Recent advances in the biosynthesis and industrial biotechnology of Gamma-amino butyric acid.

GABA (Gamma-aminobutyric acid), a crucial neurotransmitter in the central nervous system, has gained significant attention in recent years due to its extensive benefits for human health. The review focused on recent advances in the biosynthesis and production of GABA. To begin with, the investigation evaluates GABA-producing strains and metabolic pathways, focusing on microbial sources such as Lactic Acid Bacteria, Escherichia coli, and Corynebacterium glutamicum. The metabolic pathways of GABA are elaborated upon, including the GABA shunt and critical enzymes involved in its synthesis. Next, strategies to enhance microbial GABA production are discussed, including optimization of fermentation factors, different fermentation methods such as co-culture strategy and two-step fermentation, and modification of the GABA metabolic pathway. The review also explores methods for determining glutamate (Glu) and GABA levels, emphasizing the importance of accurate quantification. Furthermore, a comprehensive market analysis and prospects are provided, highlighting current trends, potential applications, and challenges in the GABA industry. Overall, this review serves as a valuable resource for researchers and industrialists working on GABA advancements, focusing on its efficient synthesis processes and various applications, and providing novel ideas and approaches to improve GABA yield and quality.

Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion.

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis.

Identification of carotenoids biosynthesis pathway in Schizochytrium sp. and utilization in astaxanthin biosynthesis.

Carotenoids, an important kind of natural pigments with great potential commercial value, have been widely used in nutrition and health care, cosmetics and aquaculture industries. Schizochytrium sp. is a potential cell factory for lipid nutrition chemicals production including docosahexaenoic acid and carotenoids. The purpose of this study is to mine and identify the carotenoid biosynthesis genes in Schizochytrium sp. Firstly, based on the genomic information of Schizochytrium sp., we obtained the gene sequences of a trifunctional enzyme (CrtIBY), carotene hydroxylase (CrtZ) and carotene ketolase (CrtO) in carotenoids biosynthesis pathway by bioinformatics analysis. Subsequently, using the lycopene-producing E. coli as the host, 22.77 ug/L of ß-carotene and 44.31 ug/L of zeaxanthin were synthesized by overexpression of CrtIBY and further co-expression with CrtZ from Schizochytrium sp. After that, 54.78 ug/L of astaxanthin was synthesized using hydroxylase and ketonase from Haematococcus pluvialis. The key enzymes for carotenoids biosynthesis identified in this study is of great significance for further understanding the metabolic mechanism in Schizochytrium sp, which could also provide the functional elements and theoretical support for astaxanthin production.

Accumulation and conversion of ß-carotene and astaxanthin induced by abiotic stresses in Schizochytrium sp.

Astaxanthin is a kind of ketone carotenoid belonging to tetraterpenoids with an excellent antioxidant activity and it is widely used in nutrition and health-care industries. This study aimed to explore the effect of different abiotic stresses on carotenoid production in Schizochytrium sp. Firstly, the characteristics of carotenoid accumulation were studied in Schizochytrium sp. by monitoring the change of carotenoid yields and gene expressions. Then, different abiotic stresses were systematically studied to regulate the carotenoid accumulation. Results showed that low temperature could advance the astaxanthin accumulation, while ferric ion could stimulate the conversion from carotene to astaxanthin. The glucose and monosodium glutamate ratio of 100:5 was helpful for the accumulation of ß-carotene. In addition, micro-oxygen supply conditions could increase the yield of ß-carotene and astaxanthin by 25.47% and 14.92%, respectively. This study provided the potential regulation strategies for carotenoid production which might be used in different carotenoid-producing strains.

Enhancing menaquinone-7 biosynthesis by adaptive evolution of Bacillus natto through chemical modulator.

Menaquinone-7 (MK-7) is a kind of vitamin K2 playing an important role in the treatment and prevention of cardiovascular disease, osteoporosis and arterial calcification. The purpose of this study is to establish an adaptive evolution strategy based on a chemical modulator to improve MK-7 biosynthesis in Bacillus natto. The inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), glyphosate, was chosen as the chemical modulator to perform the experiments. The final strain ALE-25-40, which was obtained after 40 cycles in 25 mmol/L glyphosate, showed a maximal MK-7 titer of 62 mg/L and MK-7 productivity of 0.42 mg/(L h), representing 2.5 and 3 times the original strain, respectively. Moreover, ALE-25-40 generated fewer spores and showed a higher NADH and redox potential. Furthermore, the mechanism related to the improved performance of ALE-25-40 was investigated by comparative transcriptomics analysis. Genes related to the sporation formation were down-regulated. In addition, several genes related to NADH formation were also up-regulated. This strategy proposed here may provide a new and alternative directive for the industrial production of vitamin K2.

