一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

1,3-丙二醇生物法生产中关键酶的研究进展

目前,国内外对于1,3-丙二醇(1,3-propanediol,1,3-PD)的生产研究正在由化学法逐渐向生物法转变。该文着重介绍了生物法生产1,3-PD的生产菌株和生物合成途径,综述了关键酶的性质特点和基因克隆表达情况,对关键酶晶体结构的相关研究成果进行了介绍,进一步探讨了运用宏基因组技术和酶分子改造技术来获得新型高性能关键酶的方法,并展望了基因工程菌的应用前景,从而推动了对1,3-PD生产途径中关键酶的了解及1,3-PD的生产应用研究。     

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。     

弗氏柠檬酸菌dhaT基因的克隆表达、序列分析、酶的纯化及特性研究

1,3-丙二醇（1,3-propanediol,1,3-PD）是一种重要的化工原料,越来越受到广泛的关注。以弗氏柠檬酸菌(Citrobacter freundii)基因组DNA为模板,通过PCR得到1,3-丙二醇氧化还原酶(1,3-propanediol dehydrogenase,PDOR) 的基因dhaT,序列显示与来源于C.freundii DSM 30040 (Genbank U09771)相应基因的相似性为78%。将此基因构建于表达载体pSE380,得到重组质粒pSE-dhaT。重组质粒转化到宿主菌E.coli JM109中进行了表达,重组酶通过镍柱及Sephacral S-300进行纯化,重组酶SDS-PAGE结果显示有非常明显的单一的42kDa特异性蛋白条带出现。以丙醛为底物测定重组酶还原反应的最适温度为37℃、最适pH为8.0,对丙醛的Km值为10.05mmol/L,最大反应速度Vmax为37.27umol/ min /mg;以1,3-PD为底物测定重组酶氧化反应的最适温度为25℃、最适pH为10.5,对1,3-PD的Km值为1.28mmol/L,最大反应速度Vmax为25.55umol/min/mg。重组酶的还原反应比活为49.50U/mg,氧化反应比活为79.72U/mg。该酶同样具有假定的结合Fe2+的G-X-X-H-X-X-A-H-X-X-G-X-X-X-X-X-P-H-G模体保守结构。此研究为工程菌高效生产1,3-PD奠定了基础。     

基于甘油的1,3-丙二醇生物合成的代谢局限及其改造策略

粗甘油是生物柴油生产中的主要副产物,一些微生物可将甘油转化为重要化工原料1,3-丙二醇(1,3-PD),而利用这些微生物野生菌株生物合成1,3-PD会存在一些局限性,如底物抑制、产物抑制等。文中从1,3-丙二醇的甘油生物转化途径与这些局限性出发,总结了生物合成中存在的问题,并针对这些问题提出了一些基于基因敲除或基因过表达等基因工程技术的改造方法,综述了利用基因工程菌生物转化甘油生成1,3-丙二醇的最新研究进展。     

利用克雷伯肺炎杆菌限制性底物发酵生产1，3-丙二醇

研究了克雷伯肺炎杆菌（Klebsiella pneumoniae）批式流加发酵生产1,3-丙二醇的发酵工艺,根据1,3-丙二醇的生产和菌体生长相关的特点,采用营养基质限制性流加的发酵工艺,通过控制氮源氯化铵以保持细胞稳定生长。结果表明：过低的氮源浓度,细胞生长受到限制,影响产物1,3-PD的合成;过高的氮源浓度,细胞比生长速率增加,但1,3-PD关于消耗甘油的得率降低,用于生长和维持代谢所消耗的甘油量增加。以0.41 g/(L·h)的氮源流加速率,残余氯化铵浓度在0.1 g/L时,转化率和生产强度最高。发酵25 h～28 h后,1,3-丙二醇最终浓度达到52.03 g/L,生产强度为2.04 g/(L·h),相对于甘油的摩尔转化率为0.66,分别比氮源限制前提高了28.0 ％、35.1 ％及29.4 ％。通过限制性流加氯化铵,控制细胞的比生长速率,使底物甘油有效转变为发酵的目标产物1,3-PD,有效实现产物1,3-PD的高生产强度以及对甘油的高转化率。     

Effects of different pH regulators on 1,3-propanediol production by Klebsiella pneumoniae in fed-batch culture

对肺炎克雷伯氏菌(Klebsiella pneumoniae)发酵生产1,3-丙二醇(1,3-Propanediol,1,3-PD)的补碱策略进行了研究.分别利用NaOH、氨水、KOH三种溶液作为pH调节剂,优化三种pH调节剂并得到按一定比例混合的混合碱.当采用混合碱调控发酵pH值为7.0时,1,3-丙二醇的产量达到了55 g/L,比无pH调控(对照)发酵过程发酵水平提高了10.6倍.     

