敲除aceE基因对猪链球菌丙酮酸代谢的影响

【目的】探究缺失编码丙酮酸脱氢酶蛋白的aceE基因对猪链球菌生长特性、三羧酸循环和丙酮酸代谢的影响。【方法】通过测量菌液的OD600值,绘制野生型菌株与aceE基因缺失突变株的生长曲线;利用试剂盒测定三羧酸循环和丙酮酸代谢旁路中乙酰CoA、琥珀酸CoA、延胡索酸、草酰乙酸、丙酮酸、乳酸和ATP的含量,通过荧光定量qRT-PCR确定柠檬酸合酶基因、苹果酸脱氢酶基因、琥珀酸脱氢酶基因、异柠檬酸脱氢酶基因、丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因的表达水平。【结果】与野生株相比,菌株ΔaceE在平台期OD600值下降;添加1g/L乙酸盐能够显著提升菌株ΔaceE平台期OD600值。菌株ΔaceE的丙酮酸含量上升,ATP含量下降;三羧酸循环代谢中乙酰CoA、琥珀酸CoA、延胡索酸含量降低;柠檬酸合酶基因和苹果酸脱氢酶基因表达水平上升,琥珀酸脱氢酶基因和异柠檬酸脱氢酶基因表达水平下调;在丙酮酸代谢旁路中丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因表达水平上升。【结论】结果显示,菌株ΔaceE三羧酸循环活性降低,虽然能够通过PDH旁路将部分丙酮酸分解为乙...     

过量表达异柠檬酸裂解酶对ldh-1谷氨酸棒状杆菌产丁二酸的影响

谷氨酸棒状杆菌SA001是缺失了乳酸脱氢酶基因 (ldhA) 的菌株。为了增加厌氧条件下经异柠檬酸到丁二酸的代谢通量,以提高丁二酸的产量。将来自大肠杆菌Escherichia coli K12的异柠檬酸裂解酶基因导入谷氨酸棒状杆菌SA001 (SA001/pXMJ19-aceA) 中。该菌经0.8 mmol/L的IPTG有氧诱导12 h后,转入厌氧发酵16 h,丁二酸的产量为10.38 g/L,丁二酸的生产强度为0.83 g/(L·h)。与出发菌株比较,异柠檬酸裂解酶的酶活提高了5.8倍,丁二酸的产量提高了48%。结果表明过量表达异柠檬酸裂解酶可以增加由乙醛酸途径流向丁二酸的代谢流。     

大肠杆菌木糖生物合成琥珀酸的代谢工程研究

目的:对大肠杆菌进行代谢网络改造,考察木糖好氧发酵生产琥珀酸的可行性。方法:以有氧条件下大肠杆菌木糖生物合成琥珀酸的代谢途径分析为基础,以大肠杆菌BL21为出发菌株,通过P1噬菌体一步敲除法敲除琥珀酸脱氢酶基因(sdhA)、磷酸转乙酰基酶基因(pta)、丙酮酸脱氢酶基因(poxB)及异柠檬酸裂解酶阻遏物基因(iclR),构建木糖好氧发酵生产琥珀酸的大肠杆菌工程菌JLS400(△poxB△pta△iclR△sdhA)。将携带磷酸烯醇式丙酮酸羧化酶基因的质粒pJW225转化到JLS400中。结果:摇瓶发酵结果表明,构建的工程菌能以木糖为碳源,在好氧发酵条件下琥珀酸产率较高,副产物仅有少量乙酸和丙酮酸。结论:基因工程大肠杆菌JLS400pJW225的构建,为有氧条件下以木糖为原料生产琥珀酸的进一步研究奠定了基础。     

