以葡萄糖为底物一步法合成γ-氨基丁酸整合型重组钝齿棒杆菌的构建

[目的]为了构建一株直接利用廉价的葡萄糖合成γ-氨基丁酸的重组钝齿棒杆菌,将来自于植物乳杆菌γ-氨基丁酸合成途径的关键酶谷氨酸脱羧酶基因(lpgad)在产谷氨酸菌株钝齿棒杆菌中进行整合表达,实现葡萄糖到GABA的一步法生产.[方法]运用PCR技术扩增得到带有tac启动子的谷氨酸脱羧酶基因tacgad.通过重叠PCR的方法获得钝齿棒杆菌精氨酸合成途径关键酶N-乙酰谷氨酸激酶(NAGK)基因内部缺失型基因△argB.利用自杀载体pK18mobsacB构建同源整合载体pK18-△argB::tacgad,以△argB的上下游序列为同源臂,通过两次同源重组将tacgad基因整合到钝齿棒杆菌基因组,同时将NAGK基因argB灭活,利用蔗糖致死基因sacB反向筛选标记筛选得到谷氨酸脱羧酶的重组钝齿棒杆菌C.crenatum △argB::tacgad.重组钝齿棒杆菌以葡萄糖为底物进行发酵,测定GABA含量.[结果]重组菌C.crenatum △argB::tacgad成功表达谷氨酸脱羧酶,同时阻断了精氨酸合成途径对谷氨酸到GABA代谢途径的竞争,粗酶液基本检测不到NAGK活性,发酵液无精氨酸合成.通过96 h发酵,重组菌可积累约8.28 g/L的GABA.[结论]本研究通过将谷氨酸脱羧酶基因定向整合到钝齿棒杆菌精氨酸合成途径的关键酶基因argB内部,成功表达谷氨酸脱羧酶的同时阻断竞争途径精氨酸的合成.本研究为实现直接利用葡萄糖合成GABA的一步法生产奠定了基础.     

L-鸟氨酸高产菌的原生质体融合育种

以谷氨酸高产菌S9114和谷氨酸棒杆菌GS538为出发菌株,应用原生质体融合技术,定向选育出一株L-鸟氨酸高产菌株RH169,该菌株能在发酵液中积累L-鸟氨酸,质量浓度可达19.3 g.L-1。     

重组大肠杆菌高效催化合成γ-氨基丁酸

[目的]实现重组大肠杆菌高效合成γ-氨基丁酸(γ-aminobutyric acid,GABA)。[方法]构建表达谷氨酸脱羧酶的基因工程菌Escherichia coli p ET-GAD,对催化工艺进行初步优化,实现高效催化L-谷氨酸脱羧反应合成GABA。[结果]在谷氨酸脱羧酶的表达过程中,维生素B6盐酸吡哆醇(PN)可以替代5-磷酸吡哆醛(PLP)作为辅酶补给,提高工程菌E. coli p ET-GAD的催化活力。在50 m L反应体系中,重组细胞浓度为8 mg/m L,底物浓度为200 mmol/L,在35℃、p H 4. 4条件下反应2 h,L-谷氨酸的转化率 98%。为了提高GABA的生产效率,采用谷氨酸/谷氨酸钠分批补料方式控制反应过程中的p H值,GABA的最终浓度达到247 g/L。[结论]重组大肠杆菌可以高效催化合成γ-氨基丁酸,为基因工程菌工业化制备GABA提供实验依据。     

