群体感应动态调控促进大肠杆菌合成酪醇

酪醇是一种多酚类天然产物,广泛应用于化工、医药和食品等领域。目前大肠杆菌(Escherichia coli)从头合成酪醇存在发酵菌体密度低和产量低等问题。为此,本研究将前期获得苯丙酮酸脱羧酶突变体ARO10F138L/D218G与不同来源的醇脱氢酶融合表达,最优组合ARO10F138L/D218G-L-YahK酪醇产量达到1.09 g/L。为进一步提高酪醇产量,敲除了4-羟基苯乙酸竞争途径关键基因feaB,使酪醇产量提高了21.15%,达到1.26g/L。针对酪醇发酵菌体密度低的问题,通过群体感应系统动态调控酪醇合成途径,减轻酪醇对底盘细胞的毒性作用,缓解生长抑制,使其产量提高了33.82%,达到1.74 g/L。在2 L发酵罐中,群体感应动态调控工程菌TRFQ5的酪醇产量达到4.22g/L,OD600值达到42.88,分别较静态诱导表达工程菌TRF5提高了38.58%和43.62%。本研究应用基因敲除技术,阻断了酪醇合成竞争途径;同时结合群体感应动态调控策略,减轻了酪醇毒性对底盘细胞的生长抑制,从而有效地提高了酪醇产量。本研究对其他高毒性化学品的生物合成具有良好的借鉴和应用价值。     

4-羟基苯乙酸-3-羟化酶A重组表达菌株的构建及其对羟基酪醇的生物转化研究

目的:构建表达4-羟基苯乙酸-3-羟化酶A(4-hydroxyphenylacetate-3-hydroxylase A,HHA)的重组菌株,进而以酪醇为底物,利用重组菌株转化生产羟基酪醇。方法:选择大肠杆菌BL21(DE3)为模板来扩增HHA基因,经酶切后连接到表达载体p ET-28a中,获得重组表达载体p ET28a-HHA,将重组载体转化到感受态细胞BL21(DE3),在重组菌液中加入适量酪醇,利用胞内的重组酶对酪醇加羟基合成羟基酪醇,细胞离心后,分别采用薄层层析法和气质联用法检测上清液中羟基酪醇的转化结果。结果:IPTG诱导表达后,经SDS-PAGE分析获得分子质量分别为58.8k Da和18.5k Da的两条蛋白质条带。薄层层析法和气质联用法均检测到催化产物羟基酪醇的生成。结论:成功构建了表达HHA的重组菌株,该菌株可有效将酪醇转化为羟基酪醇。     

一株红景天内生细菌的筛选及初步研究

【目的】从红景天根部筛选并鉴定一株产酪醇的细菌,初步研究其产酪醇特性,为寻找红景天替代资源提供新途径。【方法】用NA培养基从大花红景天根部中分离内生细菌,通过薄层层析(TLC)、高效液相色谱(HPLC)、气相色谱-质谱联用(GC-MS)筛选出产量最大的菌株,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析其分类学地位。单因素实验确定初始pH、培养温度、发酵时间及接种量对菌株产酪醇活力的影响。【结果】从大花红景天根部分离出14株内生细菌,其中8株能产酪醇,筛选出酪醇产量最大的菌株B3,经菌落形态分析、革兰氏染色分析及16S rRNA基因序列分析初步鉴定为水生拉恩氏菌(Rahnella aquatilis)。研究其发酵条件,其最适pH为6.0,最适温度为32 °C,最佳发酵时间为42 h,最佳接种量为15%。在最适发酵条件下,用改良NA培养基发酵,B3菌株酪醇的产量为15.68 mg/L。【结论】B3菌株是一株具有产酪醇能力的细菌,在最适发酵条件下酪醇产量达到15.68 mg/L,具有潜在的开发价值。     

产D-乳酸重组大肠杆菌ptsG基因的敲除及其混合糖同步发酵

为构建能够同时高效利用五碳糖和六碳糖发酵产D-乳酸的重组大肠杆菌工程菌,以能高效利用五碳糖发酵产D-乳酸的大肠杆菌工程菌E.coli JH13为出发菌株,通过Red同源重组技术敲除葡萄糖跨膜转运基因pts G。实验结果表明,pts G缺陷菌株E.coli JH15在10%混合糖(5%葡萄糖和5%木糖)培养基中发酵,可同时利用五碳糖和六碳糖以完成发酵;而对照菌葡萄糖消耗完才利用木糖,发酵结束还有18 g/L木糖残留;JH15乳酸产量为83.04 g/L,相比于对照菌株提高了25.86%;在稻草秸秆水解液中发酵,JH15同时利用葡萄糖、木糖和L-阿拉伯糖,乳酸产量为25.15 g/L,转化率为86.42%。JH15作为能利用混合糖同步发酵产D-乳酸的大肠杆菌工程菌,它的成功构建为利用廉价的木质纤维素水解物为原料发酵生产D-乳酸提供参考依据。     