光滑球拟酵母中AMP代谢对其生理功能的影响北大核心CSCD

【目的】研究光滑球拟酵母(Candida glabrata)中AMP代谢影响其碳流代谢和耐酸胁迫的生理机制。【方法】同源重组法敲除基因cgade12(ORF CAGL0K05027g)和cgade13(ORF CAGL0B02794g)构建双缺菌株cgade12Δade13Δ,与出发菌株(ATCC55)比较ATP水平、碳流代谢酶活性和中间代谢物含量变化分析和AMP代谢对其碳流代谢的影响,对比菌株有机酸胁迫下生长情况及胞内环境变化,研究AMP代谢变化对光滑球拟酵母酸胁迫耐受性的影响。【结果】与出发菌株ATCC55相比,cgade12Δade13Δ的ATP水平下降了12.50%。与ATCC55相比,cgade12Δade13Δ中柠檬酸合成酶、苹果酸脱氢酶、异柠檬酸脱氢酶、α-酮戊二酸脱氢酶的活性分别上升了31.26%、19.45%、28.96%、18.36%,柠檬酸、α-酮戊二酸、苹果酸、琥珀酸含量分别提高了44.11%、73.60%、50.00%、65.68%。胞内丙酮酸浓度下降20.00%,丙酮酸产量下降73.11%。与ATCC55相比,cgade12Δade13Δ在0.4%丙酮酸、0.6%苹果酸和0.2%乙酸胁迫下的菌体浓度分别提高了8.71%、11.21%和12.71%。在0.2%乙酸胁迫下,菌株cgade12Δade13Δ的H^+-ATPase活性、细胞膜完整度、细胞膜电势分别比出发菌株ATCC55上升了7.04%、8.71%、25.14%,ROS水平下降了19.51%。【结论】基因cgade12、cgade13的缺失导致菌株的ATP水平下降,TCA循环活性和有机酸耐受性上升。     

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。     

嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum-Δldh缺氧条件下代谢葡萄糖途径的变化

缺氧条件下嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum ATCC13870生长停滞,却能够代谢葡萄糖产生以乳酸和琥珀酸为主的有机酸。采用以sacB基因为反向筛选标记的同源重组染色体基因敲除系统,敲除嗜乙酰乙酸棒杆菌的乳酸脱氢酶基因,得到的Δldh菌株CCTCC NO.M20122041在缺氧条件下不产乳酸,葡萄糖消耗速率降低了29.3%,产琥珀酸和乙酸浓度分别提高45.6%和182%;NADH/NAD+值小于1(约0.7);磷酸烯醇式丙酮酸羧化酶和乙酸激酶的比酶活分别提高84%和12倍。说明嗜乙酰乙酸棒杆菌中乳酸合成途径的阻断驱使了琥珀酸和乙酸代谢途径加强,推测加强NADH供给和阻断乙酸产生支路可能是提高C.acetoacidophilum菌株产琥珀酸产量的有效途径。     

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%。     

枯草芽孢杆菌聚谷氨酸合成途径相关基因功能研究

聚谷氨酸(polyglutamic acid, PGA)作为一种天然多功能的聚合物,近年来成为研究的热点。由于很难通过化学方法合成,微生物发酵是目前生产聚谷氨酸的有效途径。【目的】从基因水平探究枯草芽孢杆菌聚谷氨酸合成途径中degS、degQ、degU、swrA、rocA、putM基因的功能,通过分子改造实现对代谢途径的调控。【方法】以枯草芽孢杆菌为出发菌株,通过对代谢途径中相关基因进行敲除或过表达,分别构建degS、degQ和degU基因缺失的重组菌,swrA、rocA和putM基因过表达的重组菌,借助菌株胞外聚谷氨酸积累的变化分析影响途径的关键节点。【结果】在摇瓶发酵条件下,重组菌Bacillus subtilis 168-swrA、Bacillus subtilis 168-rocA、Bacillus subtilis 168-putM的胞外聚谷氨酸含量分别是原始菌株的1.28倍、1.47倍和1.37倍。重组菌Bacillus subtilis 168-ΔdegS、Bacillus subtilis 168-ΔdegQ、Bacillus subtilis 168-ΔdegU的胞外...     

