表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸（5-aminolevulinate,ALA）由5-氨基乙酰丙酸合酶（5-aminolevulinate synthase,ALAS）催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH 6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg /L培养基。纯化的ALA纯度达到90%。     

表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸(5-aminolevulinate,ALA)由5-氨基乙酰丙酸合酶(5-aminolevulinate synthase,ALAS)催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg/L培养基。纯化的ALA纯度达到90%。     

重组Rhodobacter sphaeroides hemA表达的调控

RhodobactersphaeroideshemA编码5氨基乙酰丙酸合酶(ALAS),催化磷酸吡哆醛依赖性琥珀酰CoA和甘氨酸缩合成ALA.将R.spaeroideshemA导入E.coli进行表达,当hemA具有与lac启动子相同的转录方向时,ALAS有活性.lac启动子与hemA之间的距离会影响ALAS在不同培养基上的表达.E.coli宿主菌对ALAS表达、ALA产量有显著影响,在实验所用6种菌株中,E.coliDH1是最佳宿主菌(P<0.05).ALAS表达还与碳源有关,琥珀酸为碳源时,重组ALAS活性最高(P<0.05),以乳酸为碳源时,ALAS活性很低.重组ALAS活性也受培养基pH值影响,pH6.5时,活性最高(P<0.05).     

光合细菌嗜酸柏拉红菌5-氨基乙酰丙酸合成酶基因的克隆与原核表达

5-氨基乙酰丙酸(ALA)可作为除草剂、杀虫剂和植物生长调节剂在农业上应用,但由于其成本较高而限制了它的大面积使用。利用常规基因工程操作方法结合载体介导PCR法(Vecterette PCR)克隆了嗜酸柏拉红菌(Rhodoblastus acidophilus)的5-氨基乙酰丙酸合成酶(ALAS)基因。并将编码ALAS的基因插入到原核表达载体pQE30中,在大肠杆菌不同菌株(E.coli JM109、M15及BL21(DE3))中进行诱导表达。对产物进行SDS-PAGE分析表明,ALAS基因已在细菌中成功表达。使用Ni-NTA亲和层析法对表达的ALAS进行分离、纯化,得到大小约为44kD的ALAS蛋白。通过优化工程菌株的培养条件,建立了发酵生产ALA的方法,其胞外分泌ALA产量达5.379g/L,ALAS酶活力高达333U/min.mg。这是目前国内外利用生物法生产ALA产量最高的报道,为ALA的产业化应用打下了良好的基础。     

谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成

【背景】大肠杆菌(Escherichia coli)以谷氨酸为前体经C5途径合成有限的血红素。【目的】探究胞内谷氨酸代谢及谷氨酰-tRNA还原酶基因(hem A)过表达对5-氨基乙酰丙酸(5-Aminolevulinic Acid,ALA)和血红素合成的影响。【方法】通过Red同源重组敲除与谷氨酸代谢有关的mscS与aroG,构建hemA表达载体并导入基因缺失菌株中。【结果】mscS单敲除或mscS与aroG双敲除对菌体生长无显著影响。与出发菌株相比,单敲除与双敲除菌株的谷氨酸含量均有所增加,ALA含量略微下降,血红素含量分别增加了11.6%和35.7%。在双敲除菌株中进一步过表达hemA后,胞内血红素含量增至47.603μmol/L。【结论】通过调控谷氨酸代谢流量与过表达hemA可促进血红素的合成,该结果为增强C5途径的血红素合成提供了新的思路。     

过量表达自身hemA基因的重组大肠杆菌发酵生产5-氨基乙酰丙酸的研究

研究了优化重组大肠杆菌产5-氨基乙酰丙酸（ALA）的条件,提高大肠杆菌发酵生产AL气的产量。在测定重组大肠杆菌GT48的生长曲线的基础上,确定诱导时间,优化摇瓶发酵条件。然后,进一步在5L发酵罐上进行间歇和流加发酵研究。摇瓶实验表明,细胞培养最佳初始pH为6．5,最佳诱导时间为稳定期前期,最佳接种量为2％,过高的葡萄糖浓度对细胞生长和产物合成均有一定的抑制作用。在5L发酵罐间歇发酵中,重组菌产ALA能力达到47．8mg／L。采用流加发酵可以进一步将产物产量提高到63．8mg／L。构建的过量表达自身的hemA基因的大肠杆菌具有较高的产ALA能力,通过发酵条件优化和采用流加发酵可以提高AL气产量。     

