产丁二酸谷氨酸棒状杆菌基因缺失代谢工程菌株的构建

【目的】提高谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC13032厌氧条件下的丁二酸产量,并降低发酵产物中副产物的含量。【方法】以谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC13032为出发菌,首先敲除乳酸形成的关键酶乳酸脱氢酶基因(ldh),构建ldh缺失株谷氨酸棒状杆菌ATCC13032Δldh;然后以缺失株谷氨酸棒状杆菌ATCC13032Δldh为出发菌,敲除该菌的丙酮酸脱氢酶系的E1p酶基因(aceE),构建一株双缺失突变菌株谷氨酸棒状杆菌ATCC13032ΔldhΔaceE。【结果】与供试菌比较,谷氨酸棒状杆菌ATCC13032Δldh的丁二酸产量和转化率分别提高了94.9%和32%,并且主要的副产物乳酸产量由出发菌产量的63.5 g/L降低到很微量的程度。丙酮酸脱氢酶的失活并不能完全消除副产物乙酸的形成,但乙酸的产量较ATCC13032Δldh降低了37.9%,丁二酸的产量略有提高。【结论】该重组菌具有较强的丁二酸生产工业化潜力,并且该研究方法为微生物代谢育种提供参考。     

谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成

为阐明谷氨酸棒杆菌的乙醛酸循环与菌体的生长以及谷氨酸合成之间的关系 ,以谷氨酸棒杆菌基因组测序用典型菌株Corynebacteriumglutamicum ATCC 130 32为出发菌株 ,构建了乙醛酸循环途径缺失的谷氨酸棒杆菌突变株Corynebacteriumglutamicum WTΔA。该菌株没有异柠檬酸裂解酶活性 ,不能在以乙酸盐为唯一碳源的基本培养基上生长。与出发菌株ATCC13032相比 ,WTΔA在以葡萄糖为唯一碳源的培养基上生长时不受影响 ,说明谷氨酸棒杆菌并不需要乙醛酸循环途径提供菌体生长所需的能量和生物合成反应所需的中间产物。但是 ,与出发菌株ATCC13032相比 ,WTΔA的谷氨酸合成能力大幅下降。     

谷氨酸棒状杆菌尿素传感器的研究

基于腺酶催化尿素分解产生氨,以氨气敏电极为基础电极,用含脲酶丰富的谷氨酸棒状杆菌研制成测定尿素的微生物传感器.在30℃、pH8.0、0.1mol/L磷酸盐缓冲液中,该传感器的线性范围为1.1×10^-4～1.4×10^-2mol/L,斜率为51.2mV/decade,检测下限为1.0×10^-5mol/L,寿命可达45d.考察了传感器响应初速和底物浓度之间的关系,测定了微生物膜中脲酶的表观米氏常数K_m及最大响应初速v_m.     

谷氨酸棒状杆菌技术研发态势分析

利用美国Thomson公司旗下的Thomson Data Analyzer和微软公司的Excel等分析工具,从时间、地域、专利权人、技术领域等方面,对德温特专利数据库中全球谷氨酸棒状杆菌领域专利的生命周期、区域布局、竞争机构和技术趋势进行了分析。以期为谷氨酸棒状杆菌技术发展态势提供情报支持,辅助企业、高校制定和调整技术发展战略、完善技术发展布局,在当今激烈的市场竞争环境下保持较高的技术优势。     

谷氨酸棒状杆菌异源合成萜类化合物的研究进展

萜类化合物具有可观的商业价值,但生产过程复杂,产量低,利用微生物异源合成萜类化合物已成为热点。谷氨酸棒状杆菌内含合成萜类色素的途径,具有异源合成萜类化合物的天然优势和研究前景。首次对谷氨酸棒状杆菌合成萜类化合物进行了综述,从萜类合成途径、关键酶和全局调控机制三个方面进行了途经介绍。概述了谷氨酸棒状杆菌中单萜、倍半萜、四萜类化合物的异源合成,并对利用谷氨酸棒状杆菌高效合成萜类化合物所需解决的问题进行讨论,为谷氨酸棒状杆菌高效合成萜类化合物提供建议。     

贯叶连翘总提取物对谷氨酸棒状杆菌和粪产碱杆菌的抗菌作用

报道了经光照和未经光照处理后贯叶连翘总提取物对谷氨酸棒状杆菌和粪产碱杆菌的作用,结果表明,总提取物对谷氨酸状杆菌有强烈的抑菌和杀菌作用,对粪产碱杆菌有抑菌作用,其抑菌或杀菌作用与其浓度有关,且不需光照。     

