生物合成聚3-羟基丙酸酯的研究进展

聚3-羟基丙酸酯(P3HP)作为聚羟基脂肪酸酯家族(PHAs)中的新型热塑性塑料,具有生物降解性和生物相容性等优点。目前,未见野生微生物可以合成P3HP的报道,生产途径主要为化学法和生物法。其中,通过化学法或添加3-HP单体及其结构类似物作为前体的P3HP合成效率低、成本高且不具环保性;而通过构建和改造工程菌的生物代谢途径,能够利用廉价、可再生的碳源,已经逐渐成为研究热点。文中综述了国内外P3HP生物合成研究进展,并对甘油途径、丙二酸单酰辅酶A(Malonyl-Co A)途径和β-丙氨酸途径等合成方法进行了优缺点分析,为生物合成P3HP的深入研究奠定理论基础。     

生物合成3-羟基丙酸的代谢工程研究进展

3-羟基丙酸(3-HP)作为一种重要的平台化合物,以此为底物能够合成多种具有商业潜质的生物制品。野生菌合成3-HP产量较低,严重限制3-HP的大规模应用与生产,通过改造合成代谢通路的相关基因,构建以廉价底物为碳源的工程菌株,实现降低生产成本提高产量的目的。文中将对近年来国内外通过代谢工程合成3-羟基丙酸的研究进展进行概述,并对甘油途径、丙二酸单酰辅酶A途径、β-丙氨酸等途径合成3-HP的优缺点进行总结分析,对3-HP未来发展前景进行展望。     

新型固碳途径—3-羟基丙酸循环的研究进展

3-羟基丙酸循环是一种存在于嗜热光合绿丝菌中新型的CO2固定途径.此循环的特征代谢中间物为3-羟基丙酸,该化合物可用于合成许多重要的化工产品,具有很高的工业价值.本文介绍了3-羟基丙酸循环固碳途径的发现、反应机理的逐步阐明过程及最新的研究进展,并对其理论意义和在绿色化工中的应用前景进行了探讨.     

微生物发酵生产3-羟基丙酸

3-羟基丙酸(3-Hydroxypropionic acid,简写3-HP)是多种光学活性物质的前体,被美国能源部列为当今世界12种最具潜力的化工产品之一[1]。目前,3-HP由化学方法合成制备。虽然生产工艺一直在改进,但是由于合成难度大、产品不易分离提纯、产品得率低等原因,生产成本较高,且生产过程存在不     

氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸

3-羟基丙酸是一种潜在的重要化工产品,可作为中间体合成多种有经济价值的工业用化合物。文中利用氧化葡萄糖酸杆菌生物催化1,3-丙二醇合成3-羟基丙酸。首先在50 mL摇瓶中(转化体系为10 mL)考察细胞加入量、底物和产物浓度等对催化反应的影响。在此基础上,在2 L鼓泡塔中(转化体系为1 L),采取适当的补料方式和生物转化与分离相耦合的手段解除抑制,以提高目标产物终浓度。结果表明:高底物和产物浓度通过降低反应初速度抑制转化的进行,并确定了最佳催化反应条件为6 g/L菌体量,pH 5.5。利用流加补料方式维持反应体系中底物浓度在15~20 g/L,经过60 h的反应,3-羟基丙酸的浓度达到60.8 g/L,生产强度为1.0g/(L.h),转化率为84.3%。采用生物转化与分离相耦合的方法,经过50 h的转化反应,3-羟基丙酸的总产量达76.3 g/L,生产强度为1.5 g/(L.h),转化率83.7%。研究结果对利用氧化葡萄糖酸杆菌的不完全氧化醇类化合物特性实现其在工业生物催化中的应用具有一定的指导意义。     

重组大肠杆菌合成聚(3-巯基丙酸)和聚(3-羟基丁酸-3-巯基丙酸)的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株.前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚(羟基丁酸-戊酸)等多种生物聚酯[Liu and Steinbüchel, Appl. Environ. Microbiol. 66739-743].利用该重组大肠杆菌,通过生物催化作用合成了3-巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物.实验首先研究了3-巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚(3-巯基丙酸)可达6.7%(占细胞干重),合成聚(3-羟基丁酸-3-巯基丙酸)(分子中3-巯基丙酸3-羟基丁酸=31)可达24.3%.实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的.还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究.聚(3-巯基丙酸)或聚(3-羟基丁酸-3-巯基丙酸)等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值.     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

