Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli

Nonribosomal peptides (NRPs) are a class of microbial secondary metabolites that have a wide variety of medicinally important biological activities, such as antibiotic (vancomycin), immunosuppressive (cyclosporin A), antiviral (luzopeptin A) and antitumor (echinomycin and triostin A) activities. However, many microbes are not amenable to cultivation and require time-consuming empirical optimization of incubation conditions for mass production of desired secondary metabolites for clinical and commercial use. Therefore, a fast, simple system for heterologous production of natural products is much desired. Here we show the first example of the de novo total biosynthesis of biologically active forms of heterologous NRPs in Escherichia coli. Our system can serve not only as an effective and flexible platform for large-scale preparation of natural products from simple carbon and nitrogen sources, but also as a general tool for detailed characterizations and rapid engineering of biosynthetic pathways for microbial syntheses of novel compounds and their analogs.     

肽类抗生素生物合成基因簇在Escherichia coli中的异源表达

微生物能够产生众多结构和生物活性多样的次生代谢产物,而其生物合成基因簇的挖掘和异源表达是药物创新和产量提高的必要前提. 在过去20年里,大量重要天然产物的生物合成基因簇在微生物中被不断的发现. 在这些被挖掘的基因簇中,肽类抗生素的生物合成基因簇占了很大比重.肽类抗生素因具有抗菌、抗肿瘤、抗病毒等多种生物学活性而备受化学家和药物学家的重视. 如能了解它们的生物合成机制,实现其基因簇的异源表达,将使合理化遗传修饰生物合成通路获取结构类似物（药物开发）和提高产量成为可能. 大肠杆菌作为最广泛、最成功的表达体系,常用来表达外源基因,但一般只能表达一个或几个基因,却很少有用它来表达整个生物合成基因簇. 2001年,Khosla和Cane在E.coli中成功异源表达了一个复杂聚酮天然产物（红霉素苷原6dEB）基因簇. 这是首个有关在E.coli中异源表达天然产物生物合成基因簇的研究. 至此之后,大肠杆菌开始作为生物合成基因簇的异源表达宿主,越来越受到相关领域的重视. 紧接着核糖体肽和非核糖体肽生物合成基因簇也相继在大肠杆菌中成功异源表达. 本文对肽类抗生素生物合成基因簇在E.coli中的异源表达进行了综述.     

基于重组技术的低毒力高效大肠杆菌原核表达系统的改造与应用

[背景]大肠杆菌作为原核表达系统常用宿主菌株,具有培养成本低、周期短和操作性强等优势,但同时也存在着一些不足.[目的]降低大肠杆菌内毒素合成水平及毒力,同时提高其可溶性表达外源蛋白的能力.[方法]利用CRISPR-Cas技术敲除大肠杆菌BL21(DE3)脂多糖生物合成途径中的lpxM,改变其毒性中心类脂A的侧链结构;在...     

氨甲酰磷酸不同合成途径在大肠杆菌中的比较

【背景】氨甲酰磷酸是生物合成代谢中精氨酸与嘧啶的重要前体物质,在工业微生物生产精氨酸与嘧啶及其衍生物中发挥关键作用。【目的】在大肠杆菌Escherichia coli BW25113中比较氨甲酰磷酸不同合成途径的催化效率。【方法】在大肠杆菌Escherichia coli BW25113中过表达鸟氨酸氨甲酰基转移酶(OTC)的基础上,分别过表达大肠杆菌自身的氨基甲酸激酶(CK)和氨甲酰磷酸合酶(CPSⅡ)并表征其反应效果。通过优化底物供应(调整底物浓度与引入L-谷氨酰胺合成酶)对CK与CPSⅡ的催化反应进行优化。【结果】在大肠杆菌中过表达OTC,建立细胞水平氨甲酰磷酸检测体系。在此基础上比较不同来源的CK,发现大肠杆菌来源的CK效果最好,50mmol/LNH4HCO3条件下全细胞催化9h得到2.95±0.15mmol/LL-瓜氨酸;过表达CPSⅡ时,50mmol/LL-谷氨酰胺催化9h得到3.16±0.29 mmol/L L-瓜氨酸。通过改变底物NH4HCO3浓度和引入外源L-谷氨酰胺合成酶(GS)等方式对CK与CPSⅡ的催化反应分别进行优化后,100 mmol/L NH4HCO3条件下,L-瓜氨酸浓度分别提高至4.67±0.55mmol/L和6.12±0.38mmol/L,且过表达GS后CPSⅡ途径可以利用NH3,不需要额外添加L-谷氨酰胺。【结论】引入L-谷氨酰胺合成酶后的CPSⅡ途径合成氨甲酰磷酸的能力优于CK途径,为精氨酸、嘧啶及其衍生物的合成提供了一种更加高效的策略。     

Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. strain SCRC-2738.

