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Phospholipase D (PLD) is an essential enzyme for the enzymatic phosphatidylserine synthesis and Bacillus subtilis is a potential microbial cell factory for the PLD production, but its widespread application in the food industry is limited by low expression levels and the use of antibiotics. In this study, a multi-copy recombinant strain BS-PLD312 was constructed through screening genome location and the number of PLD expression cassettes. The enzyme activity of BS-PLD312 can reached 392.88 U/mL in the absence of antibiotics and inducers. Furthermore, a food grade reaction system within egg yolk was established and the phosphatidylserine content reached 35.13 g/L in egg yolk. This reaction system is applicable for synthesis of other PLs and regulating the composition of various phospholipids in egg yolk. The protocols established in this study shows great potential for application in industrial PLD production and expanded the practical application of PLD in food industry. • A multicopy recombinant strain was constructed through screening genome location and the number of PLD expression cassettes. • The PLD activity of recombinant strain reached 392.88 U/mL. • The recombinant strain exhibited excellent expression stability without addition of antibiotics. • A food grade biphasic reaction system was constructed for the synthesis of PS in egg yolk, achieving a yield of 35.13 g/L. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) are extensively utilized in clinical, cosmetic, and healthcare field, as well as in the treatment of thrombosis, osteoarthritis, rheumatism, and cancer. The biological production of GAGs is a strategy that has garnered significant attention due to its numerous advantages over traditional preparation methods. In this review, we embark on a journey to decode the intricate molecular symphony that orchestrates the biosynthesis of glycosaminoglycans. By unraveling the complex interplay of related enzymes and thorough excavation of the intricate metabolic cascades involved, GAGs chain aggregation and transportation, which efficiently and controllably modulate GAGs sulfation patterns involved in biosynthetic pathway, we endeavor to offer a thorough comprehension of how these remarkable GAGs are intricately assembled and pushes the boundaries of our understanding in GAGs biosynthesis. • The enzymes involved in GAGs synthesis pathways are summarized. • Microbial production systems and its synthesis mechanism are discussed. • The sulfation model involved in GAGs biosynthesis pathway was analyzed. [ABSTRACT FROM AUTHOR]

β-Nicotinamide mononucleotide (NMN) is a direct precursor of the important coenzyme NAD+ in the human body and has broad application prospects in healthcare. This study engineered an Escherichia coli BL21 (DE3) strain to efficiently biosynthesize NMN from nicotinamide (NAM) and xylose. Firstly, the nicotinamide phosphoribosyltransferase (Nampt) from Chitinophaga pinensis was selected and heterologous expressed to construct the NAD+ remediation pathway for NMN synthesis from nicotinamide (NAM) and in vivo precursor phosphoribosyl pyrophosphate (PRPP). Then, the supply of PRPP was enhanced by constructing the metabolic flux from d -xylose. To facilitate NMN accumulation, the branch pathways, including PRPP competitive pathway, carbon flow competitive pathway, and NMN downstream metabolic decomposition pathway were fine-tuned using CRISPR/Cas9 editing tools. Combined with process optimization of whole-cell biocatalytic reaction, a NMN titre of 497.5 mg·L−1 was obtained. Finally, the scale-up culture was carried out on a 5 L fermenter, and the yield reached 760.2 mg·L−1. This work achieved green biosynthesis of NMN using xylose as substrate and enhanced the productivity by systematic engineering strategies, presenting more possibilities for the synthesis of NMN from a wide range of monosaccharides. [Display omitted] [ABSTRACT FROM AUTHOR]

The constitutively expression system for P. putida nitrilase was firstly constructed to improve the nicotinic acid production and reduce the production costs. High density culture strategy was employed to enhance the biomass and nitrilase production of recombinant strain. The total nitrilase activity reached up to 654 U·mL −1 without the induction. 541 g·L −1 nicotinic acid was accumulated via fed batch mode of substrate feeding through 290 min of conversion. [ABSTRACT FROM AUTHOR]

