一株芽孢杆菌在维生素C二步发酵中对小菌的促进作用

从土壤中分离到1株能更好促使小菌生长和产酸的芽孢杆菌B601,作为伴生菌与巨大芽孢杆菌相比,在生长过程中,发酵液中B601活菌数小于巨大芽孢杆菌,而其芽孢数则多于巨大芽孢杆菌。对B601组成菌系的发酵条件进行优化,得到如下结果：100g／L L-山梨糖、6g／L尿素、10g/L玉米浆、培养温度30℃和发酵周期44h。与巨大芽孢杆菌组成菌系相比其底物,L-山梨糖质量浓度提高了25％,尿素下降了50％．玉米浆质量浓度下降了33％,温度提高了2℃,发酵周期缩短了4h。结果表明：B601作为伴生菌,与巨大芽孢杆菌相比,该菌株明显提高了发酵效率。     

酮古龙酸菌与呼吸链偶联的2-KGA代谢途径研究进展

酮古龙酸菌可将底物L-山梨糖转化为维生素C的前体2-酮基-L-古龙酸(2-KGA)。该菌共存在5种反应参与2-KGA代谢,包括:1D-山梨醇氧化为L-山梨糖;2L-山梨糖氧化为L-山梨酮;3L-山梨酮(吡喃型)氧化为2-KGA;4L-山梨酮(呋喃型)氧化为维生素C。52-KGA还原为L-艾杜糖酸。其中L-山梨糖/L-山梨酮脱氢酶(SSDH)参与反应123,L-山梨糖脱氢酶(SDH)参与反应23,L-山梨酮脱氢酶(SNDH)参与反应34,醛脱氢酶(ALDH)参与反应3,2-KGA还原酶(2-KGR)参与反应5。SDH/SSDH/ALDH属于Ⅰ型醌酶,其辅酶为1分子PQQ;SNDH属Ⅱ型醌酶,与PQQ、heme C共同构成quinohemoproteins,2种醌酶均分布于周质空间中与呼吸链相偶联,意味着这种膜上直接氧化过程伴随ATP产生,使得菌体可以利用环境中的底物实现快速供能。     

氧化葡萄糖酸杆菌中硫辛酸合成模块对维生素C一步混菌发酵的影响

在由氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌构建的维生素C两菌一步发酵体系中,为了强化氧化葡萄糖酸杆菌对普通生酮古龙酸杆菌生长和产酸的促进作用,文中在氧化葡萄糖酸杆菌中构建硫辛酸合成功能模块。由含硫辛酸功能模块的氧化葡萄糖酸杆菌和普通生酮古龙酸杆菌组成的两菌一步体系,能减轻普通生酮古龙酸杆菌单菌培养时的生长抑制,强化两菌的互作关系,使维生素C前体(2-酮基-L-古龙酸,2-KGA)的产量提高到73.34 g/L(对照组为59.09 g/L),醇酸转化率提高到86.0%。研究结果为进一步优化维生素C两菌一步发酵体系提供了新思路。     

产脂肪酶重组枯草芽胞杆菌的发酵优化

笔者所在实验室前期筛选到1株产脂肪酶粘质沙雷氏菌,克隆其脂肪酶基因,构建重组枯草芽胞杆菌Bacillus subtilis 168/pMA5-lipA,成功实现了来源于粘质沙雷氏菌的脂肪酶基因在枯草芽胞杆菌中的表达。基于以上工作基础上,对B.subtilis 168/pMA5-lipA进行了摇瓶水平上的产酶发酵优化。首先通过单因素和正交试验确定了有利于产脂肪酶的最佳培养基成分,并对发酵条件进行了优化。结果表明:优化后的培养基组分为蔗糖35 g/L,玉米浆27.5 g/L,(NH4)2SO41.25 g/L,CaCl24 g/L,pH 7.0。在最优发酵培养基的条件下,37℃、160 r/min摇床培养33 h,每毫升发酵液中重组菌脂肪酶酶活可达98.6 U,是优化前的3倍。     

混合菌发酵L-山梨糖生产Vc前体2-酮基-L-古龙酸研究进展

利用混合菌发酵L-山梨糖生产2-酮基-L-古龙酸(2-KCA),再经化学转化合成维生素C(Vc),是我国工业生产Vc的主要途径,具有简化工艺,减少污染,降低能耗等诸多优点.从菌系组合、菌种选育、代谢途径与酶学特性、工程菌构建、伴生作用机制及发酵工艺等方面出发,综述混合菌发酵L-山梨糖生产Vc前体2-KGA的研究现状和最新进展,并提出进一步研究和探索的方向.     

