培养基组分对枯草芽胞杆菌产表面活性素的影响

表面活性素(surfactin)中的C15组分是表面活性素Surfactin异构体混合物中的重要活性组分,在表面活性、溶血活性、抗病毒活性和抑菌活性方面都有突出的表现。考察培养基p H、碳源、氮源、碳氮比、金属离子以及氨基酸等主要参数对枯草芽胞杆菌发酵产物surfactin中C15组分的影响,利用优化后的培养基(葡萄糖20 g/L、NH4NO32 g/L、K2HPO43 g/L、Na H2PO410 g/L、Mg SO40.02 g/L、Leu 1 g/L和p H 8.5),枯草芽胞杆菌BS-37发酵24 h,surfactin总产量达750 mg/L,其中C15组分达510 mg/L,占surfactin总量的68%。     

大肠杆菌全细胞转化联产L-2-氨基丁酸和D-葡萄糖酸

以大肠杆菌BL21(DE3)为表达宿主,构建两株分别表达L-苏氨酸脱氨酶(LTD,基因来源大肠杆菌)和共表达亮氨酸脱氢酶(LDH,来源蜡样芽孢杆菌)/葡萄糖脱氢酶(GDH,来源枯草芽孢杆菌)的重组大肠杆菌,在此基础上,构建了一种以L-苏氨酸和D-葡萄糖为底物联产L-2-氨基丁酸(L-ABA)和D-葡萄糖酸的全细胞转化系统。通过转化条件(温度、p H、细胞通透性和菌体量)优化,并采用分批补料策略,164 g/L L-苏氨酸和248 g/L D-葡萄糖最终转化得到141.6 g/L的L-ABA和269.4 g/L的D-葡萄糖酸,时空得率分别达到7.1 g/(L?h)和13.5 g/(L?h),得率超过99%。本研究使用价格低廉的大宗化学品高效率生产出有较高附加值的产物,全细胞转化系统无需额外添加昂贵的辅酶,更适用于工业化生产。     

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。     

聚γ-谷氨酸高产菌的选育与培养基优化

利用合成培养基为筛选培养基,以枯草芽孢杆菌(Bacillus subtilis)B6-1为出发菌株,经过三轮紫外线诱变和一轮硫酸二乙酯诱变得到了聚γ-谷氨酸高产突变株枯草芽孢杆菌W003,摇瓶液体发酵的聚γ-谷氨酸产量由出发菌株的10.9 g/L提高到20.5 g/L.单因素实验结果表明,该菌产聚γ-谷氨酸的合适碳源为葡萄糖,氮源为硫酸铵.通过正交实验得到了优化的培养基配方,经36h液体发酵,聚γ-谷氨酸产量可达到45.3 g/L.     

枯草芽孢杆菌fmb60菌株非核糖体肽类化合物对铜绿微囊藻生长的抑制作用

铜绿微囊藻是一种分布广泛的水华微藻,可对人类健康和生态环境造成严重危害,而枯草芽孢杆菌作为一种生防微生物可通过非核糖体肽合成酶合成多种生物活性物质。因此,枯草芽孢杆菌fmb60非核糖体肽(Non-ribosomal peptide,NRP)类产物对铜绿微囊藻生长抑制作用的研究在水华治理方面具有重要的意义。利用基因组挖掘技术从枯草芽孢杆菌fmb60中分离鉴定出bacillibactin、表面活性素(Surfactin)和芬芥素(Fengycin)3种NRP类产物,进而通过添加不同浓度的NRP类产物研究其对铜绿微囊藻生长的影响,结果显示其对铜绿微囊藻4 d的半效应浓度值EC50.4 d为26.5 mg/L,且随着样品浓度的增加,枯草芽孢杆菌fmb60的NRP类产物对铜绿微囊藻的抑制作用增强。当向其分别加入50 mg/L的样品时,培养4 d的铜绿微囊藻Fv/Fm、Fv/Fo、Yield参数分别比对照组降低了2.8%、1.7%、2.0%。表明枯草芽孢杆菌fmb60 NRP类产物能够显著抑制铜绿微囊藻的光合作用强度及代谢合成,从而为枯草芽孢杆菌抑藻剂的开发奠定理论基础。     