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%。     

利用甘油发酵耦联生产3-羟基丙酸及1,3-丙二醇重组菌的构建及筛选

在肺炎克雷伯杆菌(Klebsiella pneumoniae)代谢甘油生产1,3-丙二醇(1,3-PD)的过程中,为了减少有毒中间产物3-羟基丙醛(3-HPA)的积累,可将其转化为3-羟基丙酸(3-HP),从而实现1,3-丙二醇和3-羟基丙酸的联产。克隆来自于酿酒酵母的NAD+依赖型的乙醛脱氢酶(ALDH)的基因aldh4,构建了表达载体pKP-aldh,转化K.pneumoniae,得到了有效表达乙醛脱氢酶的重组肺炎克雷伯杆菌(K.pneumoniae A+)。在此基础上,使用紫外诱变联合菌种驯化的方法对K.pneumoniae A+进行筛选,获得了可耐受较高3-HP浓度(≥35 g/L)的重组肺炎克雷伯杆菌K.pneumoniae A+5-3。发酵实验结果表明,K.pneumoniae A+5-3可将3-HPA转化为3-HP,能够同时利用甘油耦联生产3-HP和1,3-PD,产量分别达到5.0 g/L和74.5 g/L。     

一株1,3-丙二醇生产菌的分离鉴定及其发酵特性

从活性污泥中分离筛选得到一株能代谢甘油生产1,3-丙二醇(1,3-PD)的菌株2-1,通过形态学鉴定、生理生化试验、16S rRNA序列分析对菌株分类学地位进行鉴定,用MEGA 4.1软件构建的系统发育树显示菌株2-1与Klebsiella pneumoniae(CP001891)的亲缘关系最近。16S rDNA序列同源性比较发现,菌株2-1与模式菌株同源率为95.4%,疑似为新种。对菌株2-1在5 L发酵罐中进行发酵特性研究,分批补料发酵时得到较高的1,3-PD终浓度,达到63.5 g/L,此时生产强度为2.19 g/(L.h),底物转化率0.64 mol/mol。     

Improved 1,3-propanediol production with hemicellulosic hydrolysates (corn straw) as cosubstrate: Impact of degradation products on Klebsiella pneumoniae growth and 1,3-propanediol fermentation

To improve 1,3-propanediol (1,3-PD) production by an economic and efficient approach, hemicellulosic hydrolysates (HH) used as cosubstrate resulted in more biomass and higher reducing power for 1,3-PD production. The effects of primary degradation products such as individual sugars (xylose, glucose, mannose, arabinose and galactose) and major inhibitors (furfural, acetate and formate) on the Klebsiella pneumoiae growth and 1,3-PD production were investigated in this study. Xylose and mannose could efficiently promote the 1,3-PD production and cell growth. Furfural (0.28 g/l) and sodium acetate (1.46 g/l) in low concentration were not inhibitory to Klebsiella pneumoniae, rather they have stimulatory effect on the growth and 1,3-PD biosynthesis, especially the acetate. In fed-batch fermentation with HH as cosubstrate, the final 1,3-PD production, conversion from glycerol and productivity were 71.58 g/l, 0.65 mol/mol and 1.93 g/l/h, respectively, which were 17.8%, 25.0% and 17.7% higher than that from glycerol alone.     

1,3-Propanediol production and tolerance of a halophilic fermentative bacterium, Halanaerobium saccharolyticum subsp. saccharolyticum

1,3-Propanediol (1,3-PD) is widely used in polymer industry in production of polyethers, polyesters and polyurethanes. In this article, a study on 1,3-PD production and tolerance of Halanaerobium saccharolyticum subsp. saccharolyticum is presented. 1,3-PD production was optimized for temperature, vitamin B(12) and acetate concentration. The highest 1,3-PD concentrations and yields (0.6 mol/mol glycerol) were obtained at vitamin B?? concentration 64 μg/l and an inverse correlation between 1,3-PD and hydrogen production was observed with varying vitamin B?? concentrations. In the studied temperature range and initial acetate concentrations up to 10 g/l, no significant variations were observed in 1,3-PD production. High initial acetate (29-58 g/l) was observed to cause slight decrease in 1,3-PD concentrations produced but no effects on 1,3-PD yields (mol/mol glycerol). Initial 1,3-PD concentrations inhibited the growth of H. saccharolyticum subsp. saccharolyticum. When initial 1,3-PD concentration was raised from 1g/l to 57 g/l, a decrease of 12% to 75%, respectively, in the highest optical density was observed.     

Physiological predisposition of various Clostridium species to synthetize 1,3-propanediol from glycerol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was first described in 1881 but little attention was paid to this biosynthesis for over a century. An increasing interest in microbial 1,3-PD production is observed since late 1980s. The high growth rate of the biofuel market and the perspective of glycerol becoming abundant attract even more attention to this valuable chemical.Glycerol conversion to 1,3-PD is known to occur in Clostridia, Enterobacteriaceae and Lactobacillaceae. Some clostridial species are among the best 1,3-PD producers.This work is a review of the current state of research on Clostridium spp. strains that ferment glycerol to 1,3-PD. It focuses on the metabolic pathways and factors that influence the production of this diol. The effects of different environmental stresses on the process of 1,3-PD synthesis are also covered. Moreover, various genetic engineering methods utilized in order to improve the capabilities of bacteria used in this process are presented.     