The objective of the study was to enhance the substrate tolerance of Pseudomonas putida nitrilase via atmospheric and room temperature plasma (ARTP) and cell immobilization. The mutant library was constructed by ARTP and rapidly screened by an OPA-TCA microscale reaction. A mutant strain of mut-D3 was obtained and its optimum substrate concentration was improved to 150 mM from 100 mM. It could accumulate 189 g/L nicotinic acid (NA) from 3-cyanopyridine (3-CP), which was increased by 42% compared with that of wild type (WT). Additionally, composite immobilization of mut-D3 was performed and SA-PVA immobilized cells could catalyze 250 mM 3-CP each batch with finally accumulating 346 g/L NA, while free cells accumulated 175 g/L NA. These results indicated that the free or immobilized catalysts of mut-D3 could serve as a good choice for NA production. This is the first report on mutation breeding of nitrilase-producing microorganisms by ARTP. [ABSTRACT FROM AUTHOR]

This study reveals the genetic and biochemical changes underlying the enhanced hyaluronan (HA) biosynthesis in Streptococcus zooepidemicus. After multiple rounds of atmospheric and room temperature plasma (ARTP) mutagenesis combined with novel bovine serum albumin/cetyltrimethylammonium bromide coupled high-throughput screening assay, the HA yield of the mutant was increased by 42.9% and reached 0.813 g L−1 with a molecular weight of 0.54 × 106 Da within 18 h by shaking flask culture. HA production was increased to 4.56 g L−1 by batch culture in 5-L fermenter. Transcriptome sequencing exhibits that distinct mutants have similar genetic changes. Regulation in direction of metabolic flow into the HA biosynthesis, by enhancing genes responsible for the biosynthesis of HA including hasB , glmU and glmM , weaking downstream gene (nagA and nagB) of UDP-GlcNAc and significantly down-regulating transcription of wall-synthesizing genes, resulting in the accumulation of precursors (UDP-GlcA and UDP-GlcNAc) increased by 39.74% and 119.22%, respectively. These associated regulatory genes may provide control point for engineering of the efficient HA-producing cell factory. [Display omitted] • ARTP mutagenesis combining with novel coupled screening assay were performed. • After multiple rounds of mutagenesis, the HA yield was increased by 42.9 %. • Distinct mutants have similar genetic changes responded to HA accumulation trends. • UDP-GlcA and UDP-GlcNAc accumulation increased by 39.74 % and 119.22 %, respectively. • Relevant enzymes and genetic variation responsible for HA biosynthesis were analyzed. [ABSTRACT FROM AUTHOR]

β-Mannanases can efficiently hydrolyze both the polysaccharide mannan and manno-oligosaccharides. In order to characterize a novel mannanase for potential industrial application, an endo-1,4-β-mannanase from Bacillus subtilis YH12 was purified to homogeneity and its biochemical characterization was performed. The optimal conditions for the purified enzyme were pH 6.5 and 55 °C, and it remained stable at temperature up to 60 °C and pH of 4.5–7.5. The enzyme had specific activity on complex structure polysaccharide. And it showed high activity on locust bean gum, konjac powder, guar and fenugreek gums. Furthermore, it efficiently hydrolyzed xanthan, and carrageenan gums, and it exhibited no activity on starch, and xylan. The mannanase exhibited higher activity on galactomannan branched with (1→6)-linked α- d -galactose than glucomannan. The predominant products resulting from mannanase hydrolysis were 1–7 units of manno-oligosaccharides from locust bean gum, 2–7 units of manno-oligosaccharides from konjac powder, and 1–2 units of manno-oligosaccharides from xanthan gum. The B. subtilis YH12 mannanase encoding gene was successfully cloned. This study provided a novel mannanase that demonstrates potential application in food and animal feeds technology. [ABSTRACT FROM AUTHOR]

It has long been found that hyaluronidases exist in a variety of organisms, playing their roles in various biological processes including infection, envenomation and metabolic regulation through degrading hyaluronan. However, exploiting them as a bioresource for specific applications had not been extensively studied until the latest decades. In recent years, new application scenarios have been developed, which extended the field of application, and emphasized the research value of hyaluronidase. This critical review comprehensively summarizes existing studies on hyaluronidase from different source, particularly in their structures, action patterns, and biological functions in human and mammals. Furthermore, we give in-depth insight into the resource mining and protein engineering process of hyaluronidase, as well as strategies for their high-level production, indicating that mixed strategies should be adopted to obtain well-performing hyaluronidase with efficiency. In addition, advances in application of hyaluronidase were summarized and discussed. Finally, prospects for future researches are proposed, highlighting the importance of further investigation into the characteristics of hyaluronidases, and the necessity of investigating their products for the development of their application value. [ABSTRACT FROM AUTHOR]

