大肠杆菌中1,3-丙二醇氧化还原酶的表达与纯化

目的:在大肠杆菌中表达1,3-丙二醇氧化还原酶(PDOR),并对PDOR进行纯化.方法:从克雷伯氏肺炎杆菌(Klebsiella pneumoniae)基因组中,克隆PDOR基因dhaT.构建表达载体pDK-dhaT,在E.coli DH5α中利用IPTG诱导进行表达.细胞裂解液利用硫酸铵盐析、Sephadex G-200凝胶层析和DE23 Cellulose阴离子交换层析,进行酶蛋白分离提纯.结果:用SDS-PAGE分析表明胞内PDOR占可溶性蛋白的39.8%,酶活为14.5U/ml.纯化后酶液比酶活提高3.94倍,回收率为15.5%.结论:成功地构建了PDOR高效表达载体,并且得到了高纯度的PDOR.     

1,3-丙二醇氧化还原酶结构与功能关系的探讨

用生物信息学方法对来源于klebsiella pneumonide的1,3-丙二醇氧化还原酶(即1,3-丙二醇氧化还原酶,PDOR)进行高级结构模建,并搜索其功能位点,以所得三维结构为对象,定位其铁信号结合位点、辅酶NADP大致位置以及可能的底物结合部位;在此基础上模拟PDOR活性部位,探讨该酶的构效关系。     

克雷伯氏菌产1,3-丙二醇ldhA基因缺失菌株的构建

克雷伯氏菌(Klebsiella pneumonia)甘油歧化发酵生产1,3-丙二醇(1,3-PD)的过程中,乳酸是氧化途径最主要的副产物,乳酸的产生和积累,不仅限制了菌体本身的生长,而且严重影响了1,3-丙二醇的转化率。利用λRed重组技术对Klebsiella pneumonia中的酶乳酸脱氢酶基因(ldhA)进行改造。在λRed重组系统作用下,将带有300 bp的线性同源片段ldhA1-Cm-ldh A2与基因组DNA的同源重组,经过抗性筛选和PCR鉴定最终获得了ldhA基因缺失菌株K.pneumonia2-1ΔldhA。经过24 h发酵可知,乳酸最大产出浓度由原来的10.16 g/L降为0.49 g/L,1,3-PD由原来的78.83 g/L增长为85.76 g/L,甘油转化率由60.64%增长到65.97%,提高了5.33%。     

克雷伯杆菌甘油脱氢酶和1,3-丙二醇氧化还原酶的动力学机制

本研究主要对克雷伯杆菌甘油转化1,3-丙二醇代谢途径中的2个关键酶甘油脱氢酶(GDH)、1,3-丙二醇氧化还原酶(PDOR)反应机制和动力学进行了研究。首先,通过初速度和产物抑制动力学研究确定了GDH、PDOR双底物酶促反应机制为有序BiBi机制,明确了由反应物消耗到产物生成之间的历程。其次,建立了GDH、PDOR双底物酶促反应动力学模型,由动力学模型可知,在偶合反应中,如果GDH和PDOR酶量相同,GDH氧化反应成为限速反应,而辅酶I将主要以氧化型NAD+形式存在。动力学信息为酶法合成1,3-丙二醇和代谢工程研究提供理论指导。     

克雷伯肺炎杆菌1,3-丙二醇氧化还原酶基因克隆及表达条件研究

1,3-丙二醇氧化还原酶是甘油歧化为1,3-丙二醇的一种关键酶。本研究从克雷伯肺炎杆菌(Klebsiella pneumoniae)基因组中,用PCR方法克隆了其编码基因dhaT。TA克隆测序正确后,构建胞内表达载体pET-28a-dhaT和分泌表达载体pET-22b-dhaT,然后转化E.coliBL21(DE3)进行原核表达。表达部位确定、SDS-PAGE和酶活分析表明,该酶得到了高水平表达。其中使用pET-22b表达的目的蛋白大都是不溶的包涵体;而使用pET-28a表达的目的蛋白胞内可溶,占胞内可溶总蛋白的45%,占菌体总蛋白的25%。常规(30℃)诱导表达即呈现1,3-丙二醇氧化还原酶活性,但低温(20℃)14 h诱导显示3.7倍的酶活性。     