The current status of biotechnological production and the application of a novel antioxidant ergothioneine.

Ergothioneine is a sulfur-containing histidine derivative, that possessesexcellent antioxidant activity and has been used in the food and cosmetics industries. It plays a significant role in anti-aging and the prevention of various diseases. This review will briefly introduce the functions and applications of ergothioneine, elaborate the biosynthetic pathways of ergothioneine and describe several strategies to increase the production of ergothioneine. Then the efficient extraction and detection methods of ergothioneine will be presented. Finally, several proposals are put forward to increase the yield of ergothioneine, and the development prospects of ergothioneine will be discussed.

Transcriptomic analysis of gene expression of menaquinone-7 in Bacillus subtilis natto toward different oxygen supply.

Menaquinone-7 (MK-7) is an important kind of vitamin K2 which plays significant roles in the treatment of coagulation and osteoporosis, and prevention of cardiovascular disease. This work was purposed to study the differences of gene expression at different oxygen supply conditions in Bacillus natto. The differences of fermentation characteristics, gene expression related to MK-7 biosynthesis, spore and biofilm formation were analyzed. The yield of MK-7 increased by two fold under high oxygen supply condition of 200 rpm. Further transcriptome analysis indicated that most of the enzymes in MK-7 biosynthesis pathway were also up-regulated. Moreover, glycerol kinase, fructose-bisphosphate aldolase and phosphofructokinase in glycolysis pathway were all up-regulated indicating that high oxygen supply can increase the consumption of substrate glycerol. Meanwhile, menD, encoded the rate-limiting enzyme in the MK pathway, was obviously up-regulated by 3.49-fold while most of the enzymes related to spore formation were down regulated at 200 rpm. Besides, superoxide dismutase (SOD2), catalase (CAT), hydroperoxide reductase (AhpF) and DNA-binding protein MrgA in the antioxidant defense system were up-regulated, while superoxide dismutase (SOD1) and glutathione peroxidase (GSH-Px) were down-regulated. These results could contribute to a better understanding for the effect of oxygen on the MK-7 production in Bacillus natto, and further analyze the molecular regulation mechanism of MK-7 biosynthesis.

Regeneration Mechanism of Sulfur Absorption Via Samarium-doped Cerium Adsorbents in the Gas Atmosphere of O2/N2.

Sulfides existing in many high-temperature gas mixtures have a negative effect on various industrial applications. Ce-based adsorbents are becoming a hotspot in the high-temperature desulfurization process owing to their excellent thermal stability at high temperatures and regeneration capacity. In this study, we investigate the regeneration path of samarium-doped cerium (SDC) sorbent at high temperature. The SDC adsorbent showed a good sulfur removal ability and excellent regeneration capacity. Ce2O2S and Ce(SO4)2 are observed in the used adsorbent, and Ce2O2S is the main sulfur-containing species. The regeneration path of the Ce2O2S is the key to the regeneration mechanism of the adsorbent. There are two regeneration paths for the Ce2O2S at high temperature in O2/N2 gas mixture. In air stream, the Ce2O2S is oxidized to Ce2O2SO4 and then decomposes into CeO2 and SO2. In a 2% O2/N2 gas condition, the Ce2O2S directly generates CeO2 and elemental sulfur with O2 assistance.

Microbial production of vitamin K2: current status and future prospects.

Vitamin K2, also called menaquinone, is an essential lipid-soluble vitamin that plays a critical role in blood clotting and prevention of osteoporosis. It has become a focus of research in recent years and has been widely used in the food and pharmaceutical industries. This review will briefly introduce the functions and applications of vitamin K2 first, after which the biosynthesis pathways and enzymes will be analyzed in-depth to highlight the bottlenecks facing the microbial vitamin K2 production on the industrial scale. Then, various strategies, including strain mutagenesis and genetic modification, different cultivation modes, fermentation and separation processes, will be summarized and discussed. The future prospects and perspectives of microbial menaquinone production will also be discussed finally.

Integration of lipidomic and transcriptomic profiles reveals novel genes and regulatory mechanisms of Schizochytrium sp. in response to salt stress.