单核细胞增生李斯特菌LadR蛋白对外排泵MdrL的调控机制研究

研究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)调控子LadR对外排泵MdrL的调控作用及方式。采用同源重组技术构建ladR基因缺失菌株ΔladR;通过实时荧光定量PCR检测mdrL基因在野生株EGD-e和突变株ΔladR中的转录水平;构建重组蛋白表达菌株BL21(DE3)-ladR并纯化重组蛋白His6-LadR;通过凝胶阻滞试验检测LadR蛋白与mdrL基因启动子区域的结合活性。成功构建ladR基因缺失菌株ΔladR。与野生株EGD-e相比,突变株ΔladR中mdrL基因的转录水平提高约51倍;成功构建重组蛋白表达菌株BL21(DE3)-ladR,纯化得到浓度为1 mg/mL的重组蛋白His6-LadR;凝胶阻滞实验结果显示,重组蛋白His6-LadR可与mdrL基因的启动子区域特异性结合。Lm的调控子LadR通过与mdrL基因启动子区域特异性结合实现对外排泵MdrL的负调控。     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Effects of excess succinate and retrograde control of metabolite accumulation in yeast tricarboxylic cycle mutants

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2Δ or fum1Δ) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ~8-fold in the sdh2Δ mutant and ~4-fold in the fum1Δ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2Δ and fum1Δ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Co-disruption of the SFC1 gene encoding the mitochondrial succinate/fumarate transporter resulted in higher relative mitochondrial levels of succinate and in substantial reductions of Cit2 expression in sdh2Δsfc1Δ and fum1Δsfc1Δ strains as compared with sdh2Δ and fum1Δ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2Δ and fum1Δ strains. A defect (rtg1Δ) in the yeast retrograde response pathway, which controls expression of several mitochondrial proteins and Cit2, eliminated expression of Cit2 and reduced expression of NAD-specific isocitrate dehydrogenase (Idh) and aconitase (Aco1) in parental, sdh2Δ, and fum1Δ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2Δ and fum1Δ strains, of fumarate in the fum1Δ strain, and citrate in an idhΔ strain. Thus, the retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in the parental strain and for metabolite accumulation in TCA cycle mutants.     

代谢木糖重组谷氨酸棒杆菌的构建

为了使谷氨酸棒杆菌较好地利用木糖生产有机酸,将来自Escherichia coli K-12的木糖异构酶基因xylA构建到表达载体pXMJ19中,导入Corynebacterium glutamicum ATCC13032Δldh中,成功表达了该酶基因。结果表明:重组菌株在以木糖为唯一C源进行发酵时,木糖的消耗速率为0.54 g/(L·h),木糖异构酶比酶活约为0.54 U/mL;在以木糖和葡萄糖的混合糖为C源进行发酵时,菌株优先利用葡萄糖,在葡萄糖完全消耗后,菌株开始有效利用木糖;以木糖为唯一C源进行两阶段发酵时,琥珀酸的收率可达(0.62±0.003)g/g。     

Pentose metabolism in Zymomonas mobilis wild-type and recombinant strains

The enzyme activities of the pentose phosphate pathway in the ethanologenic, Gram-negative bacterium Zymomonas mobilis were studied in order to construct a xylose catabolic pathway. In cell-free extracts of wild-type Z. mobilis CP4, activities of the enzymes transketolase (TKT) [2 munits (U)/mg], phosphoribose epimerase (640 mU/mg), phosphoribose isomerase (1600 mU/mg) and 6-phosphogluconate dehydrogenase (2 mU/mg) were determined. However, no transaldolase activity could be detected. Recombinant strains of Z. mobilis were constructed that carried the xylAB genes of the xylose catabolic pathway from Klebsiella pneumoniae. Expression of xylose isomerase (XI, 150 mU/mg) and xylulokinase (XK) (1300 mU/mg) were found in recombinant strains but no growth on pentose as sole carbon source occurred. The xyl-recombinant cells were moreover growth-inhibited in the presence of xylose and were found to accumulate xylitol phosphate due to the subsequent action of a novel enzyme, an NADPH-dependent aldose reductase, and a side reaction of XK on xylitol. From the xylAB recombinant strains, mutants were isolated that were less inhibited and formed less xylitol phosphate when grown in the presence of xylose. The tkt gene of E. coli was cloned on the xylAB plasmid and introduced into Z. mobilis strains. This led to higher TKT activities (150 mU/mg) and, in cooperation with the enzymes XI and XK, mediated a conversion of small amounts of xylose to CO₂ and ethanol. However, no growth on xylose as sole carbon source was detected, instead sedoheptulose 7-P accumulated intracellularly. Correspondence to: G. Sprenger     