[Display omitted] • A high-efficiency expression system of PLD in B. subtilis was constructed. • Optimized culture strategy achieved a high PLD activity of 4055.9 U/mL and a production efficiency of 52.0 U/mL/h. • An optimal phosphatidic acid production of 219.1 g/L was achieved. Although Bacillus subtilis shows promise as a potential microbial cell factory for phospholipase D (PLD) expression, its production capacity remains insufficient. In this study, a secretory expression system, by co-optimization the promoter and signal peptides and employing a fed-batch fermentation strategy, was constructed to enhance expression of PLD from separate sources. The highest PLD production of 4056.9 U/mL was observed using this system, with a PLD production efficiency of 52.0 U/mL/h. Finally, a phosphatidic acid (PA) biosynthesis system was established using the constructed PLD as a catalyst, which achieved a PA yield of 219.1 g/L. This is the highest PLD production and PA yield reported globally to date. The protocol has significant potential for application for industrial PLD production as well as enzymatic phospholipids modification and also provides a valuable reference for overexpressing proteins in B. subtilis. [ABSTRACT FROM AUTHOR]

Keratinases are unique among proteolytic enzymes for their ability to degrade recalcitrant insoluble proteins, and they are of critical importance in keratin waste management. Over the past few decades, researchers have focused on discovering keratinase producers, as well as producing and characterizing keratinases. The application potential of keratinases has been investigated in the feed, fertilizer, leathering, detergent, cosmetic, and medical industries. However, the commercial availability of keratinases is still limited due to poor productivity and properties, such as thermostability, storage stability and resistance to organic reagents. Advances in molecular biotechnology have provided powerful tools for enhancing the production and functional properties of keratinase. This critical review systematically summarizes the application potential of keratinase, and in particular certain newly discovered catalytic capabilities. Furthermore, we provide comprehensive insight into mechanistic and molecular aspects of keratinases including analysis of gene sequences and protein structures. In addition, development and current advances in protein engineering of keratinases are summarized and discussed, revealing that the engineering of protein domains such as signal peptides and pro-peptides has become an important strategy to increase production of keratinases. Finally, prospects for further development are also proposed, indicating that advanced protein engineering technologies will lead to improved and additional commercial keratinases for various industrial applications. [ABSTRACT FROM AUTHOR]

Wool waste is the main by-product of the textile industry, which causes environmental problems. Therefore, the recycling of wool waste is of increasing concern and is an important tool for environmental protection and resource-efficient utilization. In this study, an eco-friendly process ensuring complete recycling of wool waste was developed using glutathione as a reducing agent. First, the main component keratin, which is an important source of renewable biomaterial, was extracted. Keratin products were characterized by infrared spectroscopy, Raman spectroscopy, and circular dichroism spectroscopy. The results indicated that glutathione treatment could cause approximately 65% weight dissolution in wool, and keratins with molecular weights of 26, 44, and 66 kDa could be obtained. Furthermore, the residues were collected and prepared as sponges. These sponges showed an excellent adsorption capacity for cationic dye methylene blue (MB). In conclusion, sustainable wool waste recycling coupled with the cleaner production of keratins and a new adsorbent was achieved; this method can turn "waste" into "wealth" and solve the environmental pressures caused by waste. To our knowledge, this is the first report to demonstrate the usefulness of glutathione in an ecofriendly process for the extraction of keratin from wool. Image 1 • A full utilization strategy was proposed for wool wastes dispose. • A novel sustainable process was developed for extracting keratin from wool wastes. • Glutathione can effectively degrade wool fibers by reducing disulfide bond. • A novel adsorbent was prepared based on the remaining wool residues. • The wool residue sponge showed excellent adsorption capability for the cationic dye. [ABSTRACT FROM AUTHOR]

