醛合成代谢流;通过阻断赖氨酸输出通道LysE,表达不同来源的四氢嘧啶操纵子ectABC,获得了四氢嘧啶生产菌株;通过强化天冬氨酸转氨酶和还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）再生途径,进一步提高四氢嘧啶产量。结果表明,构建了一株高产赖氨酸的谷氨酸棒杆菌工程菌株LYS-3,赖氨酸积累量为28.48 g/L;表达操纵子HEectABC的谷氨酸棒杆菌ECT-3四氢嘧啶产量达到17.21 g/L;过表达基因aspC和ECpntAB谷氨酸棒杆菌ECT-6,摇瓶发酵四氢嘧啶产量达到21.85 g/L。研究结果可为天冬氨酸-β-半醛衍生物的生物合成提供参考。     

代谢工程改造谷氨酸棒状杆菌促进L-异亮氨酸发酵合成的研究进展

L-异亮氨酸作为必需氨基酸具有多种生理功能,对人体和动物健康有重要的调节作用。微生物发酵是工业生产L-异亮氨酸的主要方式,而谷氨酸棒状杆菌（Corynebacterium glutamicum）是L-异亮氨酸发酵生产的主要菌株,主要通过筛选和随机诱变得到,其遗传背景不明且很难通过诱变进一步提高产量,因而通过代谢工程改造谷氨酸棒状杆菌生产L-异亮氨酸成为近年来研究热点。该文综述了近年来代谢工程改造谷氨酸棒杆菌促进L-异亮氨酸生物合成的相关研究,主要涉及代谢通量调控、解除反馈抑制、强化还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）水平以及提高产物分泌能力等方面,对进一步改造菌株和促进L-异亮氨酸发酵合成具有一定的参考意义。     

葡萄糖-6-磷酸脱氢酶基因的Candida tropicalis过量表达及其对木糖醇合成代谢的影响

研究葡萄糖-6-磷酸脱氢酶基因g6pd过量表达对Candida tropicalis木糖醇生物合成代谢的影响。克隆Candidatropicalis CT16的g6pd基因,并将其与表达载体pYES-pgk重组连接,构建重组载体pYES-pgk-g6pd,LiAc/ssDNA/PEG方法转化导入C. tropicalis CT16,筛选阳性转化子,实现g6pd基因的过量表达。结果表明：发酵62 h,阳性转化子C. tropicalis SYG5的葡萄糖-6-磷酸脱氢酶活力提高了300%,发酵液中木糖醇质量浓度达到79.90 g/L,较野生型对照菌株的木糖醇产量提高了12.41%,木糖醇产率提高了44.94%。因此,葡萄糖-6-磷酸脱氢酶在C. tropicalis木糖醇的合成代谢途径中发挥重要作用,增强g6pd基因的表达,可以明显提高菌体NADPH供应量和还原力,有利于木糖醇的生物合成。     

Metabolic physiology of aroma-producing Kluyveromyces marxianus

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway. It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways. While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.     

谷氨酸棒杆菌生产缬氨酸的代谢工程研究进展

L-缬氨酸是人体必需的三种支链氨基酸之一,在生命代谢过程中起着非常重要的作用,因此被广泛应用于食品、医药及饲料等行业。目前,L-缬氨酸主要采用微生物发酵法生产,而谷氨酸棒杆菌是最常用的生产菌种。作者分析了谷氨酸棒杆菌中L-缬氨酸的生物合成途径和代谢调控,综述了对其进行代谢工程改造来提高L-缬氨酸产量的最新研究进展。     

葡萄糖亚适量流加对谷氨酸棒杆菌产L-异亮氨酸的影响

通过动态调节葡萄糖对谷氨酸棒杆菌的供应速率,解决菌种在发酵产L-异亮氨酸不同时期对葡萄糖消耗能力差异的问题。实验结果表明,以谷氨酸棒杆菌YILM1504为供试菌株,在发酵初始葡萄糖添加量80 g/L、90%最大补糖速率的亚适量补糖工艺下,得到动态补糖速率为0（0～8 h）、5.40～7.47（8～14 h）、7.47～7.00（16～24 h）、7.00～5.20 g/（L·h）（24～40 h）,L-异亮氨酸达到44.32 g/L,糖酸转化率为19.63%。经代谢流对比分析发现,葡萄糖经戊糖磷酸途径和糖酵解途径代谢流之比为0.56,进入Ile的代谢流增强了18.92%,进入缬氨酸、亮氨酸、丙氨酸的代谢流减弱了67.12%、72.21%、30.23%,该方法针对谷氨酸棒杆菌在细胞生长阶段与产酸阶段不同的耗糖能力,提供了精细的补糖速率策略,产酸高峰期明显延长,副产物积累比例得到降低,原料利用率大幅提升。     

重组谷氨酸棒杆菌全细胞催化一步法合成高果糖浆

高果糖浆是一种可替代蔗糖的甜味剂,在食品和饮料行业应用广泛.该研究实现了经密码子优化的密苏里游动放线菌(Actinoplanes missouriensis)CICIM B0118(A)来源的葡萄糖异构酶在食品安全菌株谷氨酸棒杆菌(Corynebacterium glutamicum)13032中的异源表达,构建了重组...     

