一种简单的高产2,3-丁二醇发酵生产方法

利用一株克雷伯氏菌(Klebsiellasp.LN145)在以葡萄糖和磷酸氢二铵为主要成分的培养基中发酵生产2,3-丁二醇。在补料发酵培养过程中,通过补糖,2,3-丁二醇和3-羟基丁酮的最大产量分别达到了84.0 g/L和10.5 g/L,二醇的摩尔转化率达到理论水平的91%,转化速率达到1.8 g/(L.h)。     

生物法生产2,3-丁二醇研究进展

2,3-丁二醇是一种重要的化工原料,可广泛应用于多个领域。二战期间由于合成橡胶需要大量1,3-丁二烯,2,3-丁二醇生产空前发展。近年来,由于聚对苯二甲酸丁烯树脂、γ-丁内酯,Spandex弹性纤维及其前体的需求增长,2,3-丁二醇的需求和产量也稳步增长。多年来,生物法生产2,3-丁二醇虽然得到了广泛的研究,但一直没有实现工业化。本文从产生2,3-丁二醇的菌种及2,3-丁二醇的生理意义、代谢途径、旋光异构体的形成机理、影响发酵的因素与产物的提纯等方面对生物法生产2,3-丁二醇进行了综述并提出了生物法生产2,3-丁二醇要解决的几个问题。     

微生物法生产1,3-二羟基丙酮代谢工程研究进展

1,3-二羟基丙酮是一种重要的化工原料和医药中间体,广泛应用于化妆品、医药、食品等领域。以下综述了微生物法生产1,3-二羟基丙酮的代谢途径和关键酶,以及微生物法生产1,3-二羟基丙酮所涉及的代谢工程技术的研究进展。指出利用基因工程的方法对菌株进行改造,提高甘油脱氢酶催化活性,同时根据菌株的代谢特性,对发酵过程进行调控,提高1,3-二羟基丙酮的得率,是今后的研究方向。     

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。     

2,3-丁二醇代谢途径关键酶基因敲除对克雷伯氏菌发酵产1,3-丙二醇的影响

2,3-丁二醇是克雷伯氏菌发酵产1,3-丙二醇的主要副产物,为减少2,3-丁二醇的产生,利用Red重组技术对克雷伯氏菌2,3-丁二醇合成途径关键酶基因budC和budA进行了敲除。突变株发酵性能实验结果表明,所获得的两株突变株生长性能受到不同程度的影响;budC基因的缺失使菌株1,3-丙二醇产量提高了10%,2,3-丁二醇降低为原来的70%,而budA基因缺失则使菌株无2,3-丁二醇和1,3-丙二醇的产生,但乳酸、琥珀酸、乙醇和乙酸的产量较出发菌株都有明显增长。通过进一步对budC基因缺失菌株主要产物分析,推测在该菌中存在2,3-丁二醇回补途径,这一结果为低副产物克雷伯氏菌的改造提供了新依据。     

β-1,3-葡聚糖酶在植物抗真菌病基因工程中的研究进展

β-1,3-葡聚糖酶是植物抗真菌病的重要抗性物质之一,植物β-1,3-葡聚糖酶可由病原物(如Mg)、化学因子(如水杨酸、乙烯、赤霉素)或物理因子(如紫外线照射、机械损伤)等多种生物因子和非生物因子诱导产生.将外源β-1,3-葡聚糖酶基因导入植物,可提高植物的抗真菌病害的能力;而将β-1,3-葡聚糖酶基因与其他防卫蛋白基因同时导入植物,将更大程度的提高植物的抗真菌病能力,是植物抗真菌病防治的有效新途径.文章中主要对β-1,3-葡聚糖酶的生物学特性、植物β-1,3-葡聚糖酶基因在转基因植株中的独立表达及其与其他抗真菌病基因的协同表达等进行了综述.     