重组大肠杆菌合成聚（3-巯基丙酸）和聚（3-羟基丁酸-3-巯基丙酸）的研究

利用Clostridium acetobutylicum的丁酸激酶基因 (buk) 和磷酸转丁酰基酶基因(ptb),以及Thiocapsa pfennigii的PHA合成酶基因,设计了一条能够合成多种聚羟基烷酸的代谢途径,用构建的质粒转化大肠杆菌,获得了重组大肠杆菌菌株。前期的研究表明,在合适的前体物条件下,该重组大肠杆菌能够合成包括聚羟基丁酸、聚（羟基丁酸戊酸）等多种生物聚酯［Liu and Steinbüchel, Appl. Environ. Microbiol. 66:739743］。利用该重组大肠杆菌,通过生物催化作用合成了3巯基丙酸的同型共聚酯,同时利用该重组大肠杆菌还获得了含3-巯基丙酸单体的多种异型共聚物。实验首先研究了3巯基丙酸对大肠杆菌生长的影响,在此基础上优化了培养过程中添加3-巯基丙酸的时机和浓度,结果表明,在实验的条件下,细胞合成聚（3-巯基丙酸）可达6.7%(占细胞干重),合成聚（3-羟基丁酸—3-巯基丙酸）（分子中3-巯基丙酸:3-羟基丁酸=3:1）可达24.3%。实验进一步研究了同时或分别表达以上3个基因的重组大肠杆菌合成聚合物的能力,结果表明只有当3个基因同时表达时才能合成聚合物,说明3个基因对合成过程是必须的,从而表明了合成途径是按照设计的路线进行的。还通过GC/MS、GPC、IR等手段对合成的化合物进行了定性的研究。聚（3-巯基丙酸）或聚（3-羟基丁酸-3-巯基丙酸）等聚酯属于一类新型生物聚合物,它在分子骨架中含有硫酯键,不同于聚羟基烷酸酯的氧酯键,从而具有显著不同的物理、化学、光学等性质和具有重要的潜在应用价值。     

静息细胞转化生产3-羟基丙酸的条件优化

本文研究了静息细胞生物转化生产3-羟基丙酸的反应体系。考察了以甘油为底物,利用静息细胞转化生产3一羟基丙酸的相关因素,确定了最佳的转化条件：细胞浓度20g／L,甘油浓度20g／L,辅酶VB12浓度10mg／L,NAD＋浓度0．15mmol／L,温度35℃,反应体系为0．05mol／LpH7．0Tris—HCl缓冲液。在上述条件下反应6h后,3-羟基丙酸的产量达到为3．17g／L,底物转化率为28．33％。由上述结果可知,采用静息细胞转化法为3-HP的生物合成提供了一种可能的方法。     

3-羟基丙酸高产菌株的筛选及诱变选育

【目的】3-羟基丙酸是一种重要的化学平台化合物,期望得到一株能够高产3-羟基丙酸的菌株。【方法】从土壤及粪便筛选并对得到的菌株进行鉴定和复合诱变。【结果】得到了一株能够利用丙酸发酵生产3-羟基丙酸的酵母Y-11,经生理生化鉴定及18S rDNA序列分析确定其为Candida sp.(假丝酵母)。以Y-11为出发菌株,经紫外-亚硝基胍-60Coγ复合诱变得到了突变性状稳定且可遗传的高产菌株5-13B,其3-羟基丙酸的产量为11.78 g/L,是出发菌株的2.46倍。【结论】对出发菌株和突变株的发酵特性进行了比较,结果表明突变株的3-羟基丙酸产量、对底物丙酸的转化率、产物3-羟基丙酸的积累性能及丙酸的耐受性均优于出发菌株。     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Evolution reveals a glutathione-dependent mechanism of 3-hydroxypropionic acid tolerance

