理性设计和构建过量合成莽草酸的大肠杆菌代谢工程菌

利用代谢工程手段理性改造野生大肠杆菌的莽草酸(Shikimic acid,SA)合成途径及相关代谢节点,以构建高产莽草酸的工程菌株.根据细胞代谢网络分析,利用Red-Xer重组系统连续删除了野生型大肠杆菌CICIMB0013的莽草酸激酶基因(aroL、aroK),葡萄糖磷酸转移酶系统(PTS)的关键组分EIICBglc的编码基因(ptsG)以及奎宁酸/莽草酸脱氢酶基因(ydiB)并系统评价了基因删除对细胞的生长、葡萄糖代谢和莽草酸积累的影响.aroL、aroK的删除阻断了莽草酸进一步转化成为莽草酸-3-磷酸,初步提高莽草酸的累积.删除ptsG基因使大肠杆菌PTS系统部分缺失,细胞通过GalP-glk(半乳糖透性酶-葡萄糖激酶)途径,利用ATP将葡萄糖磷酸化后进入细胞.利用该途径运输葡萄糖能够减少PEP的消耗,使得更多的碳代谢流进入莽草酸合成途径,从而显著提高了莽草酸的产量.在此基础上删除ydiB基因,阻止了莽草酸合成的前体物质3-脱氢奎宁酸转化为副产物奎宁酸(Quinic acid,QA),进一步提高了莽草酸的累积.初步发酵显示4个基因缺失的大肠杆菌代谢工程菌生产莽草酸的能力比原始菌提高了90多倍.     

使用动态分子开关调控大肠杆菌生产莽草酸

莽草酸是大肠杆菌合成芳香族氨基酸的中间代谢物,也是抗流感药物"达菲"的重要合成前体。合成莽草酸需要截断莽草酸途径,导致芳香族氨基酸无法合成,因此面临细胞生长受到抑制的问题。使用动态调控策略通过将细胞生长和莽草酸的合成相互分离,可以提高菌株的生产性能。通过使用生长偶联型启动子和降解决定子(Degrons),组建动态分子开关。利用该动态分子开关实现细胞生长与莽草酸合成分离,在5L发酵罐中经过72h发酵得到了14.33g/L的莽草酸。结果表明,这种动态分子开关可以通过调控靶蛋白丰度来改变碳流量平衡,使菌株获得更优秀的生产性能。     

单棕榈酰莽草酸的高效液相色谱-蒸发光散射检测法定量测定

本文建立一种采用高效液相色谱-蒸发光散射检测法定量测定单棕榈酰莽草酸的方法。色谱条件:色谱柱:反相Symmetry C18柱4.6×250 mm i.d.(填料粒度5μm),柱温30℃,流动相:乙腈-水-甲酸(80∶20∶0.2);流速0.8 mL/min;ELSD漂移管温度:80℃,载气流速:2 L/min。3-,4-和5-棕榈酰莽草酸纯品采用酶法合成,合成产物经柱层析和高效液相色谱制备纯化得到纯品。在该色谱条件下,3-,4-和5-棕榈酰莽草酸在0.21~4.12μg的范围内,其质量与其峰面积线性关系良好(r=0.9991,0.9998,0.9997),回收率分别为95.2~98.7%,96.1%~98.2%,96.3%~98.5%;RSD分别为1.6%,1.8%,1.9%。实验结果表明该方法具有简便、快速、准确、重现性较好的特点,可以用于定量分析单棕榈酰莽草酸,尤其适用于非水相酶促反应合成体系中的产物检测。     

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。     

途径工程改造谷氨酸棒杆菌产莽草酸

莽草酸是一种芳香族中间代谢产物,也是合成抗禽流感药物磷酸奥司他韦的前体。目前,国内外莽草酸的生产主要依靠成本较高,周期较长的植物提取法。微生物发酵法合成莽草酸具有生产成本低、周期短等优势成为研究的热点。为了构建产莽草酸的重组谷氨酸棒杆菌,此次研究从基因组水平上对谷氨酸棒杆菌体内的莽草酸代谢途径进行代谢工程改造。通过阻断莽草酸分解代谢途径、解除反馈抑制以及阻断竞争性代谢途径的策略,实现了莽草酸产量的大幅提升。结果显示,所构建的重组谷氨酸棒杆菌SKA06经72 h摇瓶发酵,莽草酸产量达到7.61 g/L,相较出发菌种提升了68倍。并且,基于染色体工程的遗传改造策略克服了引入质粒带来传代不稳定、需要添加抗生素等问题,可以为莽草酸工程菌种的选育提供重要参考。     