高渗条件下利用蔗糖提升2-酮基-L-古龙酸生产效率

旨在进一步提升维生素C前体2-酮基-L-古龙酸(2-KLG)的生产效率。在详细考察了2-KLG工业化生产过程中渗透压变化规律的基础上,研究了高渗对混合菌系细胞生长和2-KLG合成的影响,提出蔗糖促进伴生菌巨大芽胞杆菌Bacillus megaterium生长,进而促进普通生酮古龙酸菌Ketogulonigenium vulgare生长和产酸的策略。结果表明,2-KLG的积累和碱性物质的流加使渗透压上升了832mOsmol/kg;高渗抑制了巨大芽胞杆菌的生长(15.4%),从而抑制普通生酮古龙酸菌(31.7%)的生长,导致2-KLG产量和生产强度分别下降67.5%和69.3%(以1250mOsmol/kg为例);蔗糖的添加则显著促进巨大芽胞杆菌的生长,使高渗条件下(摇瓶,1250 mOsmol/kg)2-KLG产量(40.6g/L)提高87%;在3L发酵罐中,补加10mmol/L蔗糖使2-KLG发酵周期缩短10.8%,2-KLG生产强度提高10.4%。研究成果为在环境胁迫下提高混菌生产目标代谢产物的产量提供了潜在的策略。     

L-山梨糖流加发酵高效生产2-酮基-L-古龙酸

实验充分利用混合菌系氧化葡萄糖酸杆菌(Gluconobacter oxydans)和蜡状芽孢杆菌(Bacillus cereus)混合发酵的优良特性,通过在发酵过程中间歇流加L-山梨糖的方法,实现了在自动控制温度、pH和溶氧的条件下,高效发酵L-山梨糖生成2-酮基-L-古龙酸(2-KLG)的目的。结果表明：当将L-山梨糖的终浓度调高到14％(w／v)时,2-KLG产量为130mg／mL左右,转化率达90％,发酵周期40—60h之间。结论：发酵过程中间歇流加L-山梨糖可以解除高浓度糖对产酸的抑制作用,提高了糖的转化率,但是发酵周期略有延长。     

分阶段pH调控提高2-酮基-L-古龙酸生产

为了提高酮古龙酸菌Ketogulonicigenium vulgare和巨大芽胞杆菌Bacillus megaterium生产2-酮基-L-古龙酸(2-KLG)的生产效率,分析了pH对K.vulgare和B.megaterium生长和产酸的影响,发现K.vulgare和B.megaterium的最适生长pH值分别为6.0和8.0,但是K.vulgare的糖酸转化活力在pH7.0时达到最大值,因此提出了三阶段pH控制策略(第一阶段:0～8h,pH8.0;第二阶段:8～20h,pH6.0;第三阶段:20h至发酵结束,pH7.0)以促进K.vulgare生长和2-KLG生产。结果表明,三阶段pH控制策略的实施进一步提高了2-KLG的产量(77.3g/L)、生产强度(1.38g/(L·h))和L-山梨糖消耗速率(1.42g/(L·h)),分别比恒定pH7.0时提高了9.7%、33.2%和25.7%。     

山梨酮脱氢酶模块与酮古龙酸杆菌底盘细胞的适配分析

酮古龙酸杆菌Ketogulonigenium vulgare是维生素C二步混菌发酵过程中的产酸菌。山梨酮脱氢酶(L-sorbosone dehydrogenase,缩写为SNDH)作为维生素C直接前体2-酮基-L-古龙酸(2-KGA)合成的关键酶,其作用机制并不十分清楚。借助全基因组测序抽提2个山梨酮脱氢酶基因,分别位于基因组(缩写为sndhg)和质粒(缩写为sndhp)上。通过工程化改造技术在工业产酸菌中构建山梨酮脱氢酶功能模块,比较其对2-KGA产量的影响。研究发现sndhg过表达对菌株产酸影响不明显,sndhp过表达使菌株明显产生副产物。将sndhg和sndhp分别配合辅因子PQQ合成基因pqq A,分别构建sndhg-pqq A和sndhp-pqq A模块,得到的工程菌株产酸情况与之前的结果大致相同。将4株K.vulgare工程菌株分别与内生芽孢杆菌Bacillus endophyticus混合培养传代50 d后,分离菌株进行混菌发酵,其2-KGA的转化率分别提高了15.4%、179%、0.65%和125%。表明混菌适应性进化策略是一种增加功能模块与底盘细胞适配性,进而快速获得优良性状菌种的有效方法。     