谷氨酸棒杆菌一步法发酵糖质原料生产γ-聚谷氨酸

γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用,目前主要的生产菌株是谷氨酸依赖型菌株,在生产过程中需要添加谷氨酸作为前体,因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先,从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgs BCA,在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达,结果显示,仅诱导型表达菌株可以积累γ-聚谷氨酸,产量为1.43 g/L。进一步对诱导条件进行优化,确定诱导时间为2 h,IPTG浓度为0.8 mmol/L,γ-聚谷氨酸产量为1.98g/L。在此基础上,在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgs BCA,对重组菌进行发酵,结果表明,在摇瓶发酵中γ-聚谷氨酸产量达到10.23g/L,在5L发酵罐中产量达到20.08g/L;继而对γ-聚谷氨酸进行分子量测定,结果显示,产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径,同时为开发其潜在应用奠定了基础。     

暹罗芽孢杆菌高产γ-聚谷氨酸的发酵条件优化

【背景】γ-聚谷氨酸（poly-γ-glutamic acid,γ-PGA）产生菌多为枯草芽孢杆菌（Bacillus subtilis）、解淀粉芽孢杆菌（Bacillus amyloliquefaciens）、地衣芽孢杆菌（Bacillus licheniformis）等,而暹罗芽孢杆菌（Bacillus siamensis）相关研究较少。【目的】研究暹罗芽孢杆菌产γ-PGA的液体发酵条件。【方法】以自行分离的暹罗芽孢杆菌CAU83为出发菌株进行液体发酵,通过单因素试验和正交试验法研究了碳氮源、前体物质、发酵温度及pH对菌株生产γ-PGA的影响。【结果】经摇瓶优化,γ-PGA的最适碳源、氮源和前体物质分别为乳糖30g/L、酵母提取物5g/L和L-谷氨酸钠60 g/L,最适培养条件为发酵温度37℃和pH 7.0,γ-PGA产量由8.4 g/L提升至30.1 g/L,比优化前提高了260%。经分批补料发酵,60 h时γ-PGA产量最高为59.5 g/L,比摇瓶提高了98%,产率为0.99 g/（L·h）。所产γ-PGA分子量为3.8×10⁶ Da,聚合度较高。【结论】...     

一株具有噬菌体抗性的芽孢杆菌BS-2的鉴定及葡萄糖流加工艺优化

筛选噬菌体抗性菌株以解决枯草芽孢杆菌(Bacillus subtilis)生产过程中噬菌体污染问题并提高抗性菌株的发酵水平。采用双层平板法从异常发酵液中分离并纯化以枯草芽孢杆菌为宿主的噬菌体,利用纯化得到的噬菌体筛选实验室芽孢杆菌库以得到抗性菌株,结合16S rDNA和gyrA基因序列进行系统发育分析确定其分类地位,以分批发酵的生长曲线及葡萄糖含量曲线作为基础,对发酵培养基的初糖浓度及葡萄糖的流加方式进行优化提高筛选到的噬菌体抗性菌株的发酵水平。从异常发酵液中分离到噬菌体S01及S02并在实验室芽孢杆菌库中筛选到一株噬菌体无法侵染的芽孢杆菌BS-2,结合16S rDNA及gyrA基因序列的系统发育分析将BS-2鉴定为枯草芽孢杆菌,按照葡萄糖初始浓度15 g/L,葡萄糖浓度低于5 g/L时以2 g/L·h的速度流加葡萄糖,补加量10 g/L的流加补料方法,可以将发酵水平提高至2.43×1010 CFU/mL。枯草芽孢杆菌BS-2具有较好的噬菌体抗性且经过工艺优化可以达到较高发酵水平,有较强工业化应用潜力。     

重组枯草芽孢杆菌发酵廉价原料生产普鲁兰酶

[目的]构建长野芽孢杆菌普鲁兰酶突变体枯草芽孢杆菌工程菌株,优化发酵条件,筛选廉价的培养基原料生产普鲁兰酶。[方法]利用分子生物学手段,将基因pul324和表达载体p WB980连接,构建表达质粒p WB-pul324并转化Bacillus subtilis WB600;对表达产物进行SDS-PAGE分析和初始酶活的测定。进一步优化发酵条件,对不同碳源和氮源进行发酵培养基的筛选,同时研究不同金属离子的添加和培养基初始p H、接种量对发酵产酶的影响。[结果]获得基因工程菌B.subtilis WB600/p WB-pul324,SDS-PAGE电泳结果显示在89 k Da处有特异性条带,发酵初始酶活为12.34 U/ml;筛选得到玉米淀粉水解液和玉米浆干粉为培养基最适碳源和氮源,其最适浓度分别为50 g/L和30 g/L。Mn~(2+)、Fe~(3+)、Fe~(2+)和Tween-80的添加能提高发酵产酶活力。在最适初始p H 6.5,以最适5%接种量接种于优化后的培养基中,摇瓶发酵80 h普鲁兰酶的酶活达到414.48 U/ml。[结论]实现了普鲁兰酶突变体在枯草芽孢杆菌中的高效表达,筛选获得的培养基主要原料经济低廉,经过发酵条件优化后,重组菌酶活达到414.48 U/ml,是之前研究结果(20.16U/ml)的20倍。     