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...     

利用代谢工程改造谷氨酸棒杆菌产丙酮酸研究

丙酮酸是一种重要的有机酸,可以作为前体物质,参与合成多种有机化合物,在生物体的能量代谢中发挥着重要作用。为提高丙酮酸产量,选择利用代谢工程改造谷氨酸棒杆菌(Corynebacterium glutamicum)生产丙酮酸。利用同源重组的方法,依次敲除谷氨酸棒杆菌中与丙酮酸代谢支流相关的5个关键基因(丙酮酸醌氧化还原酶基因pqo、丙酮酸羧化酶基因pyc、转氨酶基因alaT、缬氨酸-丙酮酸氨基转移酶基因avtA、丙酮酸脱氢酶基因aceE),摇瓶发酵72h后丙酮酸的产量达到14.64g/L。通过过表达编码转酮醇酶基因tkt、转醛酶基因tal、磷酸烯醇丙酮酸羧激酶基因pck,增加合成丙酮酸前体物质的供应。最终,复合培养基摇瓶发酵72h后,发酵液中丙酮酸的产量达到15.39g/L,与野生型菌株相比提高了28倍。研究旨在为利用微生物发酵生产丙酮酸提供一定的理论参考。     

谷氨酸棒杆菌L-色氨酸营养缺陷型突变 及其高产菌株的选育

目的以谷氨酸棒杆菌(Corynebacterium glutamicum)HX-22为出发菌株,研究了目标发酵产物L-色氨酸合成途径交叉反馈抑制途径代谢突变与色氨酸分解代谢类似物抗性的营养缺陷型突变诱导过程及L-色氨酸高产复合突变菌株的筛选。方法采用硫酸二乙酯、紫外线、钴60γ-射线等诱变方法交叉处理起始与突变菌株,通过营养缺陷表型和自杀代谢底物抗性的筛选方法,定向突变和选育L-色氨酸合成与分解代谢突变的色氨酸高产菌株。结果通过多次连续营养缺陷型诱变选育,筛选出一株具有分支酸-Trp/Phe/Tyr代谢途径L-Phe和L-Tyr合成缺陷型和L-Trp分解代谢自杀代谢底物抗性的L-色氨酸高产菌HX22-118(ΔPhe~--ΔTyr~--5FT~r-4FP~r-SG~r);通过连续传代10次发酵,色氨酸产酸最高达到28.4 g/L,平均27.1g/L,较出发菌株HX22产酸水平提高81.8%。结论选育出的高产菌株具有多重代谢途径突变(ΔPhe~--ΔTyr~--5FT~r-4FP~r-SG~r),突变体具有良好的遗传稳定性,具有后续工业化生产应用潜力。     

谷氨酸棒状杆菌合成L-高丝氨酸的代谢改造与发酵条件探究

目的:本研究以谷氨酸棒状杆菌ATCC 13032为底盘细胞,构建1株L-高丝氨酸合成菌株并分析溶氧环境对其产物合成的影响。方法:首先通过外源添加0～40 g/L的L-高丝氨酸分析谷氨酸棒状杆菌的产物耐受性;随后,通过基因thrB敲除阻断L-高丝氨酸的降解途径,获得谷氨酸棒状杆菌重组菌H1;在此基础上利用挡板摇瓶进行细胞培养以增强发酵过程中氧气供给能力。结果:与大肠杆菌相比,谷氨酸棒状杆菌对L-高丝氨酸具有更强耐受性。研究中通过敲除基因thrB构建了L-苏氨酸缺陷型谷氨酸棒状杆菌重组菌H1,发现基础培养基中加入0.5 g/L的L-苏氨酸后,该重组菌生长恢复正常水平。挡板摇瓶条件下重组菌H1的L-高丝氨酸产量增加至836.7 mg/L,较普通摇瓶产量44.6 mg/L提高了17.76倍。结论:通过阻断L-苏氨酸的合成,成功构建L-高丝氨酸合成菌株谷氨酸棒状杆菌H1,并且发现利用挡板摇瓶增强发酵过程中供氧能力是促进谷氨酸棒状杆菌高效合成L-高丝氨酸的有效手段,为后续提高L-高丝氨酸发酵产量提供了参考。     