In this study, the effects of salt stress on the physiological, lipidomic and transcriptomic profiles of halophilic microalga Schizochytrium sp. were investigated. In general, Schizochytrium sp. could survive under high osmotic fermentation medium containing 30 g/L NaCl, and showed a significant increase in C14:0 percentage in total fatty acids. In lipidomic analysis, C14:0 was specifically enriched in phosphatidylcholine (PC), and membrane phospholipids participated in the salt stress response mostly. Specially, one novel signal lipid N-acylphosphatidylethanolamine (NAPE) (18:0/20:3/14:0) was upregulated significantly. Transcriptomic analysis revealed glycerol-3-phosphate acyltransferase (GPAT) and phospholipase ABHD3 (PLABDH3) were involved in C14:0 metabolism and NAPE biosynthesis. Signalling pathways they mediated were activated as evident by high expression level of Myristoyl-CoA: protein N-myristoyltransferase (NMT) and NAPE-hydrolyzing PLD (NAPE-PLD). This study gives us an insight in specific responses to salt stress in Schizochytrium sp. and provides a considerable proportion of novel genes that could commendably be used for engineering modification.

Intracellular response of Bacillus natto in response to different oxygen supply and its influence on menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7) plays an important role in blood clotting, cardiovascular disease and anti-osteoporosis, and has been wildly used in the food additives and pharmaceutical industries. The aim of this study was to investigate the mechanism of menaquinone-7 biosynthesis in response to different oxygen supplies in Bacillus natto. The differences of fermentation performance, intracellular metabolites, oxidative stress reaction and enzyme activities of Bacillus natto R127 were analyzed under different KLa. Glycerol consumption rate and MK-7 yield at 24.76 min- 1 was 2.1 and 7.02 times of that at 18.23 min- 1. Oxidative stress analysis showed the cell generated more active oxygen and possessed higher antioxidant capacity at high oxygen supply condition. Meanwhile, high pyruvate kinase and high cytochrome c oxidase activities were also observed at 24.76 min- 1. Furthermore, comparative metabolomics analyses concluded series of biomarkers for high MK-7 biosynthesis and cell rapid growth. Besides, several metabolic responses including low glyceraldehyde-3-phosphate accumulation, low flux from pyruvate to lactic acid, high active TCA pathway, were also found to be associated with high MK-7 accumulation at high oxygen supply conditions. These findings provided the information for better understanding of oxygen effect on MK-7 biosynthesis and lay a foundation for further improvement of MK-7 production as well.

Fermentation performance and metabolomic analysis of an engineered high-yield PUFA-producing strain of Schizochytrium sp.

The ω-3/long-chain polyunsaturated fatty acids (LC-PUFAs) play an important role in human health, but they cannot be synthesized in sufficient amounts by the human body. In a previous study, we obtained an engineered Schizochytrium sp. strain (HX-RS) by exchanging the acyltransferase (AT) gene, and it was able to co-produce docosahexaenoic acid and eicosapentaenoic acid. To investigate the mechanism underlying the increase of PUFA content in HX-RS, the discrepancies of fermentation performance, key enzyme activities and intracellular metabolites between HX-RS and its wild-type parent strain (WTS) were analyzed via fed-batch fermentation in 5-L bioreactors. The results showed that the cell dry weight (CDW) of HX-RS was higher than that of the WTS. Metabolomics combined with multivariate analysis showed that 4-aminobutyric acid, proline and glutamine are potential biomarkers associated with cell growth and lipid accumulation of HX-RS. Additionally, the shift of metabolic flux including a decrease of glyceraldehyde-3-phosphate content, high flux from pyruvate to acetyl-CoA, and a highly active glycolysis pathway were also found to be closely related to the high PUFA yield of the engineered strain. These findings provide new insights into the effects of exogenous AT gene expression on cell proliferation and fatty acid metabolism.

Silk scaffolds with gradient pore structure and improved cell infiltration performance.

Electrospun scaffold with three-dimensional (3D) geometry and appropriate pore structure is an important challenge to mimic natural tissues such as skin, cartilage, etc. In this work, 3D silk fibroin (SF) electrospun scaffolds with gradient pore size were prepared by combining multi-step electrospinning with low temperature (LTE) collecting. The LTE electrospun scaffolds achieved 3D macro-structure with large pore size. The effects of relative humidity (RH), collecting temperature on the morphology of the scaffolds were investigated by scanning electron microscopy and computed tomography. The pore size of the scaffolds was tailored by adjusting SF concentration, electric field, flow rate, needle gauge and collector temperature during electrospinning at 50% RH. L929 cell infiltration results of the scaffolds showed that conventional electrospun scaffolds with small pore size (average diameter 5.9 ±â€¯1.4 µm) restrained cell proliferation and infiltration. On the contrary, LTE electrospun scaffolds with medium pore size (average diameter 11.6 ±â€¯1.4 µm) improved cell proliferation obviously. Large pore size scaffolds (average diameter 37.2 ±â€¯12.9 µm) was beneficial to cell infiltration depth in the thickness direction of the scaffolds. The scaffolds, which were integrated with layers of small, medium and large pores, are promising in the repair of tissue with gradient pore structures.