大肠杆菌rhtA缺失对血红素合成的影响

【背景】Escherichia coli BL21(DE3)是基因工程的常用宿主,以C5途径合成5-氨基乙酰丙酸(5-Aminolevulinicacid,ALA),ALA是合成血红素的重要前体物质,但ALA分泌对血红素合成的影响尚不清楚。【目的】阐明参与ALA外运的RhtA在血红素合成途径中的作用。【方法】利用Red同源重组,敲除Escherichia coli BL21(DE3)的rhtA,同时构建重组质粒pEA过表达血红素合成途径中的关键酶基因hemA,检测分析血红素及其前体物质含量,以及血红素合成途径中10个关键基因的表达水平。【结果】敲除rhtA对菌体生长没有显著影响,敲除菌株BL21(DE3)Δrht A与原始菌株BL21(DE3)比较,ALA的胞外含量下降23%,血红素含量提高12%,尿卟啉III (Uroporphyrin III,UIII)、粪卟啉III (Coproporphyrin III,CIII)和原卟啉IX (Protoporphyrin IX,PPIX)的含量分别提高25%、15%和18%;敲除rhtA同时过表达hemA的菌株BL21(DE3)ΔrhtA/pEA与仅过表达hemA的菌株BL21(DE3)/pEA比较,胞外ALA减少了16%,血红素含量提高了24%,UIII和CIII含量分别提高55%和64%,PPIX含量显著增加,约为4.7倍。实时定量PCR结果表明,rhtA缺失后,hemC基因转录水平下调,其余9个基因转录水平均有不同程度的上调。【结论】rhtA敲除减少了ALA的外运,使得胞内血红素产量得到提高。     

谷氨酸棒状杆菌合成5-氨基乙酰丙酸的途径构建与发酵优化

5-氨基乙酰丙酸(5-amino levulinic acid,ALA)是生物体内天然存在的一种非蛋白质氨基酸,在农业和医药等领域应用广泛。为了在谷氨酸棒状杆菌中建立ALA碳四合成途径并优化其发酵体系,首先在谷氨酸棒状杆菌中过表达沼泽红假单胞菌来源的ALA合成酶(ALAS),建立高效的ALA碳四合成途径,然后从不同发酵培养基的比较、诱导剂和底物甘氨酸的浓度以及初始接种量等不同方面对ALA摇瓶发酵工艺进行了优化。结果显示,过表达Hem A的13032/p ZWA1菌株ALA产量达到1.41 g/L,是对照菌株的67.14倍。ALA的最优摇瓶发酵条件为以酵母粉为氮源的M9培养基,采用5%的接种量0.1 mmol/L IPTG进行Hem A的诱导表达,体系中甘氨酸的浓度要控制在4 g/L,摇瓶中ALA产量可达到3.28 g/L,比优化前提高了132.62%。采用发酵优化的条件,在5 L发酵罐的放大发酵中ALA产量可达10.08 g/L,这是现有报道中谷氨酸棒状杆菌一步发酵合成ALA的最高产量。     

代谢改造重组谷氨酸棒杆菌C4途径高效合成5-氨基乙酰丙酸

5-氨基乙酰丙酸 (5-aminolevulinic acid,5-ALA) 在医药和农业等领域有着广泛作用,目前主要采用大肠杆菌或谷氨酸棒杆菌以微生物发酵法合成。为了进一步提高谷氨酸棒杆菌合成5-ALA的能力,对其C4代谢途径进行了系统代谢改造。首先分别在谷氨酸棒杆菌中异源表达荚膜红杆菌和沼泽红假单胞菌的5-氨基乙酰丙酸合成酶ALAS,选择酶活相对较高的沼泽红假单胞菌的RphemA基因作为关键合成酶基因,并筛选到能显著增强RphemA的酶活性的核糖体结合位点RBS5。重组菌株ALAS的比酶活可达 (221.87±3.10) U/mg,且5-ALA产量提高了14.3%;随后通过敲除α-酮戊二酸脱氢酶抑制蛋白基因 (odhI) 和琥珀酸脱氢酶基因 (sdhA),促进了前体琥珀酰CoA向5-ALA途径的流动;通过sRNA抑制hemB表达减少了5-ALA的降解;并且过表达半胱氨酸/O-乙酰丝氨酸转运蛋白eamA提高了5-ALA的输出效率;使用重组菌株C. glutamicum 13032/?odhI/?sdhA-sRNAhemB-RBS5RphemA-eamA摇瓶发酵,5-ALA最高产量达11.90 g/L,较出发菌株提高了57%。最后,在5 L发酵罐中进行补料分批发酵,48 h内5-ALA的产量达25.05 g/L,为目前以葡萄糖为碳源发酵的最高产量。本研究构建了高产5-ALA重组谷氨酸棒杆菌,具有良好的工业应用前景。     

重组球形红细菌5-氨基乙酰丙酸合酶同工酶hemA和hemT的特性

将编码光合细菌Rhodobactersphaeroides 5- 氨基乙酰丙酸合酶(ALAS)的同工酶基因hemA、hemT转入E .coli中进行高表达,并将高表达的同工酶进行分离、纯化.纯化的hemA是可溶的,并具有催化活性,而hemT大部分是不溶的,且在体外条件下无活性.与其它重组ALAS相比,R .sphaeroides的hemA活性表达需PLP作为催化因子,除去PLP或用硼酸钠破坏与PLP的连接,hemA活性下降90 % .hemA PLP的紫外 可见光谱分析表明hemA与PLP之间形成一个醛亚胺键,而hemT与PLP之间未形成该键.hemA对修饰组氨酸、精氨酸、胱氨酸残基的试剂很敏感,对可切割Arg15 1和Ser15 2的类胰蛋白酶也很敏感,PLP也不能阻止该酶的切割作用.抗血清试验表明,hemA、hemT的抗血清均可与小鼠的ALAS杂交,并都有一个抗原决定簇.     