产脂肪酶菌株筛选及柠檬酸杆菌产酶条件优化

脂肪酶可以催化甘油三酯水解成脂肪酸和甘油,已广泛应用在工业领域,而获得产酶微生物是研究的基础。采用油脂平板法筛选出1株脂肪酶产生菌。经16S rRNA序列分析可知,该菌株属于柠檬酸杆菌(Citrobacter werkman and Gillen)。单因素试验对其进行产酶条件优化,优化后产酶条件（g/L）：淀粉2.0,KH₂PO₄ 1.0,K₂HPO₄·3H₂O 2.2,(NH₄)₂SO₄ 1.0,MgSO₄·7H₂O 0.1,牛肉膏2.0,橄榄油10.0 mL,pH 7.5,接种量1.5%(v/v),37 ℃培养43 h。获得最大酶活为384 U/mL,是优化前的13倍。可以利用该菌制备脂肪酶。     

应用纤维素结合域标签构建谷氨酸棒状杆菌表达系统

为优化谷氨酸棒状杆菌表达系统的纯化工艺,合成里氏木霉的CBD基因,将其与谷氨酸棒状杆菌分泌表达载体pXMJ19-sp连接,构建以CBD为纯化标签的重组载体pXMJ 19-sp-CBD.在该载体中插入GFP基因并转化至谷氨酸棒状杆菌,可获得分泌表达融合蛋白GFP-CBD的重组菌.该菌经IPTG诱导后的发酵液在紫外灯下显示强烈的绿色荧光,重组蛋白的分泌表达量达200 mg/L.利用CBD标签对纤维素柱的可逆性吸附,可直接对谷氨酸棒状杆菌分泌到培养基中的重组蛋白进行纯化,从而简化工艺和降低成本,为工业化大生产奠定基础.     

谷氨酸棒杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种具有重要应用价值的四碳平台化合物。微生物法发酵生产琥珀酸以其社会、环境和经济优势展现出良好的发展前景。谷氨酸棒杆菌被广泛应用于氨基酸、核苷酸等高附加值化学品的工业化生产,在厌氧条件下细胞处于生长停滞状态,但仍能高效利用碳源合成有机酸,通过代谢工程改造的谷氨酸棒杆菌有望成为理想的琥珀酸生产菌株。结合近年来谷氨酸棒杆菌生产琥珀酸取得的最新成果,本文综述了构建高产琥珀酸工程菌株的代谢工程策略、底物的扩展利用,并展望了将来的研究方向。     

双组份转运系统BrnFE的过表达和表面活性剂的添加对谷氨酸棒杆菌发酵生产L-异亮氨酸的影响

为了提高L-异亮氨酸生产菌株Corynebacterium glutamicum LD320的产酸水平,通过改善其分泌系统,在C. glutamicum LD320中分别过表达突变型和野生型的双组份转运系统BrnFE操纵子,构建了重组菌LD320/pXMJ19-brnFE和LD320/pXMJ19-brnFE1。通过对两株重组菌的L-异亮氨酸生产分析比较,发现突变型比野生型能更有效地提高 L-异亮氨酸产量。同时对 LD320/pXMJ19-brnFE1进行表面活性剂添加实验,发现Tween-80为最佳选择,其最佳添加量为0.5 g/L,最佳添加时间为对数期的16 h。最后通过7 L发酵罐放大实验,LD320/pXMJ19-brnFE1的L-异亮氨酸产量由18.53 g/L提高到25.45 g/L,比对照组提高了37%。     

Enhanced L-cysteine production by overexpressing potential L-cysteine exporter genes in an L-cysteine-producing recombinant strain of Corynebacterium glutamicum

ABSTRACT

We identified L-cysteine exporter candidates of Corynebacterium glutamicum and investigated the effect of overexpression of the potential L-cysteine exporter genes on L-cysteine production in a recombinant strain of C. glutamicum. Overexpression of NCgl2566 and NCgl0580 resulted in enhanced L-cysteine production in an L-cysteine-producing recombinant strain of C. glutamicum.     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

Abstract Phosphoenolpyruvate (PEP) carboxykinase is present in crude extracts of Corynebacterium glutamicum grown on both glucose and lactate. Preparation of PEP carboxykinase free from interfering PEP carboxylase and oxaloacetate decarboxylase showed an absolute dependence on divalent manganese and IDP for activity in the oxaloacetate (OAA) formation. Other diphosphate nucleotides could not substitute for IDP. The enzyme activity displayed Michaelis-Menten kinetics for the substrates PEP, IDP, KHCO₃, OAA and ITP with a K _m of 0.7 mM, 0.4 mM, 12 mM, 1.0 mM, and 0.5 mM, respectively. At the optimum pH of 6.6, 850 nmol of OAA were formed per min per mg of protein. ATP inhibited PEP carboxykinase in the OAA forming reaction for 60% at 0.1 mM, indicating that the enzyme mainly functions in gluconeogenesis.     

磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响

【目的】L-缬氨酸生物合成的前体物质是丙酮酸。为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1(Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-Δpepc,并研究pepc敲除后菌株生理特性的改变。【方法】运用交叉PCR方法得到pepc基因内部缺失的同源片段Δpepc,并构建敲除质粒pK18mobsacB-Δpepc。利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-Δpepc。采用摇瓶发酵对C.glutamicum V1-Δpepc进行发酵特性的研究。对谷氨酸棒杆菌模式菌株C.glutamicum ATCC 13032、出发菌株C.glutamicum V1和敲除菌株C.glu-tamicum V1-Δpepc的丙酮酸激酶(Pyruvate kinase,PK)、丙酮酸脱氢酶(Pyruvate dehydro-genase,PDH)、丙酮酸羧化酶(Pyruvate carboxylase,PC)分别进行测定和分析。【结果】PCR验证以及PEPC酶活测定都表明筛选到pepc缺陷的突变菌株C.glutamicum V1-Δpepc,摇瓶发酵结果表明,突变菌株C.glutamicum V1-Δpepc不再积累L-缬氨酸而是积累L-精氨酸达到7.48 g/L。酶活测定结果表明出发菌株的PDH和PC酶活均低于模式菌株C.glu-tamicum ATCC13032和重组菌株C.glutamicum V1-Δpepc,出发菌株的PK与PEPC酶活与模式菌株没有较大的差异。【结论】研究表明,通过切断PEPC参与的三羧酸循环的回补途径,增加磷酸烯醇式丙酮酸向丙酮酸的流向使丙酮酸向TCA循环的流量增加,精氨酸的累积量提高。同时,以丙酮酸为前体的L-缬氨酸和丙氨酸的积累量降低。     

利用内源元件在谷氨酸棒杆菌中分泌表达木聚糖酶

以来自于谷氨酸棒杆菌内源AH6启动子和5′UTR及其前38 bp结合合适的Shine-Dalgarno (SD)序列,构建双顺反子表达载体对木聚糖酶进行表达。为了能够实现分泌表达,选取了来自谷氨酸棒杆菌的两种分泌途径的信号肽,分别为Tat型的CgR0949及Sec型的CspB信号肽。在实现分泌表达之后,对其进行5 L发酵罐的扩大培养以提高分泌量。并对纯化的木聚糖酶进行了部分酶学性质的研究,包括最适催化pH及酸碱耐受性;最适催化温度及热稳定性。结果表明:在上述表达体系中,以CgR0949为信号肽木聚糖酶不能分泌到胞外;木聚糖酶能在CspB信号肽的引导下分泌到胞外,分泌表达量为486.2 U/mL。木聚糖酶的分泌量在5 L发酵罐水平上达到1 648.7 U/mL,是摇瓶培养的3.4倍。该木聚糖酶的最适反应pH为4.5,最适温度为45℃;在pH 4–11范围内4℃处理24 h酶活保持在80%以上;在50℃前处理15 min酶活保持在95%以上,超过60℃则酶活迅速下降至20%及其以下。上述结果表明,谷氨酸棒杆菌内源元件能有效用于木聚糖酶的分泌表达,扩大培养能进一步提升木聚糖酶的分泌量。该双顺反子表达体系能为外源蛋白在谷氨酸棒杆菌中的分泌表达提供一种可用的工具。此外,通过酶学性质的研究可进一步提高木聚糖酶的催化效率。     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

谷氨酸棒杆菌metX、dapA基因敲除对苏氨酸合成的影响

谷氨酸棒杆菌中metX基因编码蛋氨酸合成途径关键酶高丝氨酸乙酰转移酶,dapA基因编码赖氨酸合成途径关键酶二氢吡啶二羧酸合成酶。为研究这两个基因缺失对苏氨酸积累的影响,以谷氨酸棒杆菌R102(AHVr)为出发菌株,通过重叠延伸PCR及同源重组技术分别构建了metX、dapA单基因缺失突变株R102ΔmetX、R102ΔdapA以及双基因缺失的突变株R102ΔmetXΔdapA。对出发菌以及上述3株重组菌进行初步摇瓶发酵试验,用HPLC法测定发酵液中苏氨酸含量。结果表明,发酵72 h后,3株重组菌的苏氨酸产量分别为2.58、2.38和3.01 g/L,比原始菌株分别提高了42.5%、31.5%和66.3%。