利用嗜盐菌Salinivibrio YS生产2,3-丁二醇和琥珀酸

以从新疆艾丁湖采集的土样中分离出的中度嗜盐菌Salinivibrio YS为研究对象,利用该菌在厌氧条件下生产2,3-丁二醇和琥珀酸,在单因素摇瓶实验基础上,确定影响产物积累的各因素及其相应条件,再利用正交试验确定这些参数的最佳水平,即温度33℃,起始pH为8.0,发酵过程pH为7.0,乙酸添加量为3 g/L,NaCl浓度为l0 g/L.利用优化条件进行3L体系的发酵放大实验,经过108 h的无氧发酵,2,3-丁二醇的产量可达35.05 g/L,而琥珀酸的含量则高达22.46 g/L,且其糖的总转化率高达约50％.首次利用嗜盐菌在厌氧条件下生产2,3-丁二醇和琥珀酸,拓展了嗜盐菌的应用,同时也为生产2,3-丁二醇和琥珀酸提供了新的思路.     

代谢工程改造枯草芽孢杆菌发酵生产乙偶姻

乙偶姻是枯草芽孢杆菌的主要代谢产物,它作为一种食用香精,广泛应用于食品、烟草、化妆品、清洁剂、酒类等行业。本研究首先在不产芽孢的枯草芽孢杆菌(BSD1,阻断了芽孢的合成途径)中敲除了2,3-丁二醇脱氢酶(BDH)的编码基因bdh A、乳酸脱氢酶(LDH)的编码基因ldh和乙酸激酶的编码基因(ACK)ack A,随后克隆了来自菌株B.subtilis168的α-乙酰乳酸合成酶(ALS)和α-乙酰乳酸脱羧酶(ALDC)基因als S和als D,并将其在上述敲除菌中过量表达,结果表明阻断副产物合成途径和加强乙偶姻合成途径关键酶的表达,会显著提高乙偶姻的产量,最终乙偶姻产量达到38.08 g/L,产率为0.45 g·L~(-1)·h~(-1),产率提高了约87.5%。     

加强表达6-磷酸果糖激酶提高Bacillus subtilis乙偶姻合成效率

乙偶姻是枯草芽孢杆菌的主要胞外产物,它是一种广泛使用的食用香精,同时也可作为重要的化学中间体,因此提高枯草芽孢杆菌乙偶姻的产量和生产效率有重要意义。本研究克隆了编码枯草芽孢杆菌6-磷酸果糖激酶(PFKA)和丙酮酸激酶(PK)的编码基因pfk A和py K,并分别将其在高产乙偶姻的枯草芽孢杆菌BM(敲除了2,3-丁二醇脱氢酶的编码基因bdh A)中过量表达。结果表明过量表达PFKA或者PK能有效提高糖酵解速率以及发酵过程的生物量,并且过量表达PFKA与过量表达PK相比更有利于乙偶姻的合成。在5 L发酵罐上对工程菌进行发酵实验发现,经底物流加发酵,乙偶姻产量达到66.8 g/L,生产效率达到0.74 g·(L·h)-1。本研究为利用枯草芽孢杆菌作为细胞工厂生产相关化合物有重要的借鉴意义。     

乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响

【目的】研究乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响。【方法】在谷氨酸棒杆菌CGF2中异源表达als SD操纵子构建乙偶姻生产菌株CGT1,考察敲除乙酸生成途径cat和pqo对乙偶姻的影响。然后引入短乳杆菌的NADH氧化酶,在优化的溶氧条件下研究其对乙偶姻产量的影响。【结果】CGT1在摇瓶发酵中可积累6.27 g/L乙偶姻,敲除cat使乙偶姻产量显著提高30.94%,达到8.21 g/L;双敲除cat和pqo没有进一步提高产量。通过优化发酵的溶氧水平,乙偶姻产量达到10.06 g/L。在高溶氧水平下引入NADH氧化酶导致菌株的生长和糖代谢速率提高,但乙偶姻产量略有降低。在分批补料发酵中,重组菌株乙偶姻产量达到40.51 g/L,产率为0.51 g/(L?h)。【结论】在谷氨酸棒杆菌中阻断乙酸合成途径cat能够有效提高乙偶姻产量,NADH氧化酶在高溶氧水平下表达不利于乙偶姻的合成,需要进一步调节表达水平以确定其效果。     