Biologically produced 3-hydroxypropionic acid (3HP) is a potential source for sustainable acrylates and can also find direct use as monomer in the production of biodegradable polymers. For industrial-scale production there is a need for robust cell factories tolerant to high concentration of 3HP, preferably at low pH. Through adaptive laboratory evolution we selected S. cerevisiae strains with improved tolerance to 3HP at pH 3.5. Genome sequencing followed by functional analysis identified the causal mutation in SFA1 gene encoding S-(hydroxymethyl)glutathione dehydrogenase. Based on our findings, we propose that 3HP toxicity is mediated by 3-hydroxypropionic aldehyde (reuterin) and that glutathione-dependent reactions are used for reuterin detoxification. The identified molecular response to 3HP and reuterin may well be a general mechanism for handling resistance to organic acid and aldehydes by living cells.     

De novo bio-production of odd-chain fatty acids in Saccharomyces cerevisiae through a synthetic pathway via 3-hydroxypropionic acid

Odd-chain fatty acids (OCFAs) and their derivatives have attracted increasing attention due to their wide applications in the chemical, fuel, and pharmaceutical industry. However, most natural fatty acids are even-chained, and OCFAs are rare. In this work, a novel pathway was designed and established for de novo synthesis of OCFAs via 3-hydroxypropionic acid (3-HP) as the intermediate in Saccharomyces cerevisiae. First, the OCFAs biosynthesis pathway from 3-HP was confirmed, followed by an optimization of the precursor 3-HP. After combining these strategies, a de novo production of OCFAs at 74.8 mg/L was achieved, and the percentage of OCFAs in total lipids reached 20.3%, reaching the highest ratio of de novo-produced OCFAs. Of the OCFAs produced by the engineered strain, heptadecenoic acid (C17:1) and heptadecanoic acid (C17:0) accounted for 12.1% and 7.6% in total lipid content, respectively. This work provides a new and promising pathway for the de novo bio-production of OCFAs.     

Coupled incremental precursor and co-factor supply improves 3-hydroxypropionic acid production in Saccharomyces cerevisiae

3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l⁻¹. Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future.     

3-羟基丙酸分批发酵过程中的pH控制策略研究

本文利用重组大肠杆菌以甘油为底物发酵合成3．羟基丙酸,考察了不同pH对3．羟基丙酸产量及菌体生长的影响,发现在pH6．5条件下,细胞比生长速率达到最大值,延迟期也相对较短;而pH7．0有利于3-羟基丙酸的合成,控制pH7．0可以使3-羟基丙酸产量达到7．39g／L。基于不同pH条件下对细胞比生长速率和3-羟基丙酸比生成速率的分析,提出3．羟基丙酸分批发酵过程中的pH控制策略,即在发酵过程前5h将pH控制在6．5,5h～15h控制pH为7．0,此时有利于细胞生长;而后在15h-25h控制pH为7．5,25h后控制pH为7．0,从而使细胞具有较高的3．羟基丙酸比合成速率。在此控制策略下经过34h发酵3-羟基丙酸的终产量达到8．76g／L,比pH7．0条件下的3-羟基丙酸产量提高了18．54％。     

Production of 3-hydroxypropionic acid through propionaldehyde dehydrogenase PduP mediated biosynthetic pathway in Klebsiella pneumoniae

The pduP gene encodes a propionaldehyde dehydrogenase (PduP) was investigated for the role in 3-hydroxypropionic acid (3-HP) glycerol metabolism in Klebsiella pneumoniae. The enzyme assay showed that cell extracts from a pduP mutant strain lacked measurable dehydrogenase activity. Additionally, the mutant strain accumulated the cytotoxic intermediate metabolite 3-hydroxypropionaldehyde (3-HPA), causing both cell death and a lower final 3-HP titer. Ectopic expression of pduP restored normal cell growth to mutant. The enzymatic property of recombinant protein from Escherichia coli was examined, exhibiting a broad substrate specificity, being active on 3-HPA. The present work is thus the first to demonstrate the role of PduP in glycerol metabolism and biosynthesis of 3-HP.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.