大肠杆菌工程菌ptsG基因敲除及其缺陷株混合糖同型乙醇发酵

为实现可同时利用木糖和葡萄糖进行生产发酵,以产乙醇的大肠杆菌工程菌SZ470为出发菌株(△pflB,△frdABCD,△ackA,△ldhA),采用同源重组技术,敲除葡萄糖转运基因ptsG,以构建不受葡萄糖抑制效应影响的菌株SZ470P.SZ470P在5％混合糖(2.5％木糖和2.5％葡萄糖)培养基中能同时利用葡萄糖和木糖进行发酵,葡萄糖消耗量是13 g/L,为对照菌株SZ470的一半;木糖消耗量是20 g/L,是SZ470的3.8倍;乙醇的最高产量为15.01 g/L,转化率为89.13％,比SZ470提高了14.32％.结果表明,工程菌SZ470P可同时利用葡萄糖和木糖发酵生产高产量的乙醇.     

好氧发酵生产琥珀酸工程菌株的构建

通过分析大肠杆菌的碳源代谢途径, 利用基因敲除手段, 以Escherichia coli MG1655为出发菌株, 成功构建了琥珀酸好氧发酵生产工程菌E. coli QZ1111 (MG1655?ptsG?poxB?pta?iclR?sdhA)。检测结果表明该菌株能以葡萄糖为碳源, 在好氧发酵且不表达任何异源基因的条件下大量积累琥珀酸。摇瓶试验证明, 琥珀酸发酵产量达到26.4 g/L, 乙酸盐作为唯一检测到的副产物产量为2.3 g/L。二者浓度比达到11.5:1。     

利用大肠杆菌工程菌廉价高效生产聚羟基丁酸酯

利用大肠杆菌生产聚羟基脂肪酸酯是近来国际上生物可降解塑料的研究热点,本研究通过对适宜于聚羟基脂肪酸酯生产的大肠杆菌菌株的选择和碳源利用试验,初步确立了大肠杆菌代谢工程改造生产聚羟基脂肪酸酯的基础。并在此基础上,通过对大肠杆菌磷酸烯醇式丙酮酸葡萄糖转移酶系统的改造和工程菌环境诱导系统的应用,解决了大肠杆菌工程菌无法同时利用多种碳源合成聚羟基脂肪酸酯的难题。发酵试验证明,工程化改造的大肠杆菌利用廉价底物在5L发酵罐中分批培养32h后,菌体终浓度能够达到8.24g/L,聚羟基脂肪酸酯占细胞干重的84.6%。     

利用代谢工程改善大肠杆菌的3-脱氢莽草酸生产

3-脱氢莽草酸是芳香族氨基酸合成代谢途径中的一种重要中间产物。除可作为一种高效的抗氧化剂,还可用于合成己二酸、香草醛等一些重要的化工产品,具有重要的应用价值。相关研究证明具有去酪氨酸反馈抑制的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶基因aroFFBR以及转酮醇酶基因tktA可以有效影响3-脱氢莽草酸的过量合成。通过增加aroFFBR和tktA串联过量表达的拷贝数,可使工程菌株在摇瓶发酵条件下3-脱氢莽草酸产量提高2.93倍。通过同源重组无痕基因敲除技术依次敲除出发菌大肠杆菌Escherichia coli AB2834的乳酸、乙酸、乙醇等副产物合成途径中的重要基因ldhA、ackA-pta和adhE,可使工程菌株的3-脱氢莽草酸产量进一步提高,达到了1.83 g/L,是初始出发菌株大肠杆菌E.coli AB2834产量的6.7倍。利用5 L发酵罐进行分批补料发酵,62 h后工程菌株3-脱氢莽草酸产量达到了25.48 g/L。本研究可为构建有应用前景的3-脱氢莽草酸生产菌株提供重要参考。     