大肠杆菌产L-苯丙氨酸发酵调控及代谢通量分析

考察了外源添加中间代谢产物、维生素B1和硫酸镁对大肠杆菌发酵产L-苯丙氨酸的影响,结果表明,添加1g/L柠檬酸三钠、1g/Lα-酮戊二酸、150mg/L维生素B1及3g/L硫酸镁均对L-苯丙氨酸的合成有利。根据构建的大肠杆菌合成L-苯丙氨酸的生化反应网络,利用代谢通量分析其原因。结果表明,这些物质的添加均可以调节G6P和PEP节点处的代谢通量分布,为L-苯丙氨酸的合成提供更多的前体物质赤藓糖四磷酸(E4P)、磷酸烯醇式丙酮酸(PEP)和还原力NADPH。通过补料分批发酵实验得出,优化后菌体对总葡萄糖的消耗提高了24.49%,菌体终浓度提高了23.50%,L-苯丙氨酸的终浓度提高了62.87%,L-苯丙氨酸的收率提高了30.88%,乙酸的合成降低了56.51%。     

脂肽-糖脂混合生物表面活性剂产生菌筛选和优化培养

结合脂肽和糖脂的性能优势,致力于产脂肽-鼠李糖脂混合型生物表面活性剂的新菌株选育和培养条件优化。采用血平板溶血圈法初筛菌株、改进排油圈法快速检测产量以及飞行时间质谱鉴定产物结构。对优选菌株的碳源、氮源和磷酸盐缓冲液、重要金属离子浓度等进行了单因子和正交试验,优化了培养基和培养条件。采用高压液相色谱和蒽酮比色法定量分析了产物组成。筛选获得了同时积累糖脂和脂肽的新菌株,鉴定命名为芽胞杆菌Bacillus subtilis THY-7。摇瓶分批培养48 h,细胞OD600为37.0,产物浓度2.4 g/L,分别是优化前的3.4倍和3.1倍。发酵罐补料分批培养,泡沫中产物浓度达到4.5 g/L,且74％为表面活性素,22％为鼠李糖脂。B. subtilis THY-7是具有脂肽-鼠李糖脂高产潜力的优选菌株。     

大肠杆菌角鲨烯合成途径的构建与调控

角鲨烯因其具有良好的抗氧化功能而被广泛应用于食品、医药、化妆品、工业应用等领域。本实验在大肠杆菌中构建角鲨烯合成途径,通过对其合成途径中关键限速酶(1-脱氧-D-木酮糖-5-磷酸合酶和异戊烯基二磷酸异构酶)过表达的方法进行初步调控,使角鲨烯的产量提升了近三倍。之后采用单因素试验对其发酵培养基和培养条件进行优化,以此来提高角鲨烯的产量。优化发酵条件后,使用最优发酵培养基——TB培养基,在最佳发酵条件:37℃,220r/min培养至OD600约为1.2时加入终浓度为0.1mmol/L的IPTG诱导剂,25℃条件下诱导48h,角鲨烯产量可达73.88mg/L。     

Optimizing iron supplement strategies for enhanced surfactin production with Bacillus subtilis

Supplement of Fe(2+) into fermentation medium was utilized as a tool to optimize the iron-mediated enhancement of surfactin production from Bacillus subtilis ATCC 21332. Up to 3000 mg L(-)(1) of surfactin was produced using an iron-enriched minimal salt (MS) medium amended with an optimal Fe(2+) dosage of 4.0 mM, leading to 8-fold and 10-fold increase in cell concentration and surfactin yield, respectively, as compared to those without Fe(2+) supplement. In addition to resulting in an optimal production yield of surfactin, a supplement of 4.0 mM of Fe(2+) also propelled maximum overall surfactin production rate to a highest value of 24 mg L(-)(1) h(-)(1). Our results also show that production of surfactin followed a growth-associated kinetic model. The best yield coefficient estimated from the model was ca. 162 mg surfactin (g dry cell)(-)(1). The supernatant of the iron-enriched culture of B. subtilis ATCC 21332 exhibited the ability to emulsify kerosene and achieved a maximum emulsion index (E(24)) of 80% for culture supplemented with 4.0 mM of Fe(2+). Comparison of emulsion index and the corresponding surfactin production indicates that the emulsification activity was essentially contributed by surfactin.     