大肠杆菌ptsHIcrr操纵子的快速敲除及敲除菌生长性能测定

敲除大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统(简称PTS系统)ptsHIcrr操纵子,考察敲除菌株生长特性并将其与ptsG敲除菌进行比较。利用I-SceⅠ特异性切割和Red同源重组方法成功构建了大肠杆菌DH5α△ptsHIcrr敲除菌。在LB培养基中,DH5α△ptsHIcrr的生长行为与DH5α和DH5α△ptsG明显不同,其最高菌密度是DH5α和DH5α△ptsG的近2倍,而DH5α△ptsG生长行为与DH5α无明显差异。但在含1%葡萄糖的LB中,DH5α△ptsHIcrr和DH5α△ptsG均表现出生长优势,最高菌密度依次是DH5α的2.8和2倍;培养液中最终乙酸含量分别是DH5α的12.2%、47%。在M9修饰培养基中,DH5α△ptsHIcrr比生长速率(1/h)和比葡萄糖消耗速率[g/(g.h)]明显低于DH5α,并略低于DH5α△ptsG。结果说明,ptsHIcrr操纵子敲除菌改变了葡萄糖的代谢速率,并呈现与ptsG基因敲除菌不同的代谢特点。     

响应曲面法优化马尾松中莽草酸的超声提取工艺

以马尾松松针为原料,采用超声波提取法从松针粉中提取莽草酸,通过考察料液比((VH2O∶m松针粉,mL:g)、提取时间、提取温度及超声波功率等因素对松针中总莽草酸含量的影响,并在单因素试验的基础上,选取料液比、提取时间、超声波功率3个变量,进行Box-Behnken中心组合设计优化,获得马尾松松针中莽草酸的最佳提取工艺参数为料液比1∶26,提取时间为46min,超声波功率为359 W,此条件下莽草酸的提取率为1.948%。     

八角茴香春果和秋果以及不同部位的莽草酸含量分析

采用HPLC法对不同季节和不同部位的八角茴香药材主成分莽草酸的含量进行分析,发现枝条、叶片、春果、秋果的莽草酸含量依次为2.64%、4.94%、8.63%、12.16%;色谱条件:Venusil HILIC(5μm,4.6 mm×250 mm)色谱柱,流动相:乙腈-0.5%三氟乙酸(95:5),流速1.0 mL·min~(-1),检测波长为210 nm。莽草酸进样量在0.001～6μg范围内线性关系良好。莽草酸的线性回归相关系数r=0.9984,平均回收率为98.4%,相对标准偏差(RSD)为1.26%。该研究为制定八角茴香药材质量检测标准提供了简便、快速、准确的方法。     

D-核糖生产菌的选育

将枯草芽胞杆菌通过紫外线诱变得到了莽草酸缺陷突变株,在２８株突变株中有１０株积累Ｄ－核糖。这些菌株均属戊糖磷酸途径的非氧化支路缺失突变株。对这些菌株的产核糖能力进行了验证、培养基中芳香族氨基酸的浓度影响Ｄ－核糖的积累     

基于rhaSR嵌合操纵子的构建及其在大肠杆菌中表达的研究

通过PCR等重组DNA技术,构建了含rhaSR启动子表达调控元件、RhaR基因、报告基因gst(谷胱甘肽-S-转移酶)的两个嵌合操纵子,并插入大肠杆菌表达载体pALEX中构成pALEX-PR1和pALEX-PR2。其中pALEX-PR2的RhaR基因上游为原有的SD序列,而pALEX-PR1的RhaR基因上游则插入了增强的SD序列。把这两个重组表达质粒分别转入大肠杆菌BL21(DE3)中,报告基因gst能够在L-鼠李糖诱导下表达,其表达量是非诱导条件下的4～5倍,且pALEX-PR1的表达量是pALEX-PR2的3.14倍。以上结果表明,gst的表达既受L-鼠李糖诱导,同时又受RhaR的正调控。SDS-PAGE结果显示,GST占大肠杆菌培养物总可溶蛋白的5.41%(W/W),平均1L培养物可获得3.0mg纯化的GST。酶活性分析表明,所构建的嵌合操纵子表达的GST保持了正确的构型且具有很高的活性。     

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.     

Homolactate fermentation by metabolically engineered Escherichia coli strains

We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter . h). Ca(OH)(2) was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N(2) for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. (13)C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter . h during the anaerobic phase.     

Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of L-lactic acid

Conversion of lignocellulose to lactic acid requires strains capable of fermenting sugar mixtures of glucose and xylose. Recombinant Escherichia coli strains were engineered to selectively produce L-lactic acid and then used to ferment sugar mixtures. Three of these strains were catabolite repression mutants (ptsG ⁻) that have the ability to simultaneously ferment glucose and xylose. The best results were obtained for ptsG ⁻ strain FBR19. FBR19 cultures had a yield of 0.77 (g lactic acid/g added sugar) when used to ferment a 100 g/l total equal mixture of glucose and xylose. The strain also consumed 75% of the xylose. In comparison, the ptsG ⁺ strains had yields of 0.47–0.48 g/g and consumed 18–22% of the xylose. FBR19 was subsequently used to ferment a variety of glucose (0–40 g/l) and xylose (40 g/l) mixtures. The lactic acid yields ranged from 0.74 to 1.00 g/g. Further experiments were conducted to discover the mechanism leading to the poor yields for ptsG ⁺ strains. Xylose isomerase (XI) activity, a marker for induction of xylose metabolism, was monitored for FBR19 and a ptsG ⁺ control during fermentations of a sugar mixture. Crude protein extracts prepared from FBR19 had 10–12 times the specific XI activity of comparable samples from ptsG ⁺ strains. Therefore, higher expression of xylose metabolic genes in the ptsG ⁻ strain may be responsible for superior conversion of xylose to product compared to the ptsG ⁺ fermentations. Received 14 December 2000/ Accepted in revised form 28 June 2002     

