Biocatalytic production of D- p -hydroxyphenylglycine by optimizing protein expression and cell wall engineering in Escherichia coli Abstract D-p-hydroxyphenylglycine (D-HPG) functions as an intermediate and has important value in antibiotic industries. The high pollution and costs from chemical processes make biotechnological route for D-HPG highly desirable. Here, a whole-cell transformation process by D-hydantoinase(Hase) and D-carbamoylase(Case) was developed to produce D-HPG from DL-hydroxyphenylhydantoin(DL-HPH) in Escherichia coli. The artificially designed ribosome binding site with strong intensity significantly facilitated the protein expression of limiting step enzyme Case. Next, the cell wall permeability was improved by disturbing the peptidoglycan structure by overproduction of D,D-carboxypeptidases without obviously affecting cell growth, to increase the bioavailability of low soluble hydantoin substrate. By fine-tuning regulation of expression level of D,D-carboxypeptidase DacB, the final production yield of D-HPG increased to 100% with 140 mM DL-HPH substrate under the optimized transformation conditions. This is the first example to enhance bio-productivity of chemicals by cell wall engineering and creates a new vision on biotransformation of sparingly soluble substrates. Additionally, the newly demonstrated ‘hydroxyl occupancy’ phenomenon when Case reacts with hydroxyl substrates provides a referential information for the enzyme engineering in future.

Deterministic and stochastic processes driving the shift in the prokaryotic community composition in wastewater treatment plants of a coastal Chinese city Abstract Wastewater treatment plants (WWTPs) rely mainly on the microbial assemblages to contribute significantly for the removal of organic pollutants and nutrients. However, limited information is available on the ecological driving forces underlying the turnover of prokaryotic communities across wastewater treatment processes (i.e., from influents (IFs) and effluents (EFs)) within WWTPs. Here, we used a combination of the 16S rRNA gene amplicon sequencing and a quantitative ecological null model analysis to explore the ecological processes governing the turnover of the prokaryotic communities and the dominant taxonomic taxa across wastewater treatment processes of five full-scale WWTPs in China. Our results indicated that a significant variation in the composition of prokaryotic communities and the dominant taxa between IFs and EFs. The analysis of the environmental sources of indicator OTUs showed that a relatively lower abundance of the sludge/sewage and human guts associated OTUs in EFs than in IFs. Ecological null models revealed that among the ecological processes, deterministic processes were dominant in controlling the turnover of the overall communities from IFs to EFs, whereas the relative importance of deterministic processes varied among the dominant taxa (i.e., Bacteroidetes > Proteobacteria > Gammaproteobacteria > Firmicutes > Betaproteobacteria). However, the assembly of IF and EF communities was influenced mainly by the deterministic and stochastic processes, respectively. In addition, our results indicated that EF communities have a higher phylogenetic diversity than those of the IF communities, but the abundance of prokaryotic 16S rRNA genes was lower in EFs than in IFs. Overall, our study provides a novel insight of the assembly mechanisms underlying the turnover of prokaryotic communities during wastewater treatment processes.

A microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli Abstract Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.

Chemical composition, antibacterial properties, and mechanism of Smilax china L. polyphenols Abstract This work aimed at investigating the chemical composition, antibacterial properties, and effect mechanism of Smilax china L. polyphenols (SCLP). SCLP was extracted and purified, and then, its eighteen polyphenolic compounds were identified by LC-MS/MS analysis. SCLP exhibited antibacterial activity against five bacteria (Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, and Escherichia coli) with minimum inhibitory concentration in a range of 195.31 to 781.25 μg/mL. Escherichia coli and Staphylococcus aureus showed a higher sensitivity to SCLP. Notably, when combined with antibiotics, the SCLP–thiamphenicol and SCLP–gatifloxacin combinations showed additional properties against Escherichia coli and Staphylococcus aureus, while SCLP–streptomycin and SCLP–penicillin combinations exhibited dramatically synergistic effects. In addition, the changes in permeability and integrity of the cell membrane and cell wall were observed by measuring UV absorption, extracellular AKP concentration, FTIR spectroscopy, and scanning electron microscopy. It is speculated that the mechanism of action of SCLP on bacteria may be described as destruction of bacterial cell wall and cell membrane. In conclusion, SCLP was a potential natural antimicrobial substance with strong antimicrobial activity, which may reduce the use of antibiotics or combat drug-resistant bacteria through synergistic combination with antibiotics.

Unique processes yielding pure azaphilones in Talaromyces atroroseus Abstract Azaphilones are a class of fungal pigments, reported mostly in association with Monascus species. In Asian countries, they are used as food colourants under the name of “red yeast rice” and their production process is well described. One major limitation of current production techniques of azaphilones is that they always occur in a mixture of yellow, orange and red pigments. These mixtures are difficult to control and to quantify. This study has established a controlled and reproducible cultivation protocol to selectively tailor production of individual pigments during a submerged fermentation using another fungal species capable of producing azaphilone pigments, Talaromyces atroroseus, using single amino acids as the sole nitrogen source. The produced azaphilone pigments are called atrorosins and are amino acid derivatives of the known azaphilone pigment Penicillium purpurogenum–orange (PP-O), with the amino acid used as nitrogen source incorporated into the core skeleton of the azaphilone. This strategy was successfully demonstrated using 18 proteinogenic amino acids and the non-proteinogenic amino acid ornithine. Two cultivation methods for production of the pure serine derivative (atrorosin S) have been further developed, with yields of 0.9 g/L being obtained. Yielding pure atrorosins through switching from KNO3 to single amino acids as nitrogen source allows for considerably easier downstream processing and thus further enhances the commercial relevance of azaphilone producing fungal cell factories.