微生物发酵法生产1,3-丙二醇的研究新进展

1,3-丙二醇(1,3-PD)是一种重要的化工原料,广泛应用于医药、化工、食品及化妆品等行业,同时1,3-PD是合成聚对苯二甲酸丙二酯(PTT)的重要单体,市场需求量逐年增多。基于生态友好型、生产安全和可持续发展的要求,利用微生物转化可再生资源来生产1,3-PD受到了人们的广泛重视。综述了微生物发酵法生产1,3-PD的菌株、代谢途径、发酵和下游分离工艺及其新进展,并对工业生产中利用生物技术生产1,3-PD的未来前景和挑战进行了探讨。     

1-辛烯-3-醇防治采后桃果实软腐病的作用

【目的】桃果实易受匍枝根霉(Rhizopus stolonifer)侵染引起软腐病,导致果实采后腐烂损失严重。目前人工合成的化学杀菌剂是控制桃果实采后病害的主要方法,但长期使用容易带来食品安全隐患、病原菌抗药性和环境污染等问题。通过研究生物源抑菌成分1-辛烯-3-醇对桃果实软腐病的控制作用,为减少化学农药使用和控制采后桃果实软腐病提供理论基础。【方法】使用1-辛烯-3-醇熏蒸接种匍枝根霉(R.stolonifer)后的桃果实,对果实抗病相关基因表达和酶活性进行测定。通过离体试验,研究1-辛烯-3-醇熏蒸对匍枝根霉(R.stolonifer)菌丝和孢子的影响。【结果】55.80μg/mL 1-辛烯-3-醇熏蒸处理可以显著降低桃果实的发病率和病斑直径(P<0.05),提高几丁质酶(chitinase,CHI)和β-1,3葡聚糖酶(β-1,3-glucanase,GLU)的活性以及病程相关基因非表达子1(nonexpressor of pathogenesis-related protein 1,NPR1)、病程相关蛋白1(pathogenesis-related protein 1,PR1)、CHI和GLU的基因表达量。离体试验结果显示,1-辛烯-3-醇可抑制平板上匍枝根霉(R.stolonifer)菌丝的生长,使菌丝体细胞结构遭到破坏,同时显著降低麦角固醇含量(P<0.05),抑制孢囊孢子的萌发和芽管伸长,并通过破坏孢子的膜结构,引起活性氧(reactive oxygen species,ROS)暴发与线粒体损伤。【结论】以上结果证实,1-辛烯-3-醇熏蒸处理不仅能直接破坏匍枝根霉(R.stolonifer)的菌丝与孢子,还可通过诱导桃果实的系统获得性抗性(systemic acquired resistance,SAR)抑制采后软腐病的蔓延。     

1,3-丙二醇生产的研究进展

作为一种重要的化工材料,1,3-丙二醇凭借其自身的优点,在工业生产中具有很高的应用价值。但其传统的生产方法,操作繁琐、技术难度大、设备投资高,已无法满足对1,3-丙二醇的日益增长的需要;而微生物发酵法又面临着产率低、发酵条件难以控制等弊病。相反,基因工程技术在1,3-丙二醇生产过程中扮演着越来越重要的角色。简要综述了1,3-丙二醇研究及生产工艺的进展。     

1,3-丙二醇发酵过程中乳酸抑制的研究

目的:研究乳酸对克雷伯氏肺炎杆菌(Klebsiella pneumonia)产1,3-丙二醇的影响。方法:通过在摇瓶和反应器水平下分析不同菌株(包含无乳酸、2,3-丁二醇产生的基因敲除菌)的乳酸代谢特性。结果:前期添加6 g/L的乳酸使1,3-丙二醇的产量降低了19%,而发酵10h后添加乳酸几乎不表现出抑制作用。通过对乳酸敲除菌株的代谢分析发现,发酵后期能够消耗培养基中的乳酸,这在一定程度上也反映了菌体发酵后期对乳酸的耐受性。结论:乳酸的抑制作用主要发生在1,3-丙二醇发酵的前期。解除了一株无副产物2,3-丁二醇生产株前期乳酸的过早积累后,1,3-丙二醇的的产量提高了56%。     

Microbial production of diols as platform chemicals: recent progresses

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.     

Stereoselective biosynthesis of chloroarylpropane diols by the basidiomycete Bjerkandera adusta: exploring the roles of amino acids, pyruvate, glycerol and phenyl acetyl carbinol