Construction of an expression vector for propionibacteria and its use in production of 5-aminolevulinic acid by Propionibacterium freudenreichii

Several promoters from Propionibacterium freudenreichii subsp. shermanii were isolated using a promoter probe vector, pCVE1, containing the Streptomyces cholesterol oxidase gene (choA) as a reporter gene. Three of four promoters isolated exhibiting a strong activity in Escherichia coli also expressed a strong activity in P. freudenreichii subsp. shermanii IFO12426. Using two promoters with a strong activity and a previously constructed shuttle vector, pPK705, shuttling between E. coli and Propionibacterium. we constructed expression vectors for propionibacteria. To overproduce 5-aminolevulinic acid (ALA), which is the first intermediate in the synthesis of porphyrins, the ALA synthase gene (hemA) from Rhodobacter sphaeroides was recombined with the expression vectors. The activity of ALA synthase in the recombinant P freudenreichii subsp. shermanii increased about 70-fold that in the strain without a vector. The recombinant Propionibacterium produced ALA at a maximum concentration of 8.6 mM in the absence of levulinic acid, an inhibitor of ALA dehydratase, with 1% glucose as a carbon source. The recombinant P. freudenreichii accumulated 18.8 mmol/g cells ALA in the presence of 1 mM levulinic acid and 30 mM glycine. The construction of an efficient expression vector will facilitate genetic studies of a vitamin B12 producer, Propionibacterium.     

5-aminolevulinic acid biosynthesis in Escherichia coli coexpressing NADP-dependent malic enzyme and 5-aminolevulinate synthase

5-aminolevulinate (ALA) synthase (E.C. 2.3.1.37), which mediates the pyridoxal phosphate-dependent condensation of glycine and succinyl-CoA, encoded by the Rhodobacter sphaeroides hemA gene, enables Escherichia coli strains to produce ALA at a low level. To study the effect of the enhanced C4 metabolism of E. coli on ALA biosynthesis, NADP-dependent malic enzyme (maeB, E.C. 1.1.1.40) was coexpressed with ALA synthase in E. coli. The concentration of ALA was two times greater in cells coexpressing maeB and hemA than in cells expressing hemA alone under anaerobic conditions with medium containing glucose and glycine. Enhanced ALA synthase activity via coupled expression of hemA and maeB may lead to metabolic engineering of E. coli capable of large-scale ALA production.     

Asn-150 of Murine Erythroid 5-Aminolevulinate Synthase Modulates the Catalytic Balance between the Rates of the Reversible Reaction

5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5′-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO₂, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn-85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function, and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. Although wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn-150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, thus ensuring that ALA is predominantly available for heme biosynthesis.     

Molecular dynamics analysis of the structural and dynamic properties of the functionally enhanced hepta-variant of mouse 5-aminolevulinate synthase

Heme biosynthesis, a complex, multistage, and tightly controlled process, starts with 5-aminolevulinate (ALA) production, which, in metazoa and certain bacteria, is a reaction catalyzed by 5-aminolevulinate synthase (ALAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme. Functional aberrations in ALAS are associated with several human diseases. ALAS can adopt open and closed conformations, with segmental rearrangements of a C-terminal, 16-amino acid loop and an α-helix regulating accessibility to the ALAS active site. Of the murine erythroid ALAS (mALAS2) forms previously engineered to assess the role of the flexible C-terminal loop versus mALAS2 function one stood out due to its impressive gain in catalytic power. To elucidate how the simultaneously introduced seven mutations of this activity-enhanced variant affected structural and dynamic properties of mALAS2, we conducted extensive molecular dynamics simulation analysis of the dimeric forms of wild-type mALAS2, hepta-variant and Rhodobacter capsulatus ALAS (aka R. capsulatus HemA). This analysis revealed that the seven simultaneous mutations in the C-terminal loop, which extends over the active site of the enzyme, caused the bacterial and murine proteins to adopt different conformations. Specifically, a new β-strand in the mutated ‘loop’ led to interaction with two preexisting β-strands and formation of an anti-parallel three-stranded β-sheet, which likely endowed the murine hepta-variant a more ‘stable’ open conformation than that of wild-type mALAS2, consistent with a kinetic mechanism involving a faster closed-to-open conformation transition and product release for the mutated than wild-type enzyme. Further, the dynamic behavior of the mALAS2 protomers was strikingly different in the two dimeric forms.     

Fish oil feeding up-regulates the expression of 5-aminolevulinate synthase 2 mRNA in rat brain

In this study, we investigated the effect of fish oil on gene expression in the cerebral cortex, and found that 5-aminolevulinate synthase 2 (ALAS2) mRNA expression was up-regulated by fish oil feeding. ALAS2 promoter activity was found to be regulated by retinoic acid. Our results suggest that fish oil modulates neuronal functions via heme synthesis.