• A promoter screening system of keratinase expression was constructed. • Promoter engineering significantly improved the keratinase activity by 16-fold. • Scale-up fermentation resulted in the highest keratinase activity of 16,860 U/mL. • The keratinase showed potential in biodegradation of feather wastes. Keratinases are promising alternatives over ordinary proteases in several industrial applications due to their unique properties compared with their counterparts in the protease categories. However, their large-scale industrial application is limited by the low expression and poor fermentation efficiency of keratinase. Here, we demonstrate that the expression level of keratinase can be improved by constructing a more efficient enzyme expression system hereby enables the highest production titer as regarding recombinant keratinase production to date. Specially, ten promoters were evaluated and the aprE promoter exhibits a significant promotion of keratinase (kerBv) titer from 165 U/mL to 2605 U/mL in Bacillus subtilis. The batch fermentation mode resulted in a maximum keratinase activity of 7176 U/mL at 36 h in a 5-L fermenter. Furthermore, the extracellular keratinase activity attained up to 16,860 U/mL via fed-batch fermentation within 30 h. The combination of keratinase with l -cysteine brings about 66.4 % degree of degradation of feather. Our work provides a new insight into the development of efficient keratinase fermentation processes with B. subtilis cell factory. [ABSTRACT FROM AUTHOR]

• Synthetic biology strategies facilitate the efficient production of hyaluronic acid. • Microbial production systems and its synthesis mechanism are summarized. • The fermentation strategies and its application of hyaluronic acid are discussed. Owing to its outstanding water-retention ability, viscoelasticity, biocompatibility and non-immunogenicity, Hyaluronic acid (HA), a natural linear polymer alternating linked by d -glucuronic acid and N-acetylglucosamine, has been widely employed in cosmetic, medical and clinical applications. With the development of synthetic biology and bioprocessing optimization, HA production via microbial fermentation is an economical and sustainable alternative over traditional animal extraction methods. Indeed, recently Streptococci and other recombinant systems for HA synthesis has received increasing interests due to its technical advantages. This review summarizes the production of HA by microorganisms and demonstrates its synthesis mechanism, focusing on the current status in various production systems, as well as common synthetic biology strategies include driving more carbon flux into HA biosynthesis and regulating the molecular weight (MW), and finally discusses the major challenges and prospects. [ABSTRACT FROM AUTHOR]

Large-scale submerged fermentation (SmF) of Antrodia camphorata ( A. camphorata ) usually encounters challenges including tedious preparation of mycelial inoculum, long fermentation period (10–14 d), and poor repeatability. Here we developed an asexual reproduction-based repeated batch fermentation (RBF) process for bioactive metabolites production by A. camphorata ATCC 200183. Compared with traditional batch fermentation, production time was shortened to 58 d from 80 d (overall time for eight cycles) using the RBF process established in this study, and accordingly, the productivities of bioactive metabolites (including antrodins) were improved by 40–60%. Kinetic parameters ( α is 2.1–18.7 times as β ) indicated that the cell growth was the major contribution for bioactive metabolites production. The RBF shows excellent batch-repeatability (Pearson correlation coefficient of 0.998 ± 0.001), together with advantages of energy-efficient, low cost, and labor-saving, RBF process can be implemented to SmF by other filamentous fungi. [ABSTRACT FROM AUTHOR]

Hydroxylation of DHEA to 7α,15α-diOH-DHEA was catalyzed by NADPH-dependent cytochrome P450 monooxygenase from Colletotrichum lini . By adding coenzyme precursor nicotinic acid, the NADPH/NADP ratio was significantly increased, and the 7α,15α-diOH-DHEA molar conversion was enhanced from 37.37% to 50.85%. To improve the availability of intracellular NADPH, a dual cosubstrate-coupled system consisting of nicotinic acid and glucose was investigated in C. lini . Using 20 mM nicotinic acid and 15 g/L glucose as cosubstrate for NADPH regeneration, the 7α,15α-diOH-DHEA molar conversion was dramatically increased by 74.58%. The conversion course was simultaneously shortened by 30 h. Moreover, a fed-batch transformation model was established to diminish DHEA toxicity to C. lini and further increase DHEA concentration. The maximum concentration of DHEA was elevated to 15 g/L using a three-batch transformation in a coenzyme regeneration system, and 7α,15α-diOH-DHEA production of 11.21 g/L could be achieved after 60 h of biotransformation. These results demonstrated that this strategy was promising for steroids hydroxylation. [ABSTRACT FROM AUTHOR]