Bjerkandera adusta produces many chlorometabolites including chlorinated anisyl metabolites (CAMs) and 1-arylpropane-1,2-diols (1, 2, 3, 4) as idiophasic metabolic products of L-phenylalanine. These diols are stereoselectively biosynthesized from a C7-unit (benzylic, from L-phenylalanine) and a C2-unit, of unknown origin, as predominantly erythro (1R,2S) enantiomers. Of the labeled amino acids tested as possible C2-units, at the 4-10 mM level, none were found to efficiently label the 2,3-propane carbons of the diols. However, glycine (2-13C), L-serine (2,3,3-d3) and L-methionine (methyl-d3) entered the biomethylation pathway. Neither pyruvate (2,3-13C2), acetate (1,2-13C2), acetaldehyde (d4) nor ethanol (ethyl-d5) labeled the 2,3-propane carbons of the diols at the 4-10 mM level. Pyruvate (2,3-13C2) and L-serine (2,3,3-d3) (which also entered the biomethylation pathway) did, however, effectively label the 2,3-propane carbons of the alpha-ketols and diols at the 40 mM level as evidenced by mass spectrometry. Glycerol (1,1,2,3,3-d5) also appeared to label one of the 2,3-propane carbons (ca. 5% as 2H2 in the C3 side chain) as suggested by mass spectrometric data and also entered the biomethylation pathway, likely via amino acid synthesis. Glycerol (through pyruvate), therefore, likely supplies C2 and C3 of the propane side chain with arylpropane diol biosynthesis. Incubation of B. adusta with synthetic [2-2H1, 2-18O]-glycerol showed that neither 2H nor 18O were incorporated in the alpha-ketols or diols. The oxygen atom on the C2 of the ketols/diols, therefore, does not appear to come from the oxygen atom on the C2 of glycerol. Glycerol, however, can readily form L-serine (which can then form pyruvate via PLP/serine dehydratase and involve transamination washing out the 18O label and providing the oxygen from water), and can then go on to label the C2-unit. Labeled alpha-ketol, phenyl acetyl carbinol (5) (PAC; ring-d(5), 2,3-13C2 propane) cultured with B. adusta leads to stereospecific reduction to the (1R,2S)-diol (6) (ring-d5 and 2,3-13C2); in all other metabolites produced, the 2,3-13C2) label is washed out. Incubation of the fungus with 4-fluorobenzaldehyde (13) produces a pooling of predominantly erythro (1R,2S) 1-(4'-fluorophenyl)-1,2-propane diol (18 as diacetate) (through the corresponding alpha-ketols 16, 17). Blocking the para-position with fluorine thus appears to prevent ring oxygenation and also chlorination, forcing the conclusion that para-ring oxygenation precedes meta-chlorination.     

Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol

1,3-Propanediol and 2,3-butanediol are two promising chemicals which have a wide range of applications and can be biologically produced. The separation of these diols from fermentation broth makes more than 50% of the total costs in their microbial production. This review summarizes the present state of methods studied for the recovery and purification of biologically produced diols, with particular emphasis on 1,3-propoanediol. Previous studies on the separation of 1,3-propanediol primarily include evaporation, distillation, membrane filtration, pervaporation, ion exchange chromatography, liquid–liquid extraction, and reactive extraction. Main methods for the recovery of 2,3-butanediol include steam stripping, pervaporation, and solvent extraction. No single method has proved to be simple and efficient, and improvements are especially needed with regard to yield, purity, and energy consumption. Perspectives for an improved downstream processing of biologically produced diols, especially 1,3-propanediol are discussed based on our own experience and recent work. It is argued that separation technologies such as aqueous two-phase extraction with short chain alcohols, pervaporation, reverse osmosis, and in situ extractive or pervaporative fermentations deserve more attention in the future.     

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ _max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l⁻¹). For glycerol at 20 g l⁻¹, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l⁻¹. However, for an initial 1,3-propanediol concentration of 50 g l⁻¹ and glycerol at 70 g l⁻¹, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l⁻¹(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l⁻¹). The addition of 1,2-propanediol or 2,3-butanediol (50 g l⁻¹) in the presence of glycerol (50–100 g l⁻¹), showed that 2-diols reduced the μ _max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered. Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000     

The pH effects on the distribution of 1,3-propanediol and 2,3-butanediol produced simultaneously by using an isolated indigenous Klebsiella sp. Ana-WS5

Several carbon sources were investigated for the production of 1,3-propanediol (PDO) and 2,3-butanediol (BDO) simultaneously, using an isolated indigenous Klebsiella sp. Ana-WS5. The results indicate that glycerol is a suitable carbon source for both BDO and PDO production. Further investigation suggests that adjustment of the pH could alter the metabolic pathway, which affects the ratio of PDO and BDO obtained. The batch with pH controlled at 7.0 had the highest total diol (PDO + BDO) productivity of 0.86 g/L h and the highest PDO/BDO of 7.67, as compared to a batch with pH controlled at 6.0. However, the batch without pH control could achieve a maximum total diol concentration of 48.1 ± 1.6 g/L and the highest yield of 86 % (total diols produced/glycerol consumed). The effects of pH control on the distribution of PDO and BDO concluded in this study could be further applied to the process design for enhancing PDO or BDO production.     