谷胱甘肽对VC一步发酵作用的研究

谷胱甘肽（GSH）能有效促进酮古龙酸杆菌的生长。就GSH对氧化葡萄糖酸杆菌和酮古龙酸杆菌一步混菌发酵的作用进行了探索,为进一步阐明维生素C一步发酵过程中氧化葡萄糖酸杆菌和酮古龙酸杆菌的关系并提供发酵工艺优化的依据。研究发现,在5L的发酵罐中,外加1mg/ml的GSH对混菌的发酵有着显著的促进作用,2-酮-L-古龙酸（2-KGA）产量提高了22.8%。通过16S rDNA荧光定量PCR法测菌数,发现GSH的添加使酮古龙酸杆菌的生长提高到148%,但抑制氧化葡萄糖酸杆菌的生长,使其生物量下降到61%。运用代谢组学方法分析发现,GSH能促进酮古龙酸杆菌的磷酸戊糖、三羧酸循环、硫酸盐等代谢,同时减缓氧化葡萄糖酸杆菌对L-山梨糖的消耗,以促进整个混菌体系的发酵效率。     

玉米浆对Vc二步发酵产2-酮基-L-古龙酸影响的研究

通过在培养基中添加不同量的玉米浆,研究其对氧化葡萄糖酸杆菌（俗称小菌）生产Vc前体2-酮基-L-古龙酸的影响,并研究玉米浆成分中的12种主要氨基酸对小菌产酸的影响。结果表明：每100 mL发酵培养基中添加2.5 g左右过滤除菌玉米浆时,2-酮基-L-古龙酸产量高达26.84 mg/mL,小菌活菌数为不添加玉米浆时小菌单菌发酵下的9.74倍。过量玉米浆抑制小菌产酸。12种氨基酸单独与氧化葡萄糖酸杆菌发酵培养及全部混合后与氧化葡萄糖酸杆菌发酵培养对产酸及菌体生长无影响。     

Integrated proteomic and metabolomic analysis of an artificial microbial community for two-step production of vitamin C

An artificial microbial community consisted of Ketogulonicigenium vulgare and Bacillus megaterium has been used in industry to produce 2-keto-gulonic acid (2-KGA), the precursor of vitamin C. During the mix culture fermentation process, sporulation and cell lysis of B. megaterium can be observed. In order to investigate how these phenomena correlate with 2-KGA production, and to explore how two species interact with each other during the fermentation process, an integrated time-series proteomic and metabolomic analysis was applied to the system. The study quantitatively identified approximate 100 metabolites and 258 proteins. Principal Component Analysis of all the metabolites identified showed that glutamic acid, 5-oxo-proline, L-sorbose, 2-KGA, 2, 6-dipicolinic acid and tyrosine were potential biomarkers to distinguish the different time-series samples. Interestingly, most of these metabolites were closely correlated with the sporulation process of B. megaterium. Together with several sporulation-relevant proteins identified, the results pointed to the possibility that Bacillus sporulation process might be important part of the microbial interaction. After sporulation, cell lysis of B. megaterium was observed in the co-culture system. The proteomic results showed that proteins combating against intracellular reactive oxygen stress (ROS), and proteins involved in pentose phosphate pathway, L-sorbose pathway, tricarboxylic acid cycle and amino acids metabolism were up-regulated when the cell lysis of B. megaterium occurred. The cell lysis might supply purine substrates needed for K. vulgare growth. These discoveries showed B. megaterium provided key elements necessary for K. vulgare to grow better and produce more 2-KGA. The study represents the first attempt to decipher 2-KGA-producing microbial communities using quantitative systems biology analysis.     

OPTIMIZATION OF FERMENTATION MEDIUM FOR ACETOIN PRODUCTION BY Bacillus subtilis SF4-3 USING STATISTICAL METHODS

To improve the acetoin-producing ability of Bacillus subtilis SF4-3, isolated from “natto,” a Japanese traditional food, the fermentation medium was optimized in shake-flask fermentation by statistically designed methods. Based on results of the single-factor experiment, orthogonal experiment, and Plackett–Burman design, yeast extract, corn steep liquor, and urea were identified as showing significant influence on the acetoin production. Subsequently, the optimum combination of the three factors was investigated by the Box–Behnken design (BBD) of response surface methodology (RSM) in order to further enhance the acetoin production. The maximum acetoin yield of 45.4 g/L was predicted when the concentrations of yeast extract, corn steep liquor, and urea were 8.5 g/L, 14.6 g/L, and 3.8 g/L, respectively. The results were further confirmed in triplicate experiments using the optimized medium (glucose 160 g/L, yeast extract 8.5 g/L, corn steep liquor 14.6 g/L, urea 3.8 g/L, manganese sulfate 0.05 g/L, ferrous sulfate 0.05 g/L), and an acetoin yield of 46.2 g/L was obtained in the validation experiment, which was in agreement with the prediction. After the optimization of medium components, an increase of 36.28% in acetoin production was achieved in comparison to that at the initial medium levels.     