产肌醇毕赤酵母细胞工厂的优化

【背景】肌醇是一种B族维生素,广泛应用于食品、医药、饲料等领域。微生物发酵法是最具前景的肌醇生产方法,但使用大肠杆菌生产的肌醇在食品及医药领域中的使用受到限制。毕赤酵母作为生物安全菌株是工业上生产异源蛋白的良好宿主,其本身含有天然的肌醇合成途径,具有被改造成为高效生产肌醇细胞工厂的潜力。【目的】通过代谢工程改造毕赤酵母工程菌株,降低副产物的生成并提高肌醇的产量。【方法】以实验室前期构建的产肌醇毕赤酵母工程菌株为出发菌株,确定副产物阿拉伯糖醇、核糖醇和甘露糖合成相关基因。通过关键基因敲除、发酵液中葡萄糖浓度控制降低副产物的产量。通过过表达甘油转运蛋白、甘油激酶和甘油-3-磷酸脱氢酶基因实现产肌醇毕赤酵母对甘油和葡萄糖的共利用,得到重组菌Z10。经过发酵条件优化,进一步提高Z10的肌醇产量。【结果】在最优条件下,重组菌Z10的肌醇产量达到36.7 g/L,是目前酵母类细胞工厂生产肌醇的最高值,副产物总产量与出发菌株相比降低了63.1%。【结论】在毕赤酵母中建立了降低阿拉伯糖醇、核糖醇和甘露糖合成的有效策略,并通过甘油、葡萄糖共利用及相对应的发酵条件优化提高了肌醇产量,为肌醇及其他高价值生物...     

重组大肠杆菌高密度发酵产甲羟戊酸动力学

为提高甲羟戊酸产量,探究其发酵生产规律,以重组大肠杆菌YJM16为供试菌株,进行5 L发酵罐匀速补料的高密度发酵实验,最终细胞产量达到54.48 g/L,甲羟戊酸产量达到40.43 g/L,产率为20.2%。然后根据Logistic方程、Luedeking-Piret方程和类似Luedeking-Piret方程,拟合出重组大肠杆菌高密度发酵过程中菌体生长、甲羟戊酸合成、基质消耗的动力学模型及模型参数。结果表明,甲羟戊酸的合成与重组大肠杆菌的生长速率及菌体积累量均有关。菌体生长、底物消耗模型和产物形成模型拟合度R2分别达到了0.931 9、0.957 8和0.975 1,可用于描述利用重组大肠杆菌高密度发酵生产甲羟戊酸过程。     

Robust production of gamma-amino butyric acid using recombinant Corynebacterium glutamicum expressing glutamate decarboxylase from Escherichia coli

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30°C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1mM PLP, fermentation was performed in a flask at 30°C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source.     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Metabolic engineering of Saccharomyces cerevisiae for hydroxytyrosol overproduction directly from glucose

Hydroxytyrosol (HT) is one of the most powerful dietary antioxidants with numerous applications in different areas, including cosmetics, nutraceuticals and food. In the present work, heterologous hydroxylase complex HpaBC from Escherichia coli was integrated into the Saccharomyces cerevisiae genome in multiple copies. HT productivity was increased by redirecting the metabolic flux towards tyrosol synthesis to avoid exogenous tyrosol or tyrosine supplementation. After evaluating the potential of our selected strain as an HT producer from glucose, we adjusted the medium composition for HT production. The combination of the selected modifications in our engineered strain, combined with culture conditions optimization, resulted in a titre of approximately 375 mg l⁻¹ of HT obtained from shake-flask fermentation using a minimal synthetic-defined medium with 160 g l⁻¹ glucose as the sole carbon source. To the best of our knowledge, this is the highest HT concentration produced by an engineered S. cerevisiae strain.     

幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺的研究

通过不同培养基、不同葡萄糖浓度、不同溶氧条件、补料与非补料对幽门螺杆菌尿素酶B亚单位(UreB)基因工程菌的菌体生长与外源蛋白表达量的影响的比较 ,建立了稳定、适宜的幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺。多批实验结果证明 ,菌体单产可达 86 g/L ,目的蛋白的表达率为 38.2 %。     

Functional expression of prokaryotic and eukaryotic genes in Escherichia coli for conversion of glucose to p-hydroxystyrene

The chemical monomer p-hydroxystyrene (pHS) is used for producing a number of important industrial polymers from petroleum-based feedstocks. In an alternative approach, the microbial production of pHS can be envisioned by linking together a number of different metabolic pathways, of which those based on using glucose for carbon and energy are currently the most economical. The biological process conserves petroleum when glucose is converted to the aromatic amino acid L-tyrosine, which is deaminated by a tyrosine/phenylalanine ammonia-lyase (PAL/TAL) enzyme to yield p-hydroxycinnamic acid (pHCA). Subsequent decarboxylation of pHCA gives rise to pHS. Bacteria able to efficiently decarboxylate pHCA to pHS using a pHCA decarboxylase (PDC) include Bacillus subtilis, Pseudomonas fluorescens and Lactobacillus plantarum. Both B. subtilis and L. plantarum possess high levels of pHCA-inducible decarboxylase activity and were chosen for further studies. The genes encoding PDC in these organisms were cloned and the pHCA decarboxylase expressed in Escherichia coli strains co-transformed with a plasmid encoding a bifunctional PAL/TAL enzyme from the yeast Rhodotorula glutinis. Production of pHS from glucose was ten-fold greater for the expressed L. plantarum pdc gene (0.11mM), compared to that obtained when the B. subtilis PDC gene (padC) was used. An E. coli strain (WWQ51.1) expressing both tyrosine ammonia-lyase(PAL) and pHCA decarboxylase (pdc), when grown in a 14L fermentor and under phosphate limited conditions, produced 0.4g/L of pHS from glucose. We, therefore, demonstrate pHS production from an inexpensive carbohydrate feedstock by fermentation using a novel metabolic pathway comprising genes from E. coli, L. plantarum and R. glutinis.     

代谢工程改造大肠杆菌生产辅酶Q10

辅酶Q10(CoQ10)是一种脂溶性抗氧化剂,具有提高人体免疫力、延缓衰老和增强人体活力等功能,广泛应用于制药行业和化妆品行业。微生物发酵法能可持续性生产辅酶Q10,具有越来越多的商业价值。本研究首先将来自类球红细菌的十聚异戊二烯焦磷酸合成酶基因(dps)整合到大肠杆菌ATCC 8739染色体上,敲除内源的八聚异戊二烯焦磷酸合成酶基因(ispB),使内源的辅酶Q8合成途径被辅酶Q10合成途径取代,得到稳定生产辅酶Q10的菌株GD-14,其辅酶Q10产量达0.68 mg/L,单位细胞含量达0.54 mg/g DCW。随后用多个固定强度调控元件在染色体上对MEP途径的关键基因dxs和idi基因以及ubiCA基因进行组合调控,将辅酶Q10单位细胞含量提高2.46倍(从0.54到1.87 mg/g)。进一步引入运动发酵单胞菌Zymomonas mobilis的Glf转运蛋白代替自身的磷酸烯醇式丙酮酸:碳水化合物磷酸转移酶系统(PTS),使辅酶Q10产量进一步提高16%。最后,对高产菌株GD-51进行分批补料发酵,辅酶Q10产量达433 mg/L,单位细胞含量达11.7 mg/g DCW。这是目前为止文献报道的大肠杆菌产辅酶Q10最高菌株。     

Enhanced production of (R)-1,2-propanediol by metabolically engineered Escherichia coli

1,2-Propanediol (1,2-PD) is a major commodity chemical currently derived from propylene. Previously, we have demonstrated the production of enantiomerically pure (R)-1,2-propanediol from glucose by an engineered E. coli expressing genes for NADH-linked glycerol dehydrogenase and methylglyoxal synthase. In this work, we investigate three methods to improve 1,2-PD in E. coli. First, we investigated improving the host by eliminating production of a byproduct, lactate. To do this, we constructed strains with mutations in two enzymes involved in lactate production, lactate dehydrogenase and glyoxalase I. (Surprisingly, when mutations were made in its ability to produce lactate, one strain of E. coli [MM294], produced a small amount of 1,2-PD without any added genes.) Second, we constructed a complete pathway to 1,2-PD from the glycolytic intermediate, dihydroxyacetone phosphate. Our previous 1, 2-PD producing strains relied on at least one endogenous E. coli activity and only produced 0.7 g/L of 1,2-PD. The complete pathway involved the coexpression of methylglyoxal synthase (mgs), glycerol dehydrogenase (gldA), and either yeast alcohol dehydrogenase (adhI) or E. coli 1,2-propanediol oxidoreductase (fucO). Third, we investigated bioprocessing improvements by carrying out a fed-batch fermentation with the best engineered strain (expressing mgs, gldA, and fucO). A final titer of 4.5 g/L of (R)-1,2-PD was produced, with a final yield of 0.19 g of 1,2-PD per gram of glucose consumed. This work provides a basis for further strain and process improvement.     