γ-氨基丁酸代谢影响拟南芥叶片节律性运动

为阐明γ-氨基丁酸(γ-aminobutyric acid,GABA)和生物节律调控的关系,本文以GABA合成(谷氨酸脱羧酶gad等)和代谢(GABA转氨酶GABA-T/pop2、谷氨酸脱氢酶gdh等)突变体,以拟南芥叶片节律性运动为监测指标,探讨了GABA代谢与叶片节律性运动的关系。结果显示,GABA合成突变体(gad1-3)和双突变体gad1/gad2中叶片节律性运动的振幅低于野生型,在gad1、gad2和gad1/gad2中叶片运动的振幅变化明显;pop2突变体中,叶片运动的节律性变化的振幅明显低于对照,gdh1/gdh2突变体中,叶片运动呈现非节律性变化的特点;此外,外源γ-氨基丁酸(1.0 mmol·L-1)不同程度提高拟南芥3种生态型(Col、Ler、Ws)叶片节律性运动的振幅,其中Col生态型和Ws生态型叶片振幅变化明显;在生物钟核心基因突变体toc1、lhy、cca1中,叶片运动呈现非节律性变化模式;但外源GABA的添加能够提高这些突变体叶片节律性运动的振幅或者恢复叶片的节律性。上述结果表明GABA代谢平衡直接和间接影响生物钟节律,外源或内源GABA的合成或代谢突变主要影响到叶片节律性运动的振幅。     

混菌发酵生产γ-聚谷氨酸的工艺条件优化

采用谷氨酸棒杆菌S9114和枯草芽胞杆菌NTG-4在10 L自控发酵罐上进行混菌发酵,探索混菌发酵生产γ-聚谷氨酸的可行性并进行工艺优化。结果表明:温度、接种量、pH及溶氧对聚谷氨酸发酵有较大影响,发酵前期维持32℃,6 h提温至37℃变温控制,谷氨酸棒杆菌和枯草芽胞杆菌接种量分别为5%和0.5%,pH 7.0,溶氧20%最有利于γ-聚谷氨酸发酵,在此条件下发酵32 hγ-聚谷氨酸最高产量为38.3 g/L。     

重组谷氨酸脱羧酶制备γ-氨基丁酸的工艺条件优化

谷氨酸脱羧酶,一种磷酸吡哆醛(PLP)依赖性酶,能专一、不可逆地催化L-谷氨酸脱羧得到γ-氨基丁酸(GABA)。构建了产Lactobacillus brevis WJH3谷氨酸脱羧酶重组大肠杆菌E.coli BL21(DE3)/p ET-24a-gad,以此作为菌种进行摇瓶发酵诱导培养,发酵过程中一次性添加0.05 mmol/L PLP培养24 h,破壁上清酶活达81.7 U/m L,是不添加PLP对照酶活的1.8倍。对酶转化L-谷氨酸钠生成GABA反应条件进行了优化,结果表明,在转化体系不添加PLP的情况下,底物谷氨酸钠浓度为250 g/L,反应初始p H5.0,温度37℃,加酶量60 U/g底物,转速200 r/min,在此条件下反应18 h,GABA转化率达到100%,为γ-氨基丁酸的工业化生产奠定基础。     

谷氨酰胺高产菌株的定向选育研究

采用谷氨酸棒杆菌S9114为出发菌株,经γ-射线—硫酸二乙酯—γ-射线诱变,磺胺胍抗性筛选后,定向选育出1株高产菌株SH77。在适宜的条件下积累谷氨酰胺平均为38.9g/L,最大达39.3g/L,比出发林提高了3.81倍。该菌株在最优化代谢控制发酵工艺条件下,谷氨酰胺产量最高达56.2g/L。     

谷氨酸棒杆菌ilvE基因的敲除对相关氨基酸合成的影响

L-缬氨酸是谷氨酸棒杆菌SYPS-062发酵生产L-丝氨酸的主要副产物.为减少L-缬氨酸的积累,利用基因重组技术敲除SYPS-062转氨酶B编码基因ilvE内部的987 bp核苷酸序列,构建了ilvE基因缺失突变株SYPS-062△ilvE.研究表明,重组菌ilvE基因的缺失直接导致了分支氨基酸(Val、Ile、Leu)的合成能力的降低,影响了菌体的生长,其中Ile成为生长限制性因子,在培养基中添加分支氨基酸能明显促进其生长.重组菌培养96 h,发酵液中L-缬氨酸含量低于0.5 g/L,与出发菌株相比,其生成率降低90％.     