产碱性蛋白酶嗜碱芽孢杆菌的筛选及其研究

利用造纸黑液对土样进行富集,筛选出3株碱性蛋白酶酶活力较高的嗜碱芽孢杆菌X1、X2、X3。对它们的生长曲线,产酶曲线,在不同C、N源、pH值、盐浓度下的产酶活力进行的研究表明:3株嗜碱芽孢杆菌(Bacillussp.JBX1、X2、X5)酶活力较高(达到100U/mL),X2最高酶活可达140U/mL。最适pH值为9.5,碳源中的蔗糖,氮源中的酵母浸提物和硝酸钠均利于产酶。X1、X5两株嗜碱芽孢杆菌均表现出较强的耐盐耐高渗透压的能力。X1在11%的NaCl浓度下生长良好,酶活仍然达到80U/mL以上。而X2和X5对温度的耐受性比较强,在经70℃处理15min后依然保持了80%以上的酶活力,所产蛋白酶为高温碱性蛋白酶,从而为进一步的应用和研究奠定了基础。     

片烟产果胶酶细菌的鉴定及酶活测定

以果胶为碳源, 对津巴布韦片烟烟叶表面产果胶酶细菌进行分离, 采用16S rDNA限制性酶切片段长度多态性分析(ARDRA)和测序方法, 结合形态学、生理生化实验, 对所分离产果胶酶菌株进行鉴定, 同时研究培养时间、温度、起始pH、接种量对菌株产酶的影响。结果表明, 从津巴布韦片烟烟叶表面分离得到的产果胶酶菌株主要为芽孢杆菌属的枯草芽孢杆菌Bacillus subtilis和产碱菌属的粪产碱菌Alcaligenes faecalis。在所分离的菌株中, 枯草芽孢杆菌T10酶活力最高, 以6%的接种量, 在温度为35 °C、起始pH为7.5条件下培养48?56 h, 其果胶酶酶活为571 U/mg, 聚半乳糖醛酸裂解酶酶活为297 U/mg。     

Peroxisomal fatty acid beta-oxidation is not essential for virulence of Candida albicans

Phagocytic cells form the first line of defense against infections by the human fungal pathogen Candida albicans. Recent in vitro gene expression data suggest that upon phagocytosis by macrophages, C. albicans reprograms its metabolism to convert fatty acids into glucose by inducing the enzymes of the glyoxylate cycle and fatty acid beta-oxidation pathway. Here, we asked whether fatty acid beta-oxidation, a metabolic pathway localized to peroxisomes, is essential for fungal virulence by constructing two C. albicans double deletion strains: a pex5Delta/pex5Delta mutant, which is disturbed in the import of most peroxisomal enzymes, and a fox2Delta/fox2Delta mutant, which lacks the second enzyme of the beta-oxidation pathway. Both mutant strains had strongly reduced beta-oxidation activity and, accordingly, were unable to grow on media with fatty acids as a sole carbon source. Surprisingly, only the fox2Delta/fox2Delta mutant, and not the pex5Delta/pex5Delta mutant, displayed strong growth defects on nonfermentable carbon sources other than fatty acids (e.g., acetate, ethanol, or lactate) and showed attenuated virulence in a mouse model for systemic candidiasis. The degree of virulence attenuation of the fox2Delta/fox2Delta mutant was comparable to that of the icl1Delta/icl1Delta mutant, which lacks a functional glyoxylate cycle and also fails to grow on nonfermentable carbon sources. Together, our data suggest that peroxisomal fatty acid beta-oxidation is not essential for virulence of C. albicans, implying that the attenuated virulence of the fox2Delta/fox2Delta mutant is largely due to a dysfunctional glyoxylate cycle.     