异源表达多巴脱羧酶促进大肠杆菌从头合成多巴胺

多巴胺是多种天然抗氧化药物生物合成的前体物质,在人体内作为神经递质调控中枢神经系统的多种生理功能,常用于多种类型休克的临床治疗。目前,通过微生物合成技术已经实现了多巴胺的从头合成,但是合成效率很低。针对该问题,在左旋多巴 (l-DOPA) 大肠杆菌工程菌基础上,利用不同拷贝数质粒表达野猪Sus scrofa来源的多巴脱羧酶基因Ssddc,实现了葡萄糖到多巴胺的生产。为了进一步提高多巴胺合成效率,从100个候选基因中筛选出5个多巴脱羧酶基因进行测试,其中来源于人Homo sapiens多巴脱羧酶基因Hsddc的工程菌摇瓶发酵的多巴胺产量最高,达到3.33 g/L;而来源于果蝇Drosophila melanogaster多巴脱羧酶基因Dmddc的工程菌摇瓶发酵的左旋多巴残余量最低,仅有0.02 g/L;这两株工程菌分批补料发酵表明,多巴胺的产量可以分别达到13.3 g/L和16.2 g/L,左旋多巴残余量分别是0.45 g/L和0.23 g/L。将多巴脱羧酶基因Dmddc和Ssddc分别整合到基因组上,获得遗传稳定的工程菌,在分批补料发酵条件下,多巴胺产量最高达到17.7 g/L,是目前国内外报道的最高产量。     

组合代谢调控提高大肠杆菌对氨基苯甲酸产量

对氨基苯甲酸是一种重要的有机合成中间体,广泛应用于医药、染料等行业。近年来对氨基苯甲酸作为一种潜在的高强度共聚物单体越来越受到重视。对氨基苯甲酸作为叶酸合成的前体之一,其合成在大肠杆菌体内由叶酸合成途径的pabA、pabB和pabC三个基因负责,催化分支酸合成对氨基苯甲酸。本研究以实验室构建的酪氨酸高产工程菌TYR002作为出发菌株,首先弱化双功能分支酸突变酶/预苯酸脱氢酶TyrA的表达,以减少酪氨酸积累,然后利用3种不同强度的组成型启动子分别调控pabA、pabB和pabC的表达。摇瓶发酵表明不同的组合调控模式下大肠杆菌发酵培养基中的对氨基苯甲酸积累量存在显著差异,最高可获得0.67 g/L的摇瓶发酵产量。进一步通过发酵条件优化和分批补料发酵,在5L发酵罐中获得了6.4g/L的对氨基苯甲酸产量。本研究为改善对氨基苯甲酸生物合成效率提供了重要理论参考。     

Use of whole cells of Pseudomonas aeruginosa for synthesis of the antioxidant hydroxytyrosol via conversion of tyrosol

For the first time, a soil bacterium, designated Pseudomonas aeruginosa, was isolated based on its ability to grow on tyrosol as a sole source of carbon and energy. During growth on tyrosol, this strain was capable of promoting the formation of a significant amount of hydroxytyrosol and trace quantities of parahydroxyphenyl acetic acid and 3,4-dihydroxyphenyl acetic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Using an optimized tyrosol concentration of 2 g liter(-1), the maximal hydroxytyrosol yield (80%) was achieved after a 7-h reaction in a growth experiment. To enhance the formation of hydroxytyrosol and prevent its degradation, a resting-cell method using P. aeruginosa was performed. The growth state of the culture utilized for biomass production, the carbon source on which the biomass was grown, the concentration of the biomass, and the amount of tyrosol that was treated were optimized. The optimal yield of hydroxytyrosol (96%) was obtained after a 7-h reaction using 4 g of tyrosol liter(-1) and 5 g of cells liter(-1) pregrown on tyrosol and harvested at the end of the exponential phase. This proposed procedure is an alternative approach to obtain hydroxytyrosol in an environmentally friendly way. In addition, the reaction is easy to perform and can be adapted to a bioreactor for industrial purposes.