All-Organic Conductive Biomaterial as an Electroactive Cell Interface.

Various attractive materials are being used in bioelectronics recently. In this paper, hydroxymethyl-3,4-ethylenedioxythiophene (EDOT-OH) has been in situ integrated and polymerized on the surface of the regenerated silk fibroin (RSF) film to construct a biocompatible electrode. In order to improve the efficiency of in situ polymerization, sodium dodecyl sulfate (SDS) was adopted as surfactant to construct a well-organized and stable poly(hydroxymethyl-3,4-ethylenedioxythiophene) (PEDOT-OH) coating, whereas ammonium persulfate was used as oxidant. The effects of dosages of surfactant and oxidant, initial pH value, and monomer concentration on the polymerization were studied. Under the optimal conditions, the RSF/PEDOT-OH film exhibited a square resistance of 3.28 × 105 Ω corresponding to a conductance of 6.1 × 10-3 S/cm. Scanning electron microscope images indicated that PEDOT-OH was deposited uniformly on the surface of the RSF film with SDS. Furthermore, Fourier transform infrared spectroscopy confirmed that interactions existed between the peptide linkages of silk fibroin (SF) macromolecules and PEDOT-OH. The RSF/PEDOT-OH film displayed favorable electrochemical stability, biocompatibility, and fastness. This study provides a feasible method to endow conductivity to RSF materials in various forms. In addition, the conductive layer and biocompatible silk substrate make the RSF/PEDOT-OH biomaterial highly suitable for potential applications in bioelectric devices, sensors, and tissue engineering.

Transcriptome and gene expression analysis of docosahexaenoic acid producer Schizochytrium sp. under different oxygen supply conditions.

BACKGROUND: Schizochytrium sp. is a promising strain for the production of docosahexaenoic acid (DHA)-rich oil and biodiesel, and has been widely used in the food additive and bioenergy industries. Oxygen is a particularly important environmental factor for cell growth and DHA synthesis. In general, higher oxygen supply favors lipid accumulation, but could lead to a reduction of the DHA percentage in total fatty acids in Schizochytrium sp. To tackle this problem, it is essential to understand the mechanisms regulating the response of Schizochytrium sp. to oxygen. In this study, we aimed to explore the acclimatization of this DHA producer to different oxygen supply conditions by examining the transcriptome changes. RESULTS: Two different fermentation processes, namely normal oxygen supply condition (shift agitation speeds from 400 rpm to 300 rpm) and high oxygen supply condition (constant agitation speeds: 400 rpm), were designed to study how the fermentation characteristics of Schizochytrium sp. HX-308 were affected by different oxygen supply conditions. The results indicated that high oxygen supply condition resulted in 49% and 37.5% improvement in the maximum cell dry weight (CDW) and total lipid concentration, respectively. However, the DHA percentage in total fatty acids decreased to 35%, which was 31.4% lower than that produced by normal oxygen supply condition. Moreover, transcriptome analysis was performed to explore the effect of the oxygen supply condition on genetic expression and metabolism. The results showed that glycolysis and pentose phosphate pathway metabolism-associated genes (hexokinase, phosphofructokinase, fructose-bisphosphate aldolase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) were substantially upregulated in response to high oxygen supply, resulting in more NADPH was available for Schizochytrium. Specially, high oxygen supply condition also led to genes (Δ6 desaturase, Δ12 desaturase, FAS, ORFA, ORFB, and ORFC) involved in fatty acid biosynthesis upregulation. In addition, a transcriptional upregulation of catalase (CAT) became apparent under high oxygen supply condition, while superoxide dismutase (SOD) and ascorbate peroxidase (APX) were found to be down-regulated. CONCLUSIONS: This study is the first to investigate the differences of gene expression at different levels of oxygen availability in the DHA producer Schizochytrium. The results of transcriptome analyses indicated that high oxygen supply condition resulting in more NADPH and acetyl-CoA production for cell growth and lipid synthesis in Schizochytrium. Δ12 desaturase and ORFC showed higher expression levels at high oxygen supply condition, which might be the key regulators for enhancing fatty acid biosynthesis in the future. These results enrich the current knowledge regarding genetic expression and provide important information to enhance DHA production in Schizochytrium sp.

Intrinsically Fluorescent Silks from Silkworms Fed with Rare-Earth Upconverting Phosphors.

