[背景]pBHR68是表达聚-3-羟基丁酸酯(Poly-3-Hydroxybutyrate,PHB)合成基因簇的高拷贝质粒,大肠杆菌K-12突变菌株S17-3在携带该质粒时生长密度高,耐低pH且在低pH条件下生长时高产可拉酸(Colanic Acid,CA).[目的]系统探究与菌种(大肠杆菌S17-3)及质粒(pBHR68)相关的高密度生长现象的分子机理,提示PHB和CA合成代谢与高密度生长的偶联机制.[方法]解析质粒的构成、CA合成途径基因组成对高密度生长现象的影响;利用全基因组同比分析寻找可能的关键突变基因;开展转录组学分析,筛查大肠杆菌S17-3及其转化子在不同培养方式中的转录组数据,通过基因敲除实现基因功能及细胞生长状态的验证.[结果]大肠杆菌S17-3的高密度生长菌与PHB合成的操纵子的过表达以及rhsA的多位点突变相关,RcsA是CA合成与高密度生长中碳代谢流调控的关键调控蛋白.在低pH培养时,敲除可拉酸合成的关键糖基转移酶导致生物量提升;此外,大肠杆菌S 17-3/pBHR68的高密度生长还可能与乳糖操纵子异常的转录调控相关,lacZ突变株高密度生长特性消失,而且无法合成可拉酸.[结论]研究分析了引起大肠杆菌S17-3高密度生长的多种因素,为大肠杆菌提高生长密度现象的进一步分析提供了重要线索,也为利用大肠杆菌S17-3的优异生理特性将其改造为寡糖合成的底盘细胞奠定了研究基础.

Catalases are a large group of enzymes that decompose hydrogen peroxide to oxygen and hydrogen, and have been applied widely in numerous areas. Bacillus subtilis ATCC 6051a is a well-known host strain for high level secretion of heterologous peptides. However, the application of 6051a was seriously hampered by insufficient transformation efficiency. In this study, D-xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, generating 164S, a mutant owns a transformation efficiency of 1 000-fold higher than its parent strain, thus allowing gene replacement by double crossover recombination using linear dsDNAs. The efficiency of the flanking arms for homologous recombination was then analyzed. We found that 400 bp was the minimal length of homologous fragments required to initiate efficient recombination in the 164S strain. In addition, DNA cassettes encoding two mesophilic catalases (Orf 2-62 and Orf 2-63) from B. licheniformis were integrated onto 164S. The catalytic properties of recombinant Orf 2-62 and Orf 2-63 were analyzed, and were found to be predominantly secreted into the fermentation broth, although they obviously lack any known secretory signal peptide. This work demonstrated that B. subtilis 164S is an excellent cell tool, not only for its superior secretion capacity, but also for its convenience in genetic modification.

Background: Xylan is the second most abundant polysaccharide biomass on the earth, the polymers have a backbone of β-1,4-linked xylose residues with various side-chain substitutions, such as arabinose, acetic acid, glucuronic acid, and other esterified groups, thus, the removal of arabinose side groups by α-L-arabinofuranosidases is helpful in various industrial processes involving xylan treatment. Bacillus subtilis ATCC 6051a is known for its excellent capacity of secretory production of recombinant peptides, however, poor experience in genetic manipulation and lack of universal expression elements impede this strain for wider application.Results: Xylose inducible comK was integrated into the genome of B. subtilis ATCC 6051a, and the transformation efficiency of the engineered strain B. subtilis 164S was increased by more than 1000 folds. B. subtilis 164S was further modified to generate B. subtilis 164T7P which incorporates a D-xylose inducible T7 RNA polymerase. The recombinant GFP expressed by 164T7P is more than thirteen times that achieved by P43 promoter, representing the most efficient expression system that had been ever reported in B. subtilis . Subsequently, abfA1 , encoding a glycoside hydrolase (GH) family 51 enzyme was cloned and overexpressed in 164T7P. The activity of recombinant α-L-arabinofuranosidase (AbfA1) reached 90.6±2.0 U mL -1 in the fermentation broth. Using p NPA as a substrate, kinetic parameters of the crude enzyme were Km of 1.4±0.1 mM and kcat of 139.4 s −1 . The optimum temperature and pH of the recombinant AbfA1 towards p NPA were observed to be 45 °C and pH 6.5, respectively.Conclusion: With enhanced cellular competence and introduction of T7 RNA polymerase, B. subtilis ATCC 6051a was engineered as a versatile cell tool for recombinant production of heterologous peptides employing T7 promoter. The novel expression kit demonstrated a very low level of leaky expression of target genes. The efficiency and applicability of the system were demonstrated by high-level production of a bacterial type α-L-arabinofuranosidase.

