Colanic acid (CA) is an extracellular polysaccharide produced by Escherichia coli that has attracted increasing interest because of its water-holding capacity, viscoelasticity, and bioactive sugar residues, which support its potential applications in the food, pharmaceutical, and materials fields. However, the regulatory basis underlying its efficient biosynthesis remains insufficiently understood. In this study, a marked increase in CA production was unexpectedly observed during construction of a 1,3-butanediol (1,3-BDO) biosynthetic pathway in E. coli S17-3. Stepwise dissection of the heterologous pathway identified the Bacillus subtilis-derived sfp gene as the key determinant responsible for this phenotype. To elucidate how sfp promotes CA biosynthesis, comparative transcriptomic analysis and functional validation were performed. Expression of sfp induced extensive metabolic reprogramming and activated the wca cluster. Further validation identified YaiY, an inner membrane protein, and YpeC, a putative envelope-associated protein, as positive contributors to CA biosynthesis. This study identifies heterologous sfp as a key factor promoting CA biosynthesis in E. coli and reveals YaiY and YpeC as previously unrecognized contributors to this process, thereby providing new insight into the regulatory basis of CA production and new targets for strain engineering.

Abstract Background l‑Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5′‑diphosphate (GDP)‑l‑fucose. Therefore, a more direct approach for biomanufacturing l‑fucose could be the enzymatic degradation of GDP‑l‑fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP‑l‑fucose. Results Herein, we constructed a de novo l‑fucose synthetic route in Bacillus subtilis by introducing heterologous GDP‑l‑fucose synthesis pathway and engineering GDP‑mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP‑l‑fucose, therefore, a substrate simulation‑based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP‑l‑fucose‑splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield l‑fucose. WcaHR36Y/N38R was found to produce 1.6 g/L l‑fucose during shake‑flask growth, which was 67.3% higher than that achieved by wild‑type WcaH. The accumulated l‑fucose concentration in a 5 L bioreactor reached 6.4 g/L. Conclusions In this study, we established a novel microbial engineering platform for the fermentation production of l‑fucose. Additionally, we found an efficient GDP‑mannose mannosyl hydrolase mutant for L‑fucose biosynthesis that directly hydrolyzes GDP‑l‑fucose. The engineered strain system established in this study is expected to provide new solutions for l‑fucose or its high value‑added derivatives production.

Citramalic acid (citramalate) is important for the chemical synthesis of methylmethacrylate, a bulk monomer used for manufacturing biodegradable polymers. Escherichia coli strains have been engineered for the biological production of citramalate since a rare mutant of citramalate synthase was invented as a mesophilic catalyzer to form the chemical. However, acidic byproducts are routinely excreted by E. coli host cells during fermentation, which could significantly reduce the carbon conversion efficiency of biosynthetic chemicals, including citramalate. Herein, an unusual mutant called S17-3 was reported, and this mutant demonstrated superb recovery efficacy from glucose to citramalate and secreted a very low level of fermentation byproducts during the fermentative production of citramalate. However, S17-3 tends to produce a large amount of colonic acid (CA). Genetically manipulating the regulators involved in CA synthesis revealed that the deletion of rcsD could significantly improve the citramalate titer. Fed-batch fermentation under optimized conditions led to a maximum titer of 46.2 g/L citramalate accumulated in the broth, with a conversion efficiency of 0.80 g citramalate per gram glucose and a close-to-the-theoretical mole transversion efficiency (mol/mol) of 0.97. This study facilitates the development of a practical bioproduction of citramalate using E. coli strains.

