High-level De novo biosynthesis of arbutin in engineered Escherichia coli

Arbutin is a hydroquinone glucoside compound existing in various plants. It is widely used in pharmaceutical and cosmetic industries owing to its well-known skin-lightening property as well as anti-oxidant, anti-microbial, and anti-inflammatory activities. Currently, arbutin is usually produced by plant extraction or enzymatic processes, which suffer from low product yield and expensive processing cost. In this work, we established an artificial pathway in Escherichia coli for high-level production of arbutin from simple carbon sources. First, a 4-hydroxybenzoate 1-hydroxylase from Candida parapsilosis CBS604 and a glucosyltransferase from Rauvolfia serpentina were characterized by in vitro enzyme assays. Introduction of these two genes into E. coli led to the production of 54.71 mg/L of arbutin from glucose. Further redirection of carbon flux into arbutin biosynthesis pathway by enhancing shikimate pathway genes enabled production of 3.29 g/L arbutin, which is a 60-fold increase compared with the initial strain. Final optimization of glucose concentration added in the culture medium was able to further improve the titer of arbutin to 4.19 g/L in shake flasks experiments, which is around 77-fold higher than that of initial strain. This work established de novo biosynthesis of arbutin from simple carbon sources and provided a generalizable strategy for the biosynthesis of shikimate pathway derived chemicals. The high titer achieved in our engineered strain also indicates the potential for industrial scale bio-manufacturing of arbutin.     

重组大肠杆菌产琥珀酸研究进展

琥珀酸作为一种优秀的C4平台化合物, 广泛用于生物高分子、食品与医药等行业, 市场潜在需求量巨大。采用微生物发酵法生产琥珀酸, 可利用廉价的可再生资源, 实现石油的原料替代, 而且过程污染小, 环境友好, 且在发酵过程中可吸收固定温室气体CO2, 开辟了其利用的新途径, 近年来引起了广泛关注。在丁二酸生产菌株中, 大肠杆菌由于其遗传背景清楚, 易操作易调 控, 培养基要求简单, 生长迅速等优点, 近年来被广泛用于研究以获得产琥珀酸优秀生产菌株。本工作系统综述了产琥珀酸大肠杆菌构建中所采用的基因工程策略及代谢工程技术, 并探讨了今后研究的方向。     

大肠杆菌生产异戊二烯的代谢工程研究进展

异戊二烯作为一种重要的化工原料,主要用于合成橡胶。此外,还广泛应用于医药或化工中间体、食品、粘合剂及航空燃料等领域。利用微生物法生产异戊二烯因具有环境友好、利用廉价的可再生原料、可持续发展等优势而成为当今研究的热点。这里介绍了大肠杆菌生产异戊二烯的代谢途径及关键酶,从代谢工程的角度出发综述了目前为提高大肠杆菌异戊二烯产量所应用到的方法和策略,并对今后的发展方向进行了展望。     

Modeling allosteric regulation of de novo pyrimidine biosynthesis in Escherichia coli

With the emergence of multifaceted bioinformatics-derived data, it is becoming possible to merge biochemical and physiological information to develop a new level of understanding of the metabolic complexity of the cell. The biosynthetic pathway of de novo pyrimidine nucleotide metabolism is an essential capability of all free-living cells, and it occupies a pivotal position relative to metabolic processes that are involved in the macromolecular synthesis of DNA, RNA and proteins, as well as energy production and cell division. This regulatory network in all enteric bacteria involves genetic, allosteric, and physiological control systems that need to be integrated into a coordinated set of metabolic checks and balances. Allosterically regulated pathways constitute an exciting and challenging biosynthetic system to be approached from a mathematical perspective. However, to date, a mathematical model quantifying the contribution of allostery in controlling the dynamics of metabolic pathways has not been proposed. In this study, a direct, rigorous mathematical model of the de novo biosynthesis of pyrimidine nucleotides is presented. We corroborate the simulations with experimental data available in the literature and validate it with derepression experiments done in our laboratory. The model is able to faithfully represent the dynamic changes in the intracellular nucleotide pools that occur during metabolic transitions of the de novo pyrimidine biosynthetic pathway and represents a step forward in understanding the role of allosteric regulation in metabolic control.     