5-酮基-D-葡萄糖酸发酵生产的工艺条件优化

葡萄糖酸氧化杆菌可将葡萄糖转化为5-酮基-D-葡萄糖酸(5-KGA),而5-KGA是重要食品添加剂L(+)-酒石酸的合成前体。为提高5-KGA产量及其对葡萄糖的转化率,对5-KGA发酵生产的工艺条件进行优化。在摇瓶水平最适的培养基和培养条件下,5-KGA最高产量为19.7 g/L,较优化前提高43.8%。在5 L发酵罐上控制恒定pH值5.5、溶氧浓度15%条件下,5-KGA产量达到46.0 g/L,较摇瓶最高产量提高1.3倍,应用葡萄糖流加工艺,5-KGA最高产量达到75.5 g/L,转化率超过70%,与已见报道的最高水平相比提高了32.0%,为实现微生物发酵生产5-KGA、进而合成L(+)-酒石酸的工业化提供了切实有效的途径。     

Fumaric Acid Production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> ATCC® 20344™ from Lignocellulosic Syrup

Powdered activated carbon-treated lignocellulosic syrup prepared from energy cane bagasse was evaluated as a potential feedstock in the production of fumaric acid by Rhizopus oryzae ATCC® 20344?. Energy cane bagasse was pretreated with dilute ammonia and enzymatically hydrolyzed with commercially available enzymes, Cellic® CTec2 and HTec2. The collected hydrolysate samples were subjected to powdered activated carbon adsorption for the removal of non-sugar compounds (i.e., organic acids, furaldehydes, total phenolic compounds) and concentrated to a final 65°Bx syrup (mostly xylose and glucose sugars). The use of lignocellulosic syrup, the effect of nitrogen source, medium additives, and initial pH in the seed culture medium on fungal morphology were investigated. The carbon to nitrogen (C/N) ratio in the acid production medium was also optimized for maximum yields in fumaric acid production. Optimum seed culture medium conditions (2.0 g/L urea, 3.0 pH) produced the desired compact, smooth, and uniform fungal pellets. Optimum acid production medium conditions (400 C/N ratio, 0.2 g/L urea) resulted in a fumaric acid production of 34.20 g/L, with a yield of 0.43 g/g and a productivity of 0.24 g/L/h. These results were comparable to those observed with the control medium (pure glucose and xylose). The present study demonstrated that lignocellulosic syrup processed from dilute ammonia pretreated energy cane bagasse has potential as a renewable carbon source for fumaric acid fermentation by Rhizopus oryzae ATCC® 20344?.     

响应面法优化枯草芽孢杆菌产γ-PGA的条件

对枯草芽孢杆菌液体发酵产γ-聚谷氨酸[γ-poly（glutamic acid）,γ-PGA]条件进行了优化。首先采用单因子实验筛选出最适碳源为玉米糖化液,氮源为蛋白胨和谷氨酸钠,无机盐为KH2PO4,MgCl,MnCl2和NaCl。在此基础上,利用Plackett-Burman设计对影响产量的12个因素进行评价,筛选出具有显著效应的因素蛋白胨、谷氨酸钠和NaCl。用最陡爬坡路径逼近最大产γ-PGA区域后,利用响应面中心组合设计对显著因素进行优化,得出蛋白胨、谷氨酸钠和NaCl的最佳质量分数分别为0.54%,8.13%和0.96%。优化后液体发酵液γ-PGA产量提高到29.00 g/L,比初始γ-PGA产量14.10 g/L提高了2倍。     

响应面法优化枯草芽孢杆菌产脂肪酶的合成培养基

对枯草芽孢杆菌(Bacillus subtilis)CICC20034利用合成培养基液体发酵产脂肪酶的条件进行了优化。首先采用单因子实验筛选出最适诱导剂为三丁酸甘油酯,氮源为尿素,碳源为葡萄糖,无机盐为MgSO4。在此基础上,利用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出具有显著效应的三丁酸甘油酯、尿素、KH2PO4和培养基起始pH值4个最显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,获得最适合成培养基组分为:葡萄糖8g/L,尿素8.57g/L,三丁酸甘油酯2.62%,KH2PO42.59g/L,MgSO4.7H2O0.5g/L,TritonX-1000.5g/L,pH9.47。优化后的B.subtilis CICC 20034胞外脂肪酶活力达0.483U/ml,比初始酶活力0.072U/ml提高了6.7倍。