Fluorescent silk fibroin (SF) fibers have great potential in biomedical application and special functions for marking and tracking. How to fabricate fluorescent SF fibers with good fluorescence stability by a simple and environmentally friendly method has yet to be explored. Here, we successfully produced fluorescent SF fibers by using silkworms as bioreactors to introduce rare-earth upconverting phosphors (UCPs) into silk fibroin. The modified silk exhibited bright green colors under 980 nm laser. This directly feeding method to produce fluorescent SF fibers is green and environmentally friendly and easy to use for mass production. Moreover, it provides an idea that SF fibers can be cooperated with more fluorescent materials which could exhibit different colors with a certain wavelength of light for broad application.

Structure and interaction of silk fibroin and graphene oxide in concentrated solution under shear.

Considering the high biocompatibility of regenerated silk fibroin (RSF) and the good enhancement effect of graphene oxide (GO), various RSF/GO composite materials have been previously investigated, and found that GO plays a vital role in the fabrication of high-performance RSF/GO materials. However, its effects on the structure of RSF solution are unclear. Therefore, in this work, we studied the rheological and optical properties, as well as the aggregation behavior of concentrated RSF/GO solution in response to applied shear. The results demonstrated that the presence of GO sheets in RSF solution increased the shear resistance, while delayed the sol-gel transition. Moreover, GO sheets were not favorable to the formation of the ordered structures of RSF. The results from small angle X-ray scattering (SAXS) of RSF/GO solution also showed that the shear process promoted the formation of RSF/GO interface. The data also provided insights into the structural evolution within the mixture solutions, which can be beneficial to the future design and fabrication of nanofiller-reinforced high-performance materials.

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers.

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables the silk proteins to be compact and ordered by shearing and elongation forces. Here, a biomimetic microfluidic channel was designed to mimic the specific geometry of the spinning duct of the silkworm. Regenerated silk fibroin (RSF) spinning doped with high concentration, was extruded through the microchannel to dry-spin fibers at ambient temperature and pressure. In the post-treated process, the as-spun fibers were drawn and stored in ethanol aqueous solution. Synchrotron radiation wide-angle X-ray diffraction (SR-WAXD) technology was used to investigate the microstructure of single RSF fibers, which were fixed to a sample holder with the RSF fiber axis normal to the microbeam of the X-ray. The crystallinity, crystallite size, and crystalline orientation of the fiber were calculated from the WAXD data. The diffraction arcs near the equator of the two-dimensional WAXD pattern indicate that the post-treated RSF fiber has a high orientation degree.

Transcriptomic Analysis of the Regulation of Lipid Fraction Migration and Fatty Acid Biosynthesis in Schizochytrium sp.

Schizochytrium sp. is the main source of docosahexaenoic acid-rich oil, which is widely used in food additive and pharmaceutical industry. In this study, using RNA-seq, comparative transcriptomic analyses were performed at four stages of DHA fermentation by Schizochytrium sp to get potential genes related to cell transition from cell growth to lipid accumulation and then to lipid turnover. 1406, 385, 1384 differently expressed genes were identified by comparisons in pairs of S2 vs S1, S3 vs S2 and S4 vs S3. Functional analysis revealed that binding and single-organism process might be involve in the cell transition from cell growth to lipid accumulation while oxidation-reduction process played an important role in the transition from lipid accumulation to lipid turnover. pfaC in the PKS pathway showed higher sensitivity to the environmental change, which might be the key regulator for enhancing PUFA biosynthesis in the future. Some other genes in signal transduction and cell transport were revealed to be related to lipid turnover, which would enrich the current knowledge regarding lipid metabolism and help to enhance the DHA production and enrich different lipid fractions by Schizochytrium in the future.

Characterization of bladder acellular matrix hydrogel with inherent bioactive factors.

Bladder acellular matrix (BAM) hydrogel may have great potential in tissue engineering due to outstanding biocompatibility and the presence of inherent bioactive factors in BAM. In this study, we prepared the BAM hydrogel by the method of enzymatic solubilization with pepsin and characterize the microrheological properties of the BAM precursor solution. The structures of the BAM hydrogel were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Furthermore, the growth factors including vascular endothelial growth factor (VEGF), platelet-derived growth factor B (PDGF-BB), keratinocyte growth factor (KGF) were quantified by ELISA. The biological performances of the hydrogels were evaluated by cultivating porcine iliac endothelial cells (PIECs) in vitro. Lyophilized BAM showed porous structure with pore diameter ranging from 50 to 100µm. BAM 4-G hydrogel (4mg/mL) with a short gelation time of 3.95±0.07min presents better thermal stability than BAM 6-G hydrogel (6mg/mL). Growth factors in the BAM hydrogel maintain valuable biological activity even after digestion process. The BAM hydrogel supported the adhesion and growth of PIECs well and has great potential for further tissue engineering.