The 38 kb eicosapentaenoic acid (EPA) biosynthesis gene cluster of Shewanella sp. strain SCRC-2738 was cloned into the cosmid vector (pEPA). A 27 kb nucleotide sequence of the XhoI to SpeI region of pEPA showed EPA production (6.3%) in E. coli JM109. Among the nine open reading frames (ORFs) in this sequence, only five (ORFs 2 and 5-8) were essential for EPA production. High levels of production (16%-22%) were found in E. coli JM109 transformed with a multicopy pNEB vector carrying only the five essential ORFs and in that transformed with a pNEB vector that integrated ORFs 3, 5, 6, 7 and 8, and vector pSTV28 that integrated the ORF2 encoding phosphopantetheinyl transferase (PPTase). Thus, production of EPA appears to be regulated by the presence of all the biosynthesis gene products and by the ratio of PPTase to the other gene products. The temperature -EPA production relationship in E. coli strain DH5alpha varied between constructs, suggesting that it is controlled not only by EPA biosynthesis enzymes but also by other factors in vivo. There was a strict upper temperature limit for EPA biosynthesis: no EPA was synthesized at 30 degrees C in E. coli transformants carrying any gene construct for EPA biosynthesis.     

Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.     

Involvement of the AtoS-AtoC signal transduction system in poly-(R)-3-hydroxybutyrate biosynthesis in Escherichia coli

The AtoS-AtoC signal transduction system in E. coli, which induces the atoDAEB operon for the growth of E. coli in short-chain fatty acids, can positively modulate the levels of poly-(R)-3-hydroxybutyrate (cPHB) biosynthesis, a biopolymer with many physiological roles in E. coli. Increased amounts of cPHB were synthesized in E. coli upon exposure of the cells to acetoacetate, the inducer of the AtoS-AtoC two-component system. While E. coli that overproduce both components of the signal transduction system synthesize higher quantities of cPHB (1.5-4.5 fold), those that overproduce either AtoS or AtoC alone do not display such a phenotype. Lack of enhanced cPHB production was also observed in cells overexpressing AtoS and phosphorylation-impaired AtoC mutants. The results were not affected by the nature of the carbon source used, i.e., glucose, acetate or acetoacetate. An E. coli strain with a deletion in the atoS-atoC locus (delta atoSC) synthesized lower amounts of cPHB compared to wild-type cells. When the delta atoSC strain was transformed with a plasmid carrying a 6.4-kb fragment encoding the AtoS-AtoC system, cPHB biosynthesis was restored to the level of the atoSC+ cells. Introduction of a multicopy plasmid carrying a functional atoDAEB operon, but not one with a promoterless operon, resulted in increased cPHB synthesis only in atoSC+ cells in the presence of acetoacetate. These results indicate that the presence of both a functional AtoS-AtoC two-component signal transduction system and a functional atoDAEB operon is critical for the enhanced cPHB biosynthesis in E. coli.     

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l(-1) (OD600 approximately 60) and the production of 700 mg l(-1) of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l(-1) of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l(-1) with E. coli as the production host.     

Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(--)-hydroxycarboxylic acids

A heterologous metabolism of polyhydroxyalkanoate (PHA) biosynthesis and degradation was established in Escherichia coli by introducing the Ralstonia eutropha PHA biosynthesis operon along with the R. eutropha intracellular PHA depolymerase gene. By with this metabolically engineered E. coli, enantiomerically pure (R)-3-hydroxybutyric acid (R3HB) could be efficiently produced from glucose. By employing a two-plasmid system, developed as the PHA biosynthesis operon on a medium-copy-number plasmid and the PHA depolymerase gene on a high-copy-number plasmid, R3HB could be produced with a yield of 49.5% (85.6% of the maximum theoretical yield) from glucose. By integration of the PHA biosynthesis genes into the chromosome of E. coli and by introducing a plasmid containing the PHA depolymerase gene, R3HB could be produced without plasmid instability in the absence of antibiotics. This strategy can be used for the production of various enantiomerically pure (R)-hydroxycarboxylic acids from renewable resources.     