Determination of kinetic parameters of 1,3-propanediol fermentation by Clostridium diolis using statistically optimized medium

1,3-propanediol (1,3-PD) is a chemical compound of immense importance primarily used as a raw material for fiber and textile industry. It can be produced by the fermentation of glycerol available abundantly as a by-product from the biodiesel plant. The present study was aimed at determination of key kinetic parameters of 1,3-PD fermentation by Clostridium diolis. Initial experiments on microbial growth inhibition were followed by optimization of nutrient medium recipe by statistical means. Batch kinetic data from studies in bioreactor using optimum concentration of variables obtained from statistical medium design was used for estimation of kinetic parameters of 1,3-PD production. Direct use of raw glycerol from biodiesel plant without any pre-treatment for 1,3-PD production using this strain investigated for the first time in this work gave results comparable to commercial glycerol. The parameter values obtained in this study would be used to develop a mathematical model for 1,3-PD to be used as a guide for designing various reactor operating strategies for further improving 1,3-PD production. An outline of protocol for model development has been discussed in the present work.     

Microbial production of 1,3-propanediol

1,3-Propanediol (1,3-PD) production by fermentation of glycerol was described in 1881 but little attention was paid to this microbial route for over a century. Glycerol conversion to 1,3-PD can be carried out by Clostridia as well as Enterobacteriaceae. The main intermediate of the oxidative pathway is pyruvate, the further utilization of which produces CO₂, H₂, acetate, butyrate, ethanol, butanol and 2,3-butanediol. In addition, lactate and succinate are generated. The yield of 1,3-PD per glycerol is determined by the availability of NADH₂, which is mainly affected by the product distribution (of the oxidative pathway) and depends first of all on the microorganism used but also on the process conditions (type of fermentation, substrate excess, various inhibitions). In the past decade, research to produce 1,3-PD microbially was considerably expanded as the diol can be used for various polycondensates. In particular, polyesters with useful properties can be manufactured. A prerequisite for making a “green” polyester is a more cost-effective production of 1,3-PD, which, in practical terms, can only be achieved by using an alternative substrate, such as glucose instead of glycerol. Therefore, great efforts are now being made to combine the pathway from glucose to glycerol successfully with the bacterial route from glycerol to 1,3-PD. Thus, 1,3-PD may become the first bulk chemical produced by a genetically engineered microorganism. Received: 12 January 1999 / Received revision: 9 March 1999 / Accepted: 14 March 1999     

Klebsiella spp as a 1, 3-propanediol producer: the metabolic engineering approach

Klebsiella spp are one of the best natural producers of 1,3-propanediol (1,3-PD). However, their usage in the biotechnological production of the diol is limited, since the species belong to the second hazard group. Nevertheless, multiple advantageous traits of Klebsiella spp justify the international effort devoted to develop a biotechnological process of 1,3-PD production with these microorganisms. Apart from the process engineering approach aiming at improvement of 1,3-PD production by Klebsiella spp, plethora of metabolic engineering approaches have been reported. Different strategies have been undertaken to genetically improve Klebsiella strains and provide them with the ability to synthesize 1,3-PD more efficiently. These include over-expression of both homologous and heterologous genes of the 1,3-PD synthesis pathway, protein and cofactor engineering, deletion of the genes involved in by-products formation. This review provides an overview of the initial and most recent reports on the metabolic engineering of Klebsiella spp with the aim of improvement of 1,3-PD biosynthesis.     

Change in proteomic profiles of genetically modified 1,3-propanediol-producing recombinant E. coli

The recombinant E. coli Delta6 mutant (galR, glpK, gldA, IdhA, lacI, tpiA) was used to produce 1,3-propanediol (PD) from glucose. The 1,3-PD production increased with feedback control of the glucose concentration using fed-batch fermentation. The maximum 1,3-PD concentration produced was 43 g/l after 60 h of fermentation. Glycerol production was minimized when controlling the glucose concentration at less than 1 g/l. The expression levels of seven enzymes related to the 1,3-PD production metabolism were compared during the cell growth phase and 1,3-PD production phase, and their expression levels all increased during 1,3-PD production, with the exception of alcohol dehydrogenase.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

Klebsiella spp as a 1, 3-propanediol producer – the metabolic engineering approach

Klebsiella spp are one of the best natural producers of 1,3-propanediol (1,3-PD). However, their usage in the biotechnological production of the diol is limited, since the species belong to the second hazard group. Nevertheless, multiple advantageous traits of Klebsiella spp justify the international effort devoted to develop a biotechnological process of 1,3-PD production with these microorganisms. Apart from the process engineering approach aiming at improvement of 1,3-PD production by Klebsiella spp, plethora of metabolic engineering approaches have been reported. Different strategies have been undertaken to genetically improve Klebsiella strains and provide them with the ability to synthesize 1,3-PD more efficiently. These include over-expression of both homologous and heterologous genes of the 1,3-PD synthesis pathway, protein and cofactor engineering, deletion of the genes involved in by-products formation. This review provides an overview of the initial and most recent reports on the metabolic engineering of Klebsiella spp with the aim of improvement of 1,3-PD biosynthesis.