Although rice husk (RH) has been used as a compost additive, the microbes present in RH are not well understood. In this study, we explored the physicochemical parameters, the microbial population and functional succession to better understand how different levels (T0: 0; T1: 20%; T2: 30%) of RH affected the performance of cow manure composting. Higher thermophilic temperatures (70 degrees C) and higher-quality compost with respect to pH, the C/N ratio, particle-size distribution and other characteristics were obtained with the addition of 20% RH. The total nitrogen and germination index (GI) of the final product (T1) increased by 48% and 175%, respectively, compared to those of T0. Moreover, fluorescence spectra indicated that the degradation of protein-type groups and the formation of humic substances increased in T1. Microbial analyses indicated that interactions between bacterial communities were also strengthened in T1. We also found that Sphaerobacter and Myceliophthora were the unique and dominant thermophilic genera of bacteria and fungi, respectively, in RH-supplemented compost. Carbohydrate metabolism and amino acid metabolism were the main metabolic pathways, and saprotrophs represented the dominant fungal trophic mode in the composting process. In T1, bacteria were mainly affected by temperature and pH, while fungi were more affected by moisture. This study found a suitable RH dosage for composting and provides a microbiological basis for RH as a compost additive. (C) 2021 Published by Elsevier B.V.

Co-composting of recycled cow manure and waste bedding material has been used to convert both agricultural wastes to biofertilizers. This study explored the succession of microbial community, metabolic function and substances conversion capacities during 60 days’ co-composting using high throughput sequencing technology. The study revealed that co-composting of cow manure and bedding material waste at a ratio of 1.32 (CM+B) had the highest efficiency among four treatments. The bacterial and fungal community diversity changed significantly during the co-composting of CM+B group, and the major phyla included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria and Ascomycota. PICRUSt and FUNGuild analysis showed that carbohydrate, lipid metabolism and especially nitrogen fixation were enhanced in the thermophilic phase, while animal and plant pathogens were not detected after the co-composting. Wood saprotrophs became the dominant fungal group (89.1%) in the maturation phase. Canonical correlation analysis (CCA) and redundancy analysis (RDA) confirmed that temperature influenced bacterial community succession more than it influenced fungal community succession. Ruminiclostridium had a significantly positive relationship with temperature (p_value < 0.05), while pH and C/N had significant effect on the fungal (p_value < 0.05), and Penicillium and Mortierella were significantly related to moisture (p_value < 0.05). This work describes an efficient methodology to deal with co-composting systems that had been successfully applied in agricultural wastes treatment, enabling further understanding in mechanisms underlying the substance conversion and the involved microbial community succession in sophisticated composting system.

[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.

alpha-L-arabinofuranosidases (EC 3.2.1.55; AFs) cause the release of arabinosyl residues from hemicellulose polymers such as xylans, and are receiving increased levels of research attention as they could be applied in a range of processes that involve the enzymatic degradation of xylans. The secretory production of bacterial AFs has not been attempted previously. In this study, we designed a unique induction system for the production of a re-combinant AF in Bacillus subtilis in order to exploit its enzymic degradation of wheat bran. We found that non-starch phytochemicals were more efficient than mxylose when inducing the expression of T7 RNA polymerase and driving the transcription of AF by the T7 promoter. The host cell, B. subtilis (ATCC 6051a-derived strain 164T7P) was engineered to incorporate a DNA cassette that expressed T7 RNA polymerase under the control of a mxylose inducible promoter (P-xylA). The T7 promoter engineered into 164T7P was initially tested and compared with P43 in terms of GFP expression; we found that the expression level of GFP by the T7 promoter was ten-fold higher than that achieved by P43. When cultured in a flask with gentle shaking, and with D-xylose as an inducer, the recombinant strain successfully expressed arbf, a family 51 (GH 51) glycoside hydrolase from Bacillus licheniformis, and secreted 141.4 +/- 4.8 U/mL of enzyme, with a Km of 1.4 +/- 0.1 mM and a kcat of 139.4 s(-1). However, the protein was devoid of a secretary signal peptide. When cultures were supplemented with wheat bran, the maximal yield of the secreted AF reached 194.8 +/- 4.1 U/mL. The results provide a foundation for the high level production of heterologous proteins using wheat bran as the inducer in B. subtilis.