转醛醇酶基因差异表达影响酵母发酵木糖及耐受乙酸性能

【目的】木糖发酵是纤维素燃料乙醇生产的一个关键瓶颈,同时木质纤维素水解液中的乙酸严重抑制酿酒酵母的木糖发酵过程,因此通过基因工程手段提高菌株对木糖的利用以及对乙酸的耐受性具有重要意义。本研究以非氧化磷酸戊糖途径(PPP途径)中关键基因转醛醇酶基因(TAL1)为研究对象,探讨了3种不同启动子PTDH3、PAHP1和PUBI4,控制其表达对菌株利用木糖和耐受乙酸的影响。【方法】通过同源重组用3种启动子替换酿酒酵母基因工程菌NAPX37的TAL1基因的启动子PTAL1,再通过孢子分离和单倍体交配构建了纯合子,利用批次发酵比较了在以木糖为唯一碳源和混合糖(葡萄糖和木糖)为碳源条件下,3种启动子控制TAL1基因表达导致的发酵和乙酸耐受能力的差异。【结果】启动子PTDH3、PAHP1和PUBI4在不同程度上提高了TAL1基因的转录水平,提高了菌株对木糖的利用速率及乙酸耐受能力,提高了菌株在60 mmol/L乙酸条件下的葡萄糖利用速率。在以木糖为唯一碳源且无乙酸存在、以及混合糖为碳源的条件下,PAHP1启动子控制TAL1表达菌株的发酵结果优于PTDH3和PUBI4启动子的菌株,PAHP1启动子控制的TAL1基因的转录水平比较合适。在木糖为唯一碳源且乙酸为30 mmol/L时,PUBI4启动子控制TAL1基因表达的菌株发酵结果则优于PAHP1和PTDH3启动子菌株,此时PUBI4启动子控制的TAL1的转录水平比较合适。【结论】启动子PTDH3、PAHP1和PUBI4不同程度地提高TAL1基因的表达,在不同程度上改善了酵母菌株的木糖发酵速率和耐受乙酸性能,改善程度受发酵条件的影响。     

Isolation and characterization of yeast mutants defective in intermediary carbon metabolism and in carbon catabolite derepression

Summary Yeast mutants deficient in the constitutive ADHI (adc1) were used for the isolation of mutants with deficiencies of the intermediary carbon metabolism, and of mutants defective in carbon catabolite derepression. Mutants were recognized by their inability to grow on YEP-glycerol and/or on ethanol synthetic complete medium. They were either defective in isocitrate lyase (icl1), succinate dehydrogenase (sdh1), or malate dehydrogenase (mdh1, mdh2), mdh-mutants could not uniformely be appointed to one of the known MDH isozymes. Homozygous mdh and sdh1 diploids are unable to sporulate.Three gene loci could be identified by mutants pleiotropically defective in many or all of the enzymes tested. In ccr1 mutants, derepression of isocitrate lyase, fructose-1,6-diphosphatase, ADHII and possibly of the cytoplasmic MDH is prevented, whereas the mitochondrial TCA-cycle enzymes, succinate dehydrogenase and malate dehydrogenase, are not significantly affected. CCR2 and CCR3 have quite similar action spectra. Both genes are obviously necessary for derepression of all enzymes tested. It could be shown that ccr1, ccr2 and ccr3 mutants are not respiratory deficient.