High-yield anthocyanin biosynthesis in engineered Escherichia coli

Anthocyanins are red, purple, or blue plant water-soluble pigments. In the past two decades, anthocyanins have received extensive studies for their anti-oxidative, anti-inflammatory, anti-cancer, anti-obesity, anti-diabetic, and cardioprotective properties. In the present study, anthocyanin biosynthetic enzymes from different plant species were characterized and employed for pathway construction leading from inexpensive precursors such as flavanones and flavan-3-ols to anthocyanins in Escherichia coli. The recombinant E. coli cells successfully achieved milligram level production of two anthocyanins, pelargonidin 3-O-glucoside (0.98 mg/L) and cyanidin 3-O-gluside (2.07 mg/L) from their respective flavanone precursors naringenin and eriodictyol. Cyanidin 3-O-glucoside was produced at even higher yields (16.1 mg/L) from its flavan-3-ol, (+)-catechin precursor. Further studies demonstrated that availability of the glucosyl donor, UDP-glucose, was the key metabolic limitation, while product instability at normal pH was also identified as a barrier for production improvement. Therefore, various optimization strategies were employed for enhancing the homogenous synthesis of UDP-glucose in the host cells while at the same time stabilizing the final anthocyanin product. Such optimizations included culture medium pH adjustment, the creation of fusion proteins and the rational manipulation of E. coli metabolic network for improving the intracellular UDP-glucose metabolic pool. As a result, production of pelargonidin 3-O-glucoside at 78.9 mg/L and cyanidin 3-O-glucoside at 70.7 mg/L was achieved from their precursor flavan-3-ols without supplementation with extracellular UDP-glucose. These results demonstrate the efficient production of the core anthocyanins for the first time and open the possibility for their commercialization for pharmaceutical and nutraceutical applications.     

Construction of a heat-inducible Escherichia coli strain for efficient de novo biosynthesis of l-tyrosine

l-Tyrosine is an important amino acid widely used in food, agriculture, and pharmaceutical industries. However, the industrial application was severely constrained due to low production. To obtain the Escherichia coli mutant producing l-tyrosine in abundance, the heat-inducible expression vector carrying the two feedback resistance enzymes (3-deoxy-7-phosphoheptulonate synthase encoded by aroG^fbr and chorismate mutase/prephenate dehydrogenase encoded by tyrA^fbr) were introduced into the phenylalanine-producing E. coli strain to enable it to synthesize l-tyrosine directly from glucose. Furthermore, the CRISPR-Cas9 technology was applied to eliminate l-phenylalanine and l-tryptophan pathways for their competition for the carbon flux. The global regulatory protein TyrR, which mediates the biosynthesis and transportation of aromatic amino acids, was also deleted to increase l-tyrosine production. Among the recombinant strains, the pheA/tyrR double-gene deletion strain had the highest yield of 5.84 g/L on shake flasks. The feeding strategies were then optimized in a 3-L fermentor. The pheA/tyrR double-gene deletion strain with the heat-inducible expression plasmid pAP-aroG^fbr-tyrA^fbr was able to produce 55.54 g/L l-tyrosine by fed-batch fermentation; the substrate conversion rate was 0.25 g/g. The recombinant strains constructed in this study could be an industrial platform for the microbial production of l-tyrosine directly from glucose.     

温度调节基因开关调控大肠杆菌发酵合成L-丙氨

【目的】L-丙氨酸的存在导致Escherichia coli的生长速率显著降低,最终会降低发酵过程中L-丙氨酸的体积合成速率。用温度调节基因开关(λpR-pL)高效、动态调控重组E. coli菌株菌体生长与L-丙氨酸合成过程,使两者相协调。【方法】以野生型E. coli B0016为出发菌株,敲除乙酸、甲酸、乙醇、琥珀酸、乳酸代谢产物合成途径以及丙氨酸消旋酶编码基因(ackA-pta、pflB、adhE、frdA、ldhA、dadX),获得菌株B0016-060B。将嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)来源的L-丙氨酸脱氢酶基因(alaD)克隆于pL启动子下游,并在B0016-060B菌株中表达,获得菌株B0016-060B/pPL-alaD,进行摇瓶和发酵罐发酵考察菌体生长和L-丙氨酸发酵性能。【结果】竞争代谢途径的敲除显著降低了副产物合成量,仅形成极少量的乙酸、琥珀酸和乙醇。28 °C下菌株B0016-060B/pPL-alaD几乎不合成L-丙氨酸,可保证菌体快速生长;而在42 °C下可高效合成L-丙氨酸。经发酵罐发酵,可合成67.2 g/L L-丙氨酸,体积生产强度达到2.06 g/(L·h)。【结论】通过发酵培养温度的简单切换,分阶段实现了细胞的快速增量和L-丙氨酸的高强度合成。     

Metabolic engineering of acetoin and meso-2, 3-butanediol biosynthesis in E. coli

The functional reconstruction of acetoin and meso-2,3-butanediol (meso-2,3-BD) biosynthetic pathways in Escherichia coli have been explored systematically. Pathway construction involved the in vsivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA⁽ ilvC( expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no coproduction of diacetyl observed. These strains were also found to produce small quantities of meso-2,3-BD, suggesting the existence of endogenous 2,3-BD dehydrogenase activity. Finally, the coexpression of bdh1 from S. cerevisiae, encoding 2,3-BD dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso-2,3-BD, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso-2,3-BD was found to be the most important consequence of ldhA deletion.     