地衣芽孢杆菌高产2,3-丁二醇菌株的选育和发酵研究

目前2,3-丁二醇生产菌株大部分为致病菌,对人类健康和环境具有一定威胁。从牛奶样品中分离到1株产2,3-丁二醇的芽孢杆菌127-7,分析其16S rRNA基因序列,确定该菌株为地衣芽孢杆菌(Bacillus licheniformis)。进一步对菌株127-7进行紫外诱变,筛选耐受高浓度葡萄糖和高产乙偶姻的菌株。摇瓶发酵结果显示,突变株BL41的2,3-丁二醇产量较出发菌株127-7提高了41.1%。对发酵副产物分析发现,不控制发酵液pH可以显著降低乳酸产量,2,3-丁二醇产量在72 h达到81.4 g/L。进一步调整补糖策略,维持最低残糖浓度为30 g/L,菌株BL41产2,3-丁二醇83.4 g/L,最高产率为1.9 g/L·h,发酵时间缩短至46 h。结果表明,地衣芽胞杆菌BL41可以作为候选菌株,用于工业规模2,3-丁二醇的生产。     

一株耐受高浓度葡萄糖且产乙偶姻菌株的筛选及产物鉴定

为获得一株耐受高浓度葡萄糖且产乙偶姻的菌株,利用高糖培养基以及乙偶姻显色液从烟田土壤中筛选到一株全新菌株;将筛选出的菌株利用全基因组测序技术和16S rDNA序列比对分析对菌株进行鉴定,并利用不同浓度葡萄糖的培养基对菌株进行耐受实验;最后,利用GC-MS(气相-质谱)将菌株的发酵产物定性分析,并对菌株进行72 h的初步发酵。结果显示,筛选获得菌株为金黄色葡萄球菌(Staphylococcus aureus),命名为PX03。葡萄糖耐受实验中,菌株PX03在含有500 g/L葡萄糖的培养基中依然能生长,OD600可达65;300 g/L葡萄糖浓度下生长最好,OD600可达100。GC-MS定性结果分析发现,该菌株可产大量的乙偶姻(22.51%)和2,3-丁二醇(21.29%),同时可产乙酸(13.97%)、3,4-二羟基-3,4-二甲基己烷-2,5-二酮(8.52%)、吡喃酮(5.24%)和3-甲基-丁酸(4.47%)等一些香气成分。对菌株的初步发酵结果显示,该菌株乙偶姻产量可达41 g/L。     

食品上的应用

924601核黄素杂文生产菌的典型代谢产物对培并物生长和维生素生成的影响「俄〕/Akimova,0.L.…厂Biotekhnologiya一1992,i一49~52〔译自DBA,1992,11(11),92一06325〕 利用核黄素杂交生产菌株枯草杆菌24(质粒pMX45)研究了不同的代谢产物(双乙酷、乙偶姻、丁二醇)对微生物生产核黄素的影响。在发酵终了时用气相色谱法检测双乙酞、乙偶姻、2,3一丁二醇、乙酸乳酸和乙酸。这些化合物对生长和核黄素生产的作用相似。双乙酞对核黄素生物合成的毒性阖值为。.029/l,乙偶姻和2,3一丁二醇的相应值分别为2和159/l。当指数生长期开始时往培养物中加…     

枯草芽孢杆菌(Bacillus subtilis)发酵生产乙偶姻的pH调控策略

【目的】为了提高Bacillus subtilis CCTCC M 208157发酵生产乙偶姻的效率。【方法】在7 L发酵罐水平上考察不同pH条件对菌株生长及乙偶姻合成的影响。【结果】pH对菌株合成乙偶姻有显著影响,pH 4.5有利于细胞合成乙偶姻,但是延迟期较长;pH 5.5时菌株生长较快,但乙偶姻的产量偏低。因此提出了两阶段pH控制策略:发酵前期(0 16 h),控制pH 5.5;发酵中后期(16 72 h),控制pH 4.5。【结论】通过此策略,菌株合成乙偶姻的能力得到进一步提高,乙偶姻的产量、产率和生产强度分别为32.7 g/L、0.41 g/g和0.91 g/(L.h),分别比初始发酵条件下提高了41%、42%和69%。     

2,3-丁二醇生产菌株生产能力的比较和培养基优化

对5株克雷伯氏肺炎杆菌 (包括两株乳酸途径被敲除的工程菌株) 发酵生产2,3-丁二醇能力进行了比较,其中K. pneumonia HR521 LDH (乳酸合成途径中ldhA基因被敲除) 具有最佳的发酵性能。通过正交试验优化了其发酵培养基的主要组分,优化后的培养基组成为：葡萄糖 90 g/L,(NH4)2HPO4 3 g/L,玉米浆 (CLSP) 6 g/L,乙酸钠 5 g/L,KCl 0.4 g/L,MgSO4 0.1 g/L,FeSO4·7H2O 0.02 g/L,MnSO4 0.01 g/L。在优化后的发酵培养基中进行摇瓶发酵,24 h发酵乙偶姻和2,3-丁二醇的终浓度为37.46 g/L,比未优化前增加了10 g/L,2,3-丁二醇得率达到了理论得率的90.53％,生产强度1.56 g/(L·h),检测不到副产物乳酸的生成,利于后提取工艺的进行和工业生产的应用。     