聚-3-羟基丁酸酯(PHB)的合成可以通过在重组大肠杆菌中过表达phaCAB操纵子实现.提升菌株的性能被认为是降低PHB生产成本的关键因素之一.在大肠杆菌S17-3中表达PHB的合成质粒pBhya-CAB,却可以自发实现菌株的高密度生长,摇瓶生长时的最高OD600可达40左右.全基因组扫描测序和转录组测序分析比对表明,一些编码碳代谢流相关蛋白如磷酸葡萄糖异构酶(pgi)和6-磷酸葡萄糖脱氢酶(zwf)发生了阅读框序列或转录水平的改变.在S17-3中敲除zwf会导致转化菌株生长密度的降低,而在模式大肠杆菌BW25113中敲除pgi则会引起转化子的生长密度变高,揭示了S17-3高密度生长与自身碳代谢流的优化密切相关.

Background: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. Result: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17- 3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. Conclusion: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified. (C) 2020 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved.

Human milk oligosaccharides (HMOs) are beneficial for infants' health and growth. As one of the most abundant oligosaccharides in human milk, 2'-fucosyllactose (2'-FL) has been approved to supplement in infant formula. Microbial synthesis of 2'-FL achieved in E. coli tends to use a T7-expression system for the heterologous expression of the fucosyltransferase and/or enzymes involved in fucose metabolism. In this paper, we report a novel bioconversion route of 2'-FL by engineering a low pH triggered colanic acid (CA) synthetic pathway, found in E. coli. S17 - 3, which supplies GDP-L-fucose for in vivo 2'-FL formation catalyzed by the heterologous alpha-1,2-fucosyltransferases. In medium added with 10 g/L lactose and 20 g/L glycerol, recombinant S17 - 3 was able to produce 0.617 g/L of 2'-FL. The concentration of 2'-FL came to 1.029 g/L when a heterologous pathway for the synthesis of polyhydroxybutyrate was additionally introduced in the engineered S17 - 3.

The biosynthesis of colanic acid (CA) in Escherichia coli was known to be activated during growth at low temperature using sub-optimal medium. However, in this study, an E. coli transformant S173-H (S17-3 with plasmid pBhya-CAB) was found to be able to excrete high amount of CA (10.39 g/L) in glucose supplemented Luria-Bertani medium (LBG) when growing at 37 °C. Inoculation of cells in low pH medium was required for the derepression of the CA regulon, another indispensable requirement was the use of high copy number plasmid for over-expression of the heterologous polyhydroxybutyrate (PHB) biosynthesis pathway in S17-3. In addition, S173-H exhibited superior growth performance in LBG, the maximal cell density (OD600) of cultures reached 40.0, far exceeding that of any known E. coli strains cultivated under similar conditions. Genomic data mining and transcriptional analysis hinted that the persistent growth or CA production might be modulated by interplaying regulation networks that signal the level of messenger substrate, acetyl-CoA or acetylphosphate. Depletion of these messenger substrates may be triggered by efficient PHB biosynthesis that links to enhanced capability in NADPH regeneration in S17-3, due to mutations on loci at pgi, csrA, or other sites.