Identification and Characterization of Coenzyme B12-Dependent Glycerol Dehydratase- and Diol Dehydratase-Encoding Genes from Metagenomic DNA Libraries Derived from Enrichment Cultures

To isolate genes encoding coenzyme B₁₂-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.     

Production of C2–C4 diols from renewable bioresources: new metabolic pathways and metabolic engineering strategies

C2–C4 diols classically derived from fossil resource are very important bulk chemicals which have been used in a wide range of areas, including solvents, fuels, polymers, cosmetics, and pharmaceuticals. Production of C2–C4 diols from renewable resources has received significant interest in consideration of the reducing fossil resource and the increasing environmental issues. While bioproduction of certain diols like 1,3-propanediol has been commercialized in recent years, biosynthesis of many other important C2–C4 diol isomers is highly challenging due to the lack of natural synthesis pathways. Recent advances in synthetic biology have enabled the de novo design of completely new pathways to non-natural molecules from renewable feedstocks. In this study, we review recent advances in bioproduction of C2–C4 diols, focusing on new metabolic pathways and metabolic engineering strategies being developed. We also discuss the challenges and future trends toward the development of economically competitive processes for bio-based diol production.     

On the analysis of long-chain alkane diols and glycerol ehters in biochemical studies

The chromatographic behavior of 1,2-, 1,3-, 1,4-, and 1,12-long-chain alkane diols and 1-O-alkylglycerols and their derivatives has been compared. Thin-layer chromatography on Silica Gel G gives poor separations of the 1,2-, 1,3-, and 1,4-alkane diols, O-alkylglycerols, and some of their isopropylidene derivatives. However, gas-liquid chromatography on 10% EGSS-X (coated on 100-120 mesh Gas-Chrom P) resolves the isopropylidenes of the alkane diols and O-alkylglycerols. We also document the formation of 1,3-alkane diols (after LiAlH(4) reduction) from 1-(14)C-labeled fatty acids incubated with mitochondrial fractions from heart and liver of rats. The labeled 1,3-alkane diol was identified by gas-liquid chromatography of its isopropylidene derivative and by its behavior after periodate oxidation. These results serve to caution investigators in the glycerol ether field against incorrect interpretation of data obtained on the incorporation of labeled fatty acids into alkyl ether bonds of glycerolipids. The methodology described points out a technique for distinguishing several types of alkane diols from O-alkylglycerols.     

1,3-Propanediol production by Klebsiella pneumoniae under different aeration strategies

1,3-Propanediol production by Klebsiella pneumoniae was studied in batch cultures under N2 flow and four airflow systems. Different byproducts were formed under different aeration conditions. An anaerobic/aerobic combined fed-batch culture was developed giving 70 g 1,3-propanediol l(-1) and 16 g 2,3-butanediol l(-1) with total diol yield of 0.6 mol(-1) glycerol.     

An approach based on liquid chromatography/electrospray ionization-mass spectrometry to detect diol metabolites as biomarkers of exposure to styrene and 1,3-butadiene

Styrene and 1,3-butadiene are important intermediates used extensively in the plastics industry. They are metabolized mainly through cytochrome P450-mediated oxidation to the corresponding epoxides, which are subsequently converted to diols by epoxide hydrolase or through spontaneous hydration. The resulting styrene glycol and 3-butene-1,2-diol have been suggested as biomarkers of exposure to styrene and 1,3-butadiene, respectively. Unfortunately, poor ionization of the diols within electrospray mass spectrometers becomes an obstacle to the detection of the two diols by liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS). We developed an LC/ESI-MS approach to analyze styrene glycol and 3-butene-1,2-diol by means of derivatization with 2-bromopyridine-5-boronic acid (BPBA), which not only dramatically increases the sensitivity of diol detection but also facilitates the identification of the diols. The analytical approach developed was simple, quick, and convincing without the need for complicated chemical derivatization. To evaluate the feasibility of BPBA as a derivatizing reagent of diols, we investigated the impact of diol configuration on the affinity of a selection of diols to BPBA using the established LC/ESI-MS approach. We found that both cis and trans diols can be derivatized by BPBA. In conclusion, BPBA may be used as a general derivatizing reagent for the detection of vicinal diols by LC/MS.