Interspecies conjugation of Escherichia coli-Streptomyces globisporus 1912 using integrative plasmid pSET152 and its derivatives]

Streptomyces globisporus 1912 produces a novel angucycline antitumor antibiotic landomycin E (LE). To study the LE biosynthetic gene cluster in detail, a system for the conjugal transfer of the integrative plasmid pSET152 from Escherichia coli into S. globisporus 1912 has been developed. It was shown that this plasmid integrates into two sites of the S. globisporus chromosome and is stably inherited under nonselective conditions. pSET152+ exconjugants of the strain 1912 are characterized by a significant decrease in LE synthesis (by 50-90%). A negative effect of pSET152 integration on antibiotic production was observed even upon the use of the recipient strain with increased LE synthesis, although in this case, the level of LE production in ex-conjugants was 120-150% of that in the original strain 1912. Based on pSET152, a vector system for gene knockouts in S. globisporus was developed. The effectivity of this system was shown in the example of disruption of the lndA gene encoding the key enzyme of LE synthesis (beta-ketoacylsynthase). Inactivation of this gene was shown to lead to the cessation of LE biosynthesis.     

产顺,顺-粘康酸细胞工厂的构建与优化

顺,顺-粘康酸是重要的平台化学品。目前,生物合成顺,顺-粘康酸还缺乏高性能菌株,已报道的主要工程菌株不仅需要诱导表达,遗传不稳定,而且发酵培养基组分复杂,不利于大规模工业化生产。构建能利用简单无机盐培养基、遗传稳定且不需要诱导表达的新型工程菌受到人们的关注。本研究在实验室前期构建的产三脱氢莽草酸工程菌株WJ060中,整合合成顺,顺-粘康酸的3个外源基因(aro Z、aro Y、cat A),并且利用3个不同强度的组成型启动子进行组合调控,成功构建了27株顺,顺-粘康酸工程菌,得到的最优工程菌MA30的产量达到1.7 g/L。为了进一步提高顺,顺-粘康酸工程菌的生产能力,利用基因组复制工程构建突变体库,结合高通量筛选方法,经过两轮筛选,成功筛选到了顺,顺-粘康酸产量提高超过8%的大肠杆菌MA30-G2。利用5 L发酵罐进行分批补料发酵,MA30-G2的顺,顺-粘康酸产量达到了11.5 g/L。本研究采用组合调控和高通量筛选相结合的策略不仅促进了顺,顺-粘康酸的生物合成,同时也为其他生物基化学品的生物制造提供了重要参考。     

Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis

Thiocoraline is a thiodepsipeptide antitumor agent that belongs to the family of bisintercalator natural products that bind duplex DNA through their two planar intercalating moieties. In thiocoraline, the 3-hydroxyquinaldic acid (3HQA) chromophores required for intercalation are derived from L-Trp. We have expressed the Micromonospora sp. ML1 tryptophan 2,3-dioxygenase(TDO) TioF, purified it from E. coli, and confirmed its role in the irreversible oxidation of L-Trp to N-formylkynurenine, the proposed first step during 3HQA biosynthesis. We have established that TioF is a catalyst with broader specificity than other TDOs, but that is less promiscuous than indoleamine 2,3-dioxygenases. TioF was found to display activity with various L-Trp analogs (serotonin, D-Trp, and indole). The TioF reaction products generated during this study will be used as substrates for subsequent analysis of the other enzymes involved in 3HQA biosynthesis.     

基于2-C-甲基-D-赤藓糖醇-4磷酸途径的产紫槐二烯大肠杆菌构建及其补糖策略优化

2-C-甲基-D-赤藻糖醇-4-磷酸(2-methyl-D-erythritol-4-phosphate, MEP) 途径是大肠杆菌Escherichiacoli 唯一的萜类前体合成途径,研究表明它比甲羟戊酸(Mevalonate, MVA)途径具有更高的理论产率。但目前有关MEP 途径的调控所知非常有限,故单独强化MEP 途径对萜类异源合成产量的提高效果并不理想。研究中通过引入外源MEP 途径基因强化E. coli 萜类合成的遗传改造策略和发酵过程补糖控制优化,尝试更有效地释放MEP 途径的潜力,建立青蒿素前体——紫槐二烯的高密度发酵过程。研究结果表明共表达阿维链霉菌Streptomyces avermitilis dxs2 基因和枯草芽胞杆菌Bacillus subtilis idi 基因可使紫槐二烯的摇瓶发酵产量比野生菌株提高12.2 倍。随后针对该菌株建立了高密度发酵过程,发现稳定期的中前期(24?72 h) 是产物合成的关键期,通过稳定期补糖速率的调整,明显改善了产物合成速度,使紫槐二烯的产量从2.5 g/L 提高到了4.85 g/L,但不影响产物积累的周期。考虑到72 h 后菌体老化可能会影响产物合成,进一步采取了调整对数期的补糖速率控制菌体生长的策略,使紫槐二烯的产量达到6.1 g/L。研究结果为基于MEP 途径的萜类异源合成工程菌构建及其发酵工艺的建立奠定了基础。     

Bifunctional intercalation and sequence specificity in the binding of quinomycin and triostin antibiotics to deoxyribonucleic acid.