高产γ-氨基丁酸的谷氨酸棒杆菌工程菌的构建和发酵条件优化

为了实现γ-氨基丁酸(GABA)在微生物中"一步法"高效生产,本研究构建出一株高产GABA的谷氨酸棒杆茵工程菌ATCC 13032/pDXW10-gadB1-gadB2,可以直接将自身合成的L-谷氨酸转变成GABA,并对该工程茵的发酵培养基和发酵条件进行了初步优化。结果表明:培养7 h~8 h的种子液按发酵起始OD_(562)=1.6转接至发酵培养基(葡萄糖、玉米浆分别100 g/L、4 g/L),10 h添加PLP至O.1 mmol/L,发酵结束后胞外GABA可达(26.39±1.68)g/L。为工业化"一步法"生产GABA提供理论和实验基础。     

酸菜中高产γ-氨基丁酸乳酸菌的筛选和鉴定

从酸菜中分离筛选出产γ-氨基丁酸(GABA)乳酸菌18株,采用改良纸层析结合高效液相色谱法测定,获得1株高产乳酸菌菌株Lb-2,该Lb-2菌株在含10g/L谷氨酸钠的TYG培养基中静置培养48h,发酵液中GABA含量可达13.4g/L。根据乳酸菌的形态特征、生理生化特征和16S rDNA基因序列分析鉴定Lb-2菌株为乳杆菌属(Lactobacillus)的短乳杆菌(Lactobacillus brevis)。     

Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30°C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1mM PLP, fermentation was performed in a flask at 30°C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source.     

Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis

γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1–gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of l-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L^?1, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L^?1 after 84-h cultivation. Under optimal urea supplementation, l-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L^?1 after 120-h flask cultivation and 26.32 g L^?1 after 60-h fed-batch fermentation. The conversion ratio of l-glutamate to GABA reached 0.60–0.74 mol mol^?1. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated l-glutamate.     

Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

γ-aminobutyric acid (GABA) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic, and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented dairy foods including cheeses and yogurt. The survey of 42 strains of the yogurt starter culture Streptococcus thermophilus by PCR techniques indicated the presence of a glutamate decarboxylase gene (gadB) in 16 strains. DNA sequencing data indicated that the GAD/GABA antiporter locus (gadB/gadC) in GAD(+) S. thermophilus strains is flanked by transposase elements (5' and 3') and positioned between the luxS (5') and the HD-superfamily hydrolase genes (3'). The PCR amplification product of a ca. 2-kb genomic fragment that included the gadB and its putative promoter region was inserted into a shuttle vector, which was used to transform Escherichia coli DH5α. Subsequently, the recombinant plasmid pMEU5a-1/gadB (7.24 kb) was electrotransformed into the GAD-negative strain S. thermophilus ST128. The ST128 transformants carrying the plasmid-encoded gadB produced functional GAD enzyme as evidenced by the conversion of glutamate to GABA at a rate similar to strains with the gadB/gadC operon located on the chromosome. The results demonstrated the potential to impart to non-GABA-producing strains of S. thermophilus and other lactic acid bacteria the GAD(+) phenotype that improves their appeal in possible applications in the development of health-promoting functional foods.     

代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸

谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT (丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶 (ilvBN) 的调节亚基进行定点突变 (ilvBN1M13),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN1M13、乙酰羟酸异构酶 (ilvC)、二羟酸脱水酶 (ilvD)、支链氨基酸氨基转移酶 (ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。     

Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum

Corynebacterium glutamicum is widely used for industrial production of various amino acids and vitamins, and there is growing interest in engineering this bacterium for more commercial bioproducts such as γ-aminobutyric acid (GABA). In this study, a C. glutamicum GABA-specific transporter (GabP(Cg)) encoded by ncgl0464 was identified and characterized. GabP(Cg) plays a major role in GABA uptake and is essential to C. glutamicum growing on GABA. GABA uptake by GabP(Cg) was weakly competed by l-Asn and l-Gln and stimulated by sodium ion (Na(+)). The K(m) and V(max) values were determined to be 41.1 ± 4.5 μM and 36.8 ± 2.6 nmol min(-1) (mg dry weight [DW])(-1), respectively, at pH 6.5 and 34.2 ± 1.1 μM and 67.3 ± 1.0 nmol min(-1) (mg DW)(-1), respectively, at pH 7.5. GabP(Cg) has 29% amino acid sequence identity to a previously and functionally identified aromatic amino acid transporter (TyrP) of Escherichia coli but low identities to the currently known GABA transporters (17% and 15% to E. coli GabP and Bacillus subtilis GabP, respectively). The mutant RES167 Δncgl0464/pGXKZ9 with the GabP(Cg) deletion showed 12.5% higher productivity of GABA than RES167/pGXKZ9. It is concluded that GabP(Cg) represents a new type of GABA transporter and is potentially important for engineering GABA-producing C. glutamicum strains.     

Overexpression of <Emphasis Type="Italic">ppc</Emphasis> or deletion of <Emphasis Type="Italic">mdh</Emphasis> for improving production of γ-aminobutyric acid in recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced l-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, l-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3?±?3.4, 24.8?±?0.7, and 25.5?±?3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7?±?0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0?±?0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.     

谷氨酸棒杆菌SYPS-062 L-丝氨酸脱水酶活性分析及其基因敲除对L-丝氨酸积累的影响

以谷氨酸棒杆菌(Corynebacterium glutamicum) SYPS-062基因组DNA为模板,扩增得到L-丝氨酸脱水酶(L-SerDH)的编码基因sdaA。将其克隆到表达载体pET-28a(+),并在E.coli BL21(DE3)中诱导表达,对纯化的L-SerDH进行了酶活测定,并与来自C.glutamicum ATCC13032的重组L-SerDH进行了比较,结果显示,两种不同菌株来源的重组L-SerDH降解L-丝氨酸的酶比活力差异并不显著。在此基础上敲除菌株SYPS-062 的sdaA基因,探讨该基因对C.glutamicum SYPS-062生长及产酸的影响。通过构建自杀型重组质粒pK18mobsacB-△sdaA,电击转入C.glutamicum SYPS-062中,以同源重组的方式获得了sdaA基因缺失突变株,并用PCR方法对突变株C.glutamicum SYPS-062△sdaA进行了验证。与出发菌株相比,突变菌株生长缓慢,单位菌体L-丝氨酸的产量(Y_P/X)提高了15.13%。     

耐温谷氨酸棒杆菌的选育及其高温谷氨酸发酵代谢流量分析

采用基因组改组的方法选育获得的一株耐温谷氨酸棒杆菌F343,并比较了F343与其出发菌株S9114在39℃发酵谷氨酸时的发酵特性和代谢流量。结果表明:耐温菌F343的比生长速率、比谷氨酸积累速率可维持在较高的水平;通过发酵中后期代谢流量分析发现耐温菌F343在磷酸烯醇式丙酮酸(PEP)节点处,磷酸烯醇式丙酮酸羧化酶(PEPc)催化的CO_2回补支路反应代谢流增加;α-酮戊二酸(KG)节点处,谷氨酸氢酶(GDH)催化的产生谷氨酸的支路代谢通量增加。此外,高温发酵谷氨酸时,耐温菌F343高温发酵谷氨酸过程产生的乳酸等副产物较出发菌株S9114少。通过改善种子质量,F343在高温发酵30 h产酸达到10.1%,较出发菌株提高67%。     

L-组氨酸高产菌株的选育

为得到L-组氨酸的高产菌株,以谷氨酸棒杆菌（Corynebacterium glutamicum）S9114为出发菌株,利用亚硝基胍（NTG）和硫酸二乙酯（DES）进行多次诱变,在D-组氨酸的抗性梯度平板上挑取正突变株,发酵检测,最终挑出一株S6（D—his＇）,可积累L-组氨酸327mg／L,比出发菌株提高47.3％。