Enhanced production of plasmid DNA by engineered Escherichia coli strains

Escherichia coli strains VH33 (PTS? GalP? strain displaying a strongly reduced overflow metabolism) and VH34 (additionally lacking the pyruvate kinase A) were evaluated for the production of a plasmid DNA (pDNA) vaccine. The parent (W3110) and mutant strains were cultured using 10 g of glucose/L. While the specific growth rates of the three strains were similar, they presented differences in the accumulation of acetate. W3110 accumulated up to 4 g/L of acetate, VH33 produced 1.4 g/L, and VH34 only 0.78 g/L. VH33 and VH34 produced 76% and 300% more pDNA than W3110. Moreover, VH34 demanded 33% less oxygen than VH33 and W3110, which can be advantageous for large-scale applications.     

Efficient free fatty acid production in engineered Escherichia coli strains using soybean oligosaccharides as feedstock

To be competitive with current petrochemicals, microbial synthesis of free fatty acids can be made to rely on a variety of renewable resources rather than on food carbon sources, which increase its attraction for governments and companies. Industrial waste soybean meal is an inexpensive feedstock, which contains soluble sugars such as stachyose, raffinose, sucrose, glucose, galactose, and fructose. Free fatty acids were produced in this report by introducing an acyl‐ACP carrier protein thioesterase and (3R)‐hydroxyacyl‐ACP dehydratase into E. coli. Plasmid pRU600 bearing genes involved in raffinose and sucrose metabolism was also transformed into engineered E. coli strains, which allowed more efficient utilization of these two kinds of specific oligosaccharide present in the soybean meal extract. Strain ML103 (pRU600, pXZ18Z) produced ～1.60 and 2.66 g/L of free fatty acids on sucrose and raffinose, respectively. A higher level of 2.92 g/L fatty acids was obtained on sugar mixture. The fatty acid production using hydrolysate obtained from acid or enzyme based hydrolysis was evaluated. Engineered strains just produced ～0.21 g/L of free fatty acids with soybean meal acid hydrolysate. However, a fatty acid production of 2.61 g/L with a high yield of 0.19 g/g total sugar was observed on an enzymatic hydrolysate. The results suggest that complex mixtures of oligosaccharides derived from soybean meal can serve as viable feedstock to produce free fatty acids. Enzymatic hydrolysis acts as a much more efficient treatment than acid hydrolysis to facilitate the transformation of industrial waste from soybean processing to high value added chemicals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:686–694, 2015     

Metabolic modeling and response surface analysis of an Escherichia coli strain engineered for shikimic acid production

Background

Classic metabolic engineering strategies often induce significant flux imbalances to microbial metabolism, causing undesirable outcomes such as suboptimal conversion of substrates to products. Several mathematical frameworks have been developed to understand the physiological and metabolic state of production strains and to identify genetic modification targets for improved bioproduct formation. In this work, a modeling approach was applied to describe the physiological behavior and the metabolic fluxes of a shikimic acid overproducing Escherichia coli strain lacking the major glucose transport system, grown on complex media.

Results

The obtained flux distributions indicate the presence of high fluxes through the pentose phosphate and Entner-Doudoroff pathways, which could limit the availability of erythrose-4-phosphate for shikimic acid production even with high flux redirection through the pentose phosphate pathway. In addition, highly active glyoxylate shunt fluxes and a pyruvate/acetate cycle are indicators of overflow glycolytic metabolism in the tested conditions. The analysis of the combined physiological and flux response surfaces, enabled zone allocation for different physiological outputs within variant substrate conditions. This information was then used for an improved fed-batch process designed to preserve the metabolic conditions that were found to enhance shikimic acid productivity. This resulted in a 40% increase in the shikimic acid titer (60 g/L) and 70% increase in volumetric productivity (2.45 gSA/L*h), while preserving yields, compared to the batch process.

Conclusions

The combination of dynamic metabolic modeling and experimental parameter response surfaces was a successful approach to understand and predict the behavior of a shikimic acid producing strain under variable substrate concentrations. Response surfaces were useful for allocating different physiological behavior zones with different preferential product outcomes. Both model sets provided information that could be applied to enhance shikimic acid production on an engineered shikimic acid overproducing Escherichia coli strain.