基于结构域活性分析的大肠杆菌T蛋白结构研究

大肠杆菌T蛋白含有三个结构域:分支酸变位酶、预苯酸脱氢酶和调节结构域。文章作者分段克隆了T蛋白的分支酸变位酶、预苯酸脱氢酶和调节结构域等片段,并对其进行了活性研究。研究发现,定位于N末端的分支酸变位酶结构域的比活性虽然不高,而稳定性较好;同时拥有调节结构域和预苯酸脱氢酶结构域的C末端片段,其预苯酸脱氢酶比活性的剩余百分率虽然高于分支酸变位酶结构域,但稳定性较差。作者进而表达了C末端切除38个氨基酸的T/1-336片段,发现预苯酸脱氢酶活性彻底丧失,而其分支酸变位酶和调节结构域的活性却基本保留。这说明T蛋白中分支酸变位酶结构域拥有一个相对独立、完整的结构,而预苯酸脱氢酶结构域和调节结构域交织共存,结构松散。     

Rapid one-step inactivation of single or multiple genes in Escherichia coli

Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid-based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose-inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β-D -1-thiogalactopyranoside-inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE–aspA and sfcA–aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.     

Biochemical genetics of aerobactin biosynthesis in Escherichia coli

Abstract Single-gene mutants of Escherichia coli defective in aerobactin biosynthesis were incubated under non-growing conditions for 2 h with radiolabelled lysine. Analysis of the intermediates produced suggested that acetylation of lysine may be the first step in aerobactin production.     

In silico design and adaptive evolution of Escherichia coli for production of lactic acid

The development and validation of new methods to help direct rational strain design for metabolite overproduction remains an important problem in metabolic engineering. Here we show that computationally predicted E. coli strain designs, calculated from a genome-scale metabolic model, can lead to successful production strains and that adaptive evolution of the engineered strains can lead to improved production capabilities. Three strain designs for lactate production were implemented yielding a total of 11 evolved production strains that were used to demonstrate the utility of this integrated approach. Strains grown on 2 g/L glucose at 37 degrees C showed lactate titers ranging from 0.87 to 1.75 g/L and secretion rates that were directly coupled to growth rates.     

大肠杆菌和志贺氏菌O-抗原糖基转移酶与聚合酶的生物信息学研究

利用生物信息学手段对大肠杆菌和志贺氏菌的 1 1 0个O 抗原糖基转移酶与 39个O 抗原聚合酶的序列进行分析 ,探讨这两种酶的序列和结构特点。统计了其序列一致性 ,密码子使用和 (G C) %含量的特点 ;讨论了O 抗原糖基转移酶和聚合酶对底物的特异性 ;推测了 6组糖基转移酶的功能 ;通过对蛋白拓扑结构的预测 ,发现O 抗原聚合酶中广泛存在一个位于细胞周质中的亲水环 (Loop) ,是可能的功能区域 ;通过对蛋白高级结构的预测 ,发现O 抗原糖基转移酶属于两个不同的蛋白超家族。     

Effect of temperature and htpR on the biosynthesis of superoxide dismutase in Escherichia coli

The synthesis of Mn- and FeSODs in response to temperature changes was examined in strains of Escherichia coli with different mutations in sod and htpR genes. Growth at or shift to elevated temperatures induced FeSOD but not MnSOD. The induction of FeSOD by heat was inhibited by chloramphenicol and was independent of the heat shock (htpR-controlled) regulon. FeSOD was more stable at 42 degrees C than was MnSOD.     

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: γ‐glutamylcysteine synthetase (γ‐GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over‐express either γ‐GCS or GS and found that γ‐GCS over‐expression resulted in inclusion body formation and impaired cell physiology, whereas GS over‐expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over‐expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild‐type cells in size (2–5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was ≈2.5 times that attained in the wild‐type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli‐based biosynthesis offers a more energy‐efficient and eco‐friendly method as opposed to chemical processes requiring high temperature and toxic solvents. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.     

工程大肠杆菌合成游离脂肪酸的研究进展

游离脂肪酸作为一种重要的平台化合物,其衍生产品被广泛应用到能源、化学工业中。作为更加可持续、绿色的生产策略,利用工程微生物合成游离脂肪酸是以石油基和动植物为原料生产脂肪酸类产品的重要补充。大肠杆菌作为经典的模式微生物,通过对其进行代谢工程改造,脂肪酸的积累已经从痕量提高到了约9g/L,展示了其作为脂肪酸合成菌株的巨大应用潜力。随着合成生物学技术的涌现,“感应-调控器”、体外重构、β氧化逆循环、异源合成途径的整合等思路的引入极大地加快了工程大肠杆菌脂肪酸合成的进化速率,并赋予大肠杆菌合成多种脂肪酸产品的能力。对近年来通过代谢工程和合成生物学手段改造大肠杆菌合成游离脂肪酸的研究进展进行综述,对其发展前景进行展望。