直接发酵法生产L-丝氨酸高产菌株的选育

为了解除高浓度L-丝氨酸对3-磷酸甘油酸脱氢酶的反馈抑制,进一步提高L-丝氨酸的产量,以一株能直接利用糖质原料发酵生产L-丝氨酸的谷氨酸棒杆菌SYPS-062-36为出发菌,经硫酸二乙酯逐级诱变处理,在D-丝氨酸抗性梯度平板上挑取正突变株,获得一株遗传性能稳定的高产菌株SYPS-062-33a-18,其可积累L-丝氨酸(11.0±0.25)g/L,较出发菌L-丝氨酸产量(6.65±0.23)g/L提高了65.4%。说明本研究不仅以D-丝氨酸作为抗性平板筛选得到高产L-丝氨酸的突变株,而且为下一步进行反向代谢工程的研究提供了良好的实验材料。     

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。     

谷氨酰胺产生菌NS611的代谢流分析

建立并完善了谷氨酸棒杆菌NS611生产谷氨酰胺的中心碳代谢网络.研究了菌体生长和生产阶段的代谢流分布,结果说明该菌葡萄糖的代谢以糖酵解途径为主,在产酸阶段丙酮酸羧化反应的代谢流量较大。NADPH不是谷氨酸棒杆菌NS611代谢的限制性因素.研究了氮饥饿处理对代谢流分布的影响,提出要提高谷氨酰胺的产量必须适当保留α-酮戊二酸的向下氧化的功能,以保证能量供给.     

链霉素抗性选育ε-聚赖氨酸高产菌株

基于核糖体工程的育种方法,通过逐级赋予小白链霉菌M-Z18链霉素抗性,以提高产生菌的ε-聚赖氨酸(ε-PL)合成能力。首先,通过筛选低浓度链霉素(1~5 MIC)抗性突变株,将M-Z18的ε-PL的摇瓶产量从1.6提高到2.43 g/L;随后,再次提升菌株的链霉素耐受性(5~10MIC),进一步增强菌株的ε-PL合成能力;最后,获得一株高产突变菌SS-19,其ε-PL产量和单位菌体合成能力分别为3.13 g/L和0.58 g/g,较出发菌M-Z18分别提高了95.63%和137.61%。研究表明,SS-19的中心碳代谢途径及ε-PL合成相关的关键酶活性有所增加,意味着ε-PL的合成代谢被明显加强。在以葡萄糖为碳源的RSM培养基中,SS-19比前期育种获得的高产菌株表现出更好的ε-PL合成能力。以上结果表明,通过逐步引入高浓度链霉素抗性的筛选方法可有效提升小白链霉菌的ε-PL产量。     

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.     

High-yield production of hydrogen by Enterobacter aerogenes mutants with decreased alpha-acetolactate synthase activity

To enhance hydrogen (H2) production from glucose by Enterobacter aerogenes HU-101, two mutants, strains VP-1 and VP-2, with decreased alpha-acetolactate synthase activity, were isolated using the Voges-Proskauer (VP) test. In pH-uncontrolled batch culture, both mutants showed a lower 2,3-butanediol yield for the glucose consumed than that shown by the wild-type strain, although glucose remained in the medium after 12 h of culture. In the same cultures, compared to the H2 yield of 0.80 mol/mol-glucose of the wild-type strain, strain VP-1 showed a high H2 yield of 1.8 mol/mol-glucose with decreased lactate and increased succinate yields, while strain VP-2 showed an H2 yield of 1.0 mol/mol-glucose with an increased lactate yield. Increasing the phosphate buffer concentration, which contributes to maintaining the pH in the medium, increased the glucose consumption by both strains. However, in a pH-controlled batch culture at neutral pH, the H2 yield of strain VP-1 was decreased to 1.2 mol/mol-glucose due to the accumulation of formate, an intermediate of the H2-producing pathway, with the yield of H2 plus formate being 1.7 mol/mol-glucose.     

基于低拷贝质粒的高产紫色杆菌素谷氨酸棒杆菌的构建和发酵

该研究以公认安全(Generally Recognized as Safe,GRAS)的谷氨酸棒杆菌(Corynebacterium glutamicum)为宿主,构建高产紫色杆菌素的重组菌株。利用谷氨酸棒杆菌天然大质粒pTET3的复制与分配元件,构建了低拷贝质粒pOK12CG1,该质粒在谷氨酸棒杆菌中的拷贝数约为6拷贝/基因组,且与谷氨酸棒杆菌常用质粒pEC-XK99E和pXMJ19兼容。以低拷贝质粒pOK12CG1为骨架构建了携带紫色杆菌素合成操纵子(vioABCDE)的质粒pCGvio,并分别以谷氨酸棒杆菌标准株ATCC 13032和插入序列(Insertion Sequence,IS)元件删除株为宿主,构建了7株合成紫色杆菌素的重组菌株。通过初步筛选,发现基于低拷贝质粒的重组菌株ATCC13032/p CGvio,其紫色杆菌素产量(508.24 mg/L)高于基于中高拷贝质粒的重组菌株ATCC 13032/pECvio(376.16 mg/L),而基于低拷贝质粒的IS元件删除重组菌株ISDM023/pCGvio紫色杆菌素产量达到了610.13mg/L。进一步采用正交实验设计对重...     

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.