Identification of major malate export systems in an engineered malate-producing Escherichia coli aided by substrate similarity search Abstract Optimization of export mechanisms for valuable extracellular products is important for the development of efficient microbial production processes. Identification of the relevant export mechanism is the prerequisite step for product export optimization. In this work, we identified transporters involved in malate export in an engineered l-malate-producing Escherichia coli strain using cheminformatics-guided genetics tests. Among all short-chain di- or tricarboxylates with known transporters in E. coli, citrate, tartrate, and succinate are most chemically similar to malate as estimated by their molecular signatures. Inactivation of three previously reported transporters for succinate, tartrate, and citrate, DcuA, TtdT, and CitT, respectively, dramatically decreased malate production and fermentative growth, suggesting that these transporters have substrate promiscuity for different short-chain organic acids and constitute the major malate export system in E. coli. Malate export deficiency led to an increase in cell sizes and accumulation of intracellular metabolites related to malate metabolism.

Production, characteristics, and biotechnological applications of microbial xylanases Abstract Microbial xylanases have gathered great attention due to their biotechnological potential at industrial scale for many processes. A variety of lignocellulosic materials, such as sugarcane bagasse, rice straw, rice bran, wheat straw, wheat bran, corn cob, and ragi bran, are used for xylanase production which also solved the great issue of solid waste management. Both solid-state and submerged fermentation have been used for xylanase production controlled by various physical and nutritional parameters. Majority of xylanases have optimum pH in the range of 4.0–9.0 with optimum temperature at 30–60 °C. For biochemical, molecular studies and also for successful application in industries, purification and characterization of xylanase have been carried out using various appropriate techniques. Cloning and genetic engineering are used for commercial-level production of xylanase, to meet specific economic viability and industrial needs. Microbial xylanases are used in various biotechnological applications like biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. This review describes production, characteristics, and biotechnological applications of microbial xylanases.

Recent studies on the biological production of D-mannose Abstract D-Mannose is an epimer of glucose at the C-2 position and exists in nature as a component of mannan. It has 60 and 86% sweetness than that of sucrose and D-glucose, respectively. Because of its low-calorie and nontoxic features, D-mannose is used widely in food, medicine, cosmetic, and food-additive industries. Besides, it exhibits many physiologic benefits on health: immune system, diabetes mellitus, intestinal diseases, and urinary tract infections. It is used as a starting material to synthesize immunostimulatory agents, anti-tumor agents, vitamins, and D-mannitol. However, D-mannose production using chemical synthesis and plant extraction cannot meet the requirements of the industry. This article presents recent research on the biological production of D-mannose. The physiologic benefits and applications of D-mannose are summarized. Besides, different D-mannose-producing enzymes from various sources are discussed in detail with regard to their biochemical characteristics, catalytic efficiency, and reaction kinetics for D-mannose production. Furthermore, attempts to use enzymatic conversion to produce D-mannose are reviewed.

Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp Abstract This study evaluated the inhibition and interaction of Bacillus velezensis BvL03 as a probiotic agent against Aeromonas hydrophila. Strain BvL03 isolated from sediment samples of fish ponds had excellent antimicrobial activity against several fish pathogenic bacteria, especially Aeromonas, including A. hydrophila, A. veronii, A. caviae, and A. sobria. The successful amplification of lipopeptide antimicrobial chemical biosynthetic genes, including iturin family (ituA, ituB, and ituD), bacillomycin family (bacA, bacD, and bacAB), surfactin family (srfAB, srfC, and srfAA), and subtilosin family (albF and sunT) from the genome of BvL03 strain, confirmed its predominant antimicrobial activity. The challenge test suggested that BvL03 significantly decreased fish mortality when challenged with A. hydrophila, which had a cumulative mortality of 12.5% in the treatment group. Toxicity and hemolytic activity of A. hydrophila after co-cultured with BvL03 were relieved as confirmed by the cell experiments, when the initial inoculated concentration of BvL03 was 109 cfu/mL or higher. Moreover, the BvL03 strain labeled with GFP protein (BvL03-GFP) and AhX040 strain labeled with mCherry protein (AhX040-mCherry) were injected into grass carps. The fluorescence levels were monitored by using In Vivo Imaging System (IVIS), in which the green color was steadily increasing, whereas the red color was gradually weakening. Whole genome sequencing revealed that strain BvL03 possesses 15 gene clusters related to antibacterial compounds, including 5 NRPS gene clusters and 3 PKS gene clusters. These results suggested that B. velezensis BvL03 has the potential to be developed as a probiotic candidate against A. hydrophila infection in aquaculture.

Insulin and its single-chain analogue Abstract Insulin therapy remains the most effective method to treat diabetes mellitus (DM), and the demand for this valuable hormone has exceeded that of any other protein-based medicine as a result of the dramatic increase in the number of diabetic patients worldwide. Understanding the structure of insulin and the interaction with its receptor is important for developing proper formulations. As a result of the relatively low thermal stability of native insulin and its two-chain analogues, the application of single-chain insulin (SCI) analogues, which can be obtained relatively easily by recombinant DNA technology or chemical synthetic methods, represents a promising alternative approach. In this review, the basic knowledge of insulin (discovery, biosynthesis, and structure) and the current model of the interaction with its receptor are outlined. Furthermore, we outline the strategies for the design and production of various SCI analogues and their reported applications.