丙酮酸对肺炎克雷伯氏菌微氧发酵甘油产1,3-丙二醇的影响

微氧条件下,考察肺炎克雷伯氏菌发酵生产1,3-丙二醇过程中柠檬酸和丙酮酸对发酵过程的影响。摇瓶实验结果表明:添加柠檬酸能抑制菌体生长和1,3-丙二醇合成;丙酮酸对菌体生长和1,3-丙二醇合成有一定的促进作用。5 L发酵罐批式发酵表明:补料培养基中加入8 g/L丙酮酸,1,3-丙二醇的产量提高了约10.8%,转化率提高了约4.4%,比生长速率提高了约10.8%。上述结果初步表明,强化能量的产生能够有效促进1,3-丙二醇的合成,可以利用分子生物学手段强化丙酮酸的产生以促进1,3-丙二醇的合成。     

提高肺炎克雷伯氏菌产1,3-丙二醇的分子生物学方法研究进展

1,3-丙二醇(1,3-propanediol,1,3-PD)可用于工业合成多种化合物,包括聚酯、聚醚和聚氨酯。发酵法生产1,3-PD具有巨大潜力。本文从代谢途径分析入手,梳理了肺炎克雷伯氏菌厌氧代谢途径的相关酶及催化作用,较详细地综述了其产1,3-PD关键酶的分子改造、基因工程菌株的构建和关键酶基因表达、副产物相关代谢酶基因敲除等方面的最新进展,并展望了其今后的发展前景。     

编码1,3-丙二醇氧化还原酶基因的克隆和表达

采用PCR法克隆了巴氏梭菌（Clostridium pasteurianum）CpN86菌株编码1,3丙二醇氧化还原酶基因（dhaT基因）;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达;分离和纯化了dhaT基因表达的重组蛋白。实验结果：（1）PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiella pneumoniae菌株dhaT基因的序列同源性为829%;（2）dhaT基因表达蛋白的酶活为108U/mg;（3）dhaT基因表达的蛋白分子量为43kD;（4）Western blot确定了dhaT基因表达的蛋白和 CpN86菌株天然蛋白有相同的抗原反应。     

pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

D-乳酸合成阻断对肺炎克雷伯氏菌的1,3-丙二醇发酵的影响

利用Red同源重组技术,快速敲除肺炎克雷伯氏菌中的编码D-乳酸脱氢酶的两个基因——ldhA和dld,获得KG1-1和KG1-2两个突变株,并研究了敲除编码D-乳酸脱氢酶基因丧失合成D-乳酸的KG1-1菌株的1,3-PD产量和菌体生长变化,实验结果表明乳酸合成缺失对现有工艺1,3-丙二醇发酵无影响。     

Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous.     

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.     

利用PDOR同工酶基因yqhD对产1,3-丙二醇克雷伯氏杆菌进行基因工程改造

由于Klebsiella pneumoniae 1,3-丙二醇合成途径中,加强甘油脱水酶基因表达,导致因NADH供应不足使3-羟基丙醛累积,并对菌体生长及1,3-丙二醇合成造成负面影响。为改善Klebsiella pneumoniae 1,3-丙二醇合成途径,本文利用PCR技术从大肠杆菌(Escherichia coli)中扩增出以NADPH 为辅酶的1,3-丙二醇氧化还原酶同工酶编码基因yqhD,从克雷伯氏杆菌中扩增出2.66kb的甘油脱水酶基因（dhaB）,构建了产1,3-丙二醇关键酶基因的串联载体pEtac-dhaB-tac-yqhD,并将其转入到野生克雷伯氏杆菌(Klebsiella pneumoniae)中,重组载体得到了表达。通过初步发酵,重组后的克雷伯氏杆菌产量比原始菌高20%左右,副产物中乙酸和丁二醇分别下降30%左右。     

Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Multi-modular engineering of 1,3-propanediol biosynthesis system in Klebsiella pneumoniae from co-substrate

Applied Microbiology and Biotechnology - 1,3-Propanediol (1,3-PDO) is a monomer for the synthesis of various polyesters. It is widely used in industries including cosmetics, solvents, and...