乙酸、糠醛和5-羟甲基糠醛对产酸克雷伯氏菌发酵生产2,3-丁二醇的影响

为了解产酸克雷伯氏菌对木质纤维素水解液中主要抑制物的耐受和代谢,考察了产酸克雷伯氏菌发酵生产2,3-丁二醇(2,3-butanediol,2,3-BDO)过程中对3种发酵抑制物乙酸、糠醛和5-羟甲基糠醛(5-hydroxymethylfurfural HMF)的耐受以及抑制物浓度的变化,检测了糠醛和HMF的代谢产物.结果表明:产酸克雷伯氏菌对乙酸、糠醛和HMF的耐受浓度分别为30 g/L、4 g/L和5 g/L.并且部分乙酸可作为生产2,3-丁二醇的底物,在0～30 g/L浓度范围内可提高2,3-丁二醇的产量.发酵过程中产酸克雷伯氏菌可将HMF和糠醛全部转化,其中约70％HMF被转化为2,5-呋喃二甲醇,30％HMF和全部糠醛被菌体代谢.研究表明在木质纤维素水解液生产2,3-丁二醇的脱毒过程中可优先考虑脱除糠醛,一定浓度的乙酸可以不用脱除.     

Bioconversion of inulin to 2,3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase

Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin.     

Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis

The present work aims to block 2,3-butanediol synthesis in acetoin fermentation of Bacillus subtilis. First, we constructed a recombinant strain BS168D by deleting the 2,3-butanediol dehydrogenase gene bdhA of the B. subtilis168, and there was almost no 2,3-butanediol production in 20?g/L of glucose media. The acetoin yield of BS168D reached 6.61?g/L, which was about 1.5 times higher than that of the control B. subtilis168 (4.47?g/L). Then, when the glucose concentration was increased to 100?g/L, the acetoin yield reached 24.6?g/L, but 2.4?g/L of 2,3-butanediol was detected at the end of fermentation. The analysis of 2,3-butanediol chiral structure indicated that the main 2,3-butanediol production of BS168D was meso-2,3-butanediol, and the bdhA gene was only responsible for (2R,3R)-2,3-butanediol synthesis. Therefore, we speculated that there may exit another pathway relating to the meso-2,3-butanediol synthesis in the B. subtilis. In addition, the results of low oxygen condition fermentation showed that deletion of bdhA gene successfully blocked the reversible transformation between acetoin and 2,3-butanediol and eliminated the effect of dissolved oxygen on the transformation.     

Isolation and identification of an acetoin high production bacterium that can reverse transform 2,3-butanediol to acetoin at the decline phase of fermentation

Acetoin (3-hydroxy-2-butanone), a very popular food spice is now used in many industries (pharmaceuticals, chemicals, paint, etc.). In this study, an acetoin high producing strain, numbered as JNA-310, was newly isolated and identified as Bacillus subtilis which is safe on food industry, based on its physiological, biological tests and 16S rDNA sequence analysis. When glucose was used as carbon source in fermentation, the fermentation characterizations of this strain were analyzed, and a new phenomenon of reverse transforming 2,3-butanediol which was synthesized from glucose in the fermentation broth to acetoin was detected. Before 96 h, glucose which was mainly transformed to 2,3-butanediol and acetoin was totally consumed, and the yield of the two products were 41.7 and 21.0 g/l respectively. Acetoin was only a by product in the fermentation broth at prophase of fermentation. At the end of fermentation, the yield of acetoin was greatly improved and the yield of 2,3-butanediol was declined and the yield of them were about 42.2 and 15.8 g/l, respectively. The results indicated that 2,3-butanediol was reversely transformed to acetoin.     

Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.

     

Mutation breeding of acetoin high producing Bacillus subtilis blocked in 2,3-butanediol dehydrogenase

Bacillus subtilis mutants were obtained after the wild strain JNA 3-10 was mutagenized by UV irradiation coupled with diethyl sulfate. A visual filter assay was employed for the qualitative identification of 2,3-butanediol dehydrogenase (BDH) blocked B. subtilis. Selected mutants were tested for the activities of acetoin reductase (AR) and BDH. According to further batch fermentation, one mutant named JNA-UD-6 that produced 24.3 % more acetoin than JNA 3-10 with the corresponding byproducts of 2,3-butanediol decreased by 39.8 % was isolated. A nonsense mutation (p.Tyr118X) that precluded the synthesis of a full-length functional AR/BDH within the bdhA gene of JNA-UD-6 was detected. Acetoin production of JNA-UD-6 was further improved to about 53.9 g/L in a 5-L fermentor with 150 g/L glucose consumed. However,a small amount of 2,3-butanediol was found in late phase of JNA-UD-6 fermentation, and it was due to the existence of a putative gene that encoding a minor AR. This work proved a strategy to efficiently breeding an acetoin high producing strain by traditional mutation methods.     