用重组大肠杆菌JM109（pKST11）转化苯生产顺-1,2-二羟基-3，5-环己二烯

顺-1,2-二羟基-3,5-环己二烯（简称DHCD）是航天业,电子工业,医药业以及精细化工业上重要的手性化合物,利用重组E.coli JM109（pKST11）,采用适时监测发酵过程中全细胞甲苯双加氧酶（Toluene dioxygenase,TDO）活性的方法,研究了发酵生产DHCD工艺中的重要影响因子IPTG以及底物苯的供给方式对DHCD产量的影响,研究结果表明：（1）发酵初期利用IPTG诱导TDO的表达,不利于细胞生长,在对数生长中期（6或8h）,采用0.5mmol/L IPTG即可诱导出TDO的最高表达。（2）发酵液中的苯对全细胞甲苯双加氧酶（TDO）的活性有抑制作用,而利用液体石蜡作为缓释剂进行两相法发酵则降低了苯的毒害,明显提高了DHCD的产量。当采用传统的通气供苯方法,DHCD的产量仅有7.5g/L;批式添加液体石蜡与苯的混溶物使DHCD的产量提高到22.6g/L,是通气供苯法的3倍;而采用流加的方式添加液体石蜡与苯的混溶物使DHCD的产量进一步提高到36.8g/L,是通气供苯法的5倍,证明通过发酵工艺的优化可以解决苯的毒害与苯作为反应底物在水相中需要一定浓度之间的矛盾,获得较好的转化结果。     

Enhanced production of surfactin from Bacillus subtilis by addition of solid carriers

Addition of a small quantity of solid porous carriers (e.g., activated carbon or expanded clay) into fermentation broth significantly increased surfactin production with Bacillus subtilis ATCC 21332. Culture medium containing 25 g L(-1) of activated carbon gave an optimal surfactin yield of 3600 mg L(-1), which was approximately 36-fold higher than that obtained from carrier-free liquid culture. The marked increase in surfactin production was primarily attributed to stimulation of cell growth due to the presence of activated carbon carriers. Concentration of limiting carbon substrate (glucose) is also an important factor affecting the production of surfactin, as an initial glucose concentration of 40 g L(-1) resulted in optimal surfactin production. An appropriate agitation rate also benefited surfactin production, as the best yield appeared at an agitation rate of 200 rpm. Surfactin was purified from fermentation broth via a series of acidic precipitation and solvent extraction. The resulting product was nearly 90% pure with a recovery efficiency of ca. 72%. The purified surfactin reduced the surface tension of water from 72 to 27 mN m(-1) with a critical micelle concentration of ca. 10 mg L(-1). The surfactin product also attained an emulsion index of 70% for kerosene and diesel at a low concentration of 100 and 600 mg L(-1), respectively.     

累积番茄红素的大肠杆菌工程菌及其培养条件的研究

噬夏孢欧文氏菌番茄红素合成相关基因crtE, crtB, crtI同时克隆进表达载体pET-15b构建pET-15bcrtIEB,将该重组质粒转化E.coliBL21(DE3)构建工程菌,IPTG诱导工程菌累积红色色素,经HPLC和吸收光谱分析,工程菌中合成的色素为番茄红素。研究了碳源、金属离子、培养温度、诱导剂浓度、诱导时间等参数对工程菌生长及色素累积的影响,确定了合适的培养条件：培养基为改良LB培养基（蛋白胨10g/L、酵母提取物5g/L、麦芽糖5g/L、MgCl2 0.1g/L,NaCl 10g/L）;起始培养温度为37℃;培养至OD600为0.6左右时加入IPTG,终浓度为0.5mmol/L,诱导温度降至30℃;诱导时间为14h。发酵完成后工程菌的生物量（干重）为3.45g/L,番茄红素的最高含量可达5.8mg/gDW。     

几种添加物对D—核糖产量的影响

研究了几种物质对D－核糖产量的影响,研究表明,当发酵培养基中玉米浆的含量为25g/L,D－核糖产量达到最高,为65g/L。发酵培养基中添加适量的粉及山梨醇亦有利于D－核糖的积累,当粉与山梨醇的添加量分别为10g/L及40g/L时,D－核糖产率分别增加7.8%、4.7%,而在发酵培养基中加入丙二酸可抑制D－核的分泌,在40ml发酵培养基中添加1ml丙二酸后,D－核糖的产率下降73.8％。甲醇也可抑制D－核糖的积累,当发酵培养基中的甲醇添加量为16g/L时,D－核糖产率下降66.2%。     

Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.