Transcriptome analysis reveals the underlying mechanism for over-accumulation of alkaline protease in Bacillus licheniformis.

AIMS: Bacillus licheniformis AQ is an industrial strain with high production of alkaline protease (AprE), which has great industrial application value. However, how to regulate the production of AprE in the process of industrial fermentation is still not completely clear. Therefore, it is important to understand the metabolic process of AprE production in the industrial fermentation medium. METHODS AND RESULTS: In this study, transcriptome sequencing of the whole fermentation course was performed to explore the synthesis and regulation mechanism of AprE in B. licheniformis AQ. During the fermentation process, the AprE got continuously accumulated, reaching a peak of 42 020 U/mL at the fermentation endpoint (48 h). Meanwhile, the highly expressed genes were observed. Compared with the fermentation endpoint, there were 61 genes in the intersection of differentially expressed genes, functioning as catabolic processes, peptidases and inhibitors, chaperones, and folding catalysts. Furthermore, the protein-protein interactions network of AprE was constructed. CONCLUSION: This study provides important transcriptome information for B. licheniformis AQ and potential molecular targets for further improving the production of AprE.

Relationship between antioxidant enzymes and sclerotial formation of Pleurotus tuber-regium under abiotic stress.

In order to explore the relationship between sclerotial formation and antioxidant enzymes under abiotic stresses, the effects of abiotic stresses including temperature, pH value, osmotic pressure, limited nitrogen, and hydrogen peroxide (H2O2) on the activities of antioxidant enzymes, ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in Pleurotus tuber-regium were studied. Meanwhile, the sclerotial formation under these abiotic stress conditions was also investigated. It was found that low temperature, weak alkaline, appropriate osmotic stress, and H2O2 can promote sclerotial formation, and sclerotial formation always tended to occur when the activities of antioxidant enzymes were at a high value. During the prolonged low temperature stress, SOD acted mainly in the early stage of stress, while POD and CAT had higher activity in the middle and late stage. Moreover, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that SOD.193 and POD.535 were significantly down-regulated in sclerotia, and CAT.1115 and POD.401 were up-regulated instead. These antioxidant enzyme genes played an important role in the sclerotial formation under low temperature stress. It is strongly suggested that antioxidant enzymes and abiotic stresses are closely related to sclerotial formation in P. tuber-regium. KEY POINTS: • Low temperature and H2O2 can promote sclerotial formation. • Sclerotia are more likely to form under high antioxidant enzyme activity. • POD.401, POD.535, SOD.193, and CAT.1115 are important for sclerotial formation.

Dynamic regulation of Monascus azaphilones biosynthesis by the binary MrPigE-MrPigF oxidoreductase system.

Monascus azaphilones (MAs) have been extensively applied as natural food coloring agents. MAs are classified into three categories: yellow MAs (YMAs), orange MAs (OMAs), and red MAs with various biological activities. However, the exact biosynthetic mechanism of OMAs and YMAs are not thoroughly elucidated. Firstly, we identified four DNA-binding residues of transcription factor MrPigB and constructed a multi-site saturation mutagenesis library of MrPigB. Then, comparative metabolite and gene expression of the mutants revealed that two oxidoreductases MrPigE and MrPigF were responsible for the formation of YMAs and OMAs. Finally, the in vitro and in vivo assays demonstrated the opposite roles of MrPigE and MrPigF in conversion of OMAs to YMAs. To our knowledge, this is the first report of a binary oxidoreductase system for dynamic regulation of fungal secondary metabolite biosynthesis. Broadly, our work also demonstrates the transcription factor engineering strategy for elucidating the biosynthetic pathway of secondary metabolite. KEY POINTS: • MrPigE converts orange Monascus azaphilones to yellow Monascus azaphilones • MrPigF oxidizes intermediates to afford orange Monascus azaphilones • MrPigE and MrPigF constitute a binary system in Monascus azaphilones biosynthesis.

A comprehensive review of spermidine: Safety, health effects, absorption and metabolism, food materials evaluation, physical and chemical processing, and bioprocessing.

Spermidine, a natural autophagy inducer, has a variety of health effects, such as antitumor, antiaging, anti-inflammation, cardiovascular protection, and neuromodulation. It has been a hot topic in the field of food processing, and current research findings suggest that spermidine-rich foods may be used in intervention and prevention of age-related diseases. In this article, recent findings on the safety, health effects, absorption and metabolism of spermidine were reviewed, and advances in food processing, including the raw materials evaluation, physical and chemical processing, and biological processing of spermidine, were highlighted. In particular, the core metabolic pathways, key gene targets, and efficient metabolic engineering strategies involved in the biosynthesis of spermidine and its precursors were discussed. Moreover, limitations and future perspectives of spermidine research were proposed. The purpose of this review is to provide new insights on spermidine from its safety to its food processing, which will advance the commercial production and applications of spermidine-rich foods and nutraceuticals.

Melanin of fungi: from classification to application.

Melanin is