2，3-丁二醇的发酵及盐析分离工艺

采用克雷伯氏菌（Klebsiella pneumoniae CICC 10011）发酵生产2,3-丁二醇,并对2,3-丁二醇的盐析分离工艺进行了考察。通过实验确定了以葡萄糖为底物微氧批式流加发酵的条件,发酵液中2,3-丁二醇和3-羟基丁酮的质量浓度分别为90．98g/L和12．40g/L,2,3-丁二醇的摩尔转化率为82．7％,生产强度达到2．1g/（L·h）。对发酵液中2,3-丁二醇的盐析分离研究表明,K2HPO4和K3PO4对2,3-丁二醇的盐析效果优于K2CO3。当发酵液浓缩70％后,加入质量分数为45％的K,HPO4,2,3-丁二醇的分配系数达到9．10,回收率为79．37％;上相中2,3-丁二醇的质量浓度达到420g／L;此时3-羟基丁酮的分配系数和回收率分别为11．9和83．48％。     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures.

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of respiratory quotient as a control parameter for optimum oxygen supply and scale-up of 2,3-butanediol production under microaerobic conditions

The respiratory quotient (RQ) was found to be a suitable control parameter for optimum oxygen supply for the production of 2,3-butanediol + acetoin under microaerobic conditions. In laboratory scale continuous cultures optimum production of 2,3-butanediol + acetoin was obtained at an RQ value between 4.0 to 4.5. This agreed well with optimum RQ value (4.0) stoichiometrically derived from the bioreactions involved. In fed-batch cultures product concentrations as high as 102.9 g/L (96.0 g/L butanediol + 6.9 g/L acetoin) can be achieved within 32 h cultivation with an RQ control algorithm for oxygen supply. Under similar conditions only 85.7 g/L product (77.6 g/L butanediol + 8.1 g/L acetoin) was obtained with control of constant oxygen supply rate throughout the cultivation.In pilot scale batch cultures under identical oxygen supply rate the achievable RQ value was found to be strongly influenced by the reactor type and scale. The initial oxygen supply rate influenced the achievable RQ as well. However, in all the reactors studied the specific product formation rate of cells in the exponential growth phase was only a function of RQ. The same optimum RQ value as found in continuous cultures was obtained. It was thus concluded that RQ can be used as a control parameter for optimum production of 2,3-butanediol + acetoin in both laboratory and pilot plant scale reactors. (c) 1994 John Wiley & Sons, Inc.     

Growth and end product formation of two psychrotrophic Lactobacillus spp. and Brochothrix thermosphacta ATCC 11509T at different pH values and temperatures

Lactobacillus viridescens, Lactobacillus sp. strain 173 (homofermentative), and Brochothrix thermosphacta ATCC 11509T were studied at different pH values and temperatures in aerobic and anaerobic batch cultures. The growth rates were higher in aerobic than in anaerobic cultures. L. viridescens grew faster at pH 5.8 than at pH 6.3, whereas the opposite was true for B. thermosphacta. Lactobacillus sp. strain 173 was inhibited in air or at 8 degrees C in anaerobic culture. B. thermosphacta did not grow in anaerobic culture at pH 5.3. The following variations in growth yields were found in the different environments studied: Lactobacillus sp. strain 173, 23 to 25 g (dry weight) per mol of glucose consumed; L. viridescens, 11 to 23 g/mol; B. thermosphacta, 16 to 38 g/mol. In air, L. viridescens produced D-lactic acid, ethanol, and acetic acid, whereas no acetic acid was produced anaerobically. Acetic acid and ethanol together constituted 41 to 48% of the total product yield irrespective of pH and temperature. Lactobacillus sp. strain 173 produced a racemic mixture of D- and L-lactic acid at pH 6.3, whereas the proportion of L-lactic acid was higher than that of D-lactic acid at pH 5.3. In air, product formation of B. thermosphacta varied from a domination of L-lactic acid to increasing yields of acetoin, acetic acid, 2,3-butanediol and isovaleric acid. The effect of pH and temperature on product formation was difficult to separate from the effect of O2 availability in aerobic cultures. However, it was indicated that more 2,3-butanediol and less acetoin were produced with a decreasing temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced 2,3-Butanediol Production by Optimizing Fermentation Conditions and Engineering Klebsiella oxytoca M1 through Overexpression of Acetoin Reductase

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.