Quinomycin C, triostin A and triostin C are peptide antibiotics of the quinoxaline family, of which echinomycin (quinomycin A) is also a member. They all remove and reverse the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs, and the unwinding angle at I 0.01 is, in all cases, almost twice that of ethidium. Thus, as with echinomycin, they can be characterized as bifunctional intercalating agents. For the triostins this conclusion has been confirmed by measurements of changes in the viscosity of sonicated rod-like DNA fragments; the helix extension was found to be almost double that expected for a simple monofunctional intercalation process. For triostin A, further evidence for bifunctionality was derived from the cross-over point of binding isotherms to nicked circular and closed circular bacteriophage-PM2DNA. Binding curves for the interaction of quinomycin C and triostin A with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis, but triostin C was too insoluble in aqueous solution to make this method applicable. For quinomycin C the highest binding constant was found with Micrococcus lysodeikticus DNA, and its pattern of specificity among natural DNA species was broadly similar to that of echinomycin, although the binding constants were 2--6 times as large. For triostin A the highest binding constant was again found for M. lysodeikticus DNA, but the specificity pattern was quite different from that of the quinomycins. In particular, triostin A bound better to poly(dA-dT) than to the poly(dG-dC) whereas this order was reversed for quinomycin C. There was also evidence that the binding to poly(dA-dT) might be co-operative in nature. No significant interaction could be detected with poly(dA).poly(dT) or with RNA from Escherichia coli. Poly(dG).poly(dC) gave variable results, depending on the source of the polymer. The different patterns of specificity displayed by the quinomycins and triostins are tentatively ascribed to differences in their conformations in solution.     

Regulation of beta-galactosidase synthesis in Escherichia coli by exogenous cyclic 3',5'-adenosine monophosphate]

The effect of cyclic 3',5'-adenosine monophosphate (cAMP) on the rate of beta-galactosidase biosynthesis was studied in the cells of Escherichia coli M-17 growing in MPB and mineral media with glucose and maltose, i.e. under the conditions of various catabolite repression, as well as upon lac-operon induction by isopropyl-beta-D-galactopyranoside (IPGP). The stimulating action of exogenous cAMP was found only in a medium with salts and glucose. The induction by IPGP was highest during the growth in a medium with glucose and maltose. When the medium contained IPGP, cAMP accelerated the enzyme synthesis in all media, but only at the early growth phases, while cAMP eliminated the effect of IPGP at the stationary phase of growth. The regulation of beta-galactosidase biosynthesis by cAMP demonstrated for the first time that this effect depended on the physiological state of E. coli: the expression of catabolite-sensitive E. coli genes was subject to both positive and negative regulation in one and the same inducible system. The effect exerted by cAMP depended on the nature of a carbon source in the growth medium.     

High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium

A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli.     

Expression of prokaryotic 1-deoxy-D-xylulose-5-phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis

Isopentenyl diphosphate (IPP) acts as the common, five-carbon building block in the biosynthesis of all isoprenoids. The first reaction of IPP biosynthesis in Escherichia coli is the formation of 1-deoxy-D-xylulose-5-phosphate, catalysed by 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). E. coli engineered to produce lycopene, was transformed with dxps genes cloned from Bacillus subtilis and Synechocystis sp. 6803. Increases in lycopene levels were observed in strains expressing exogenous DXPS compared to controls. The recombinant strains also exhibited elevated levels of ubiquinone-8. These increases corresponded with enhanced DXP synthase activity in the recombinant E. coli strains.     

利用重组大肠杆菌进行寡糖合成的研究进展

随着更多寡糖生物学活性的阐明,寡糖合成研究已成为糖生物学研究的热点之一,其中,以重组大肠杆菌作为酶盒或生物反应器,利用Leloir途径合成寡糖的方法,是近年来发展起来的一类重要的寡糖生物合成技术,并取得了较多的进展。将从细菌糖基转移酶的表达和鉴定、糖核苷酸的供给和寡糖的合成途径等几个方面,关注利用细菌功能尤其是利用重组大肠杆菌合成寡糖的研究进展,并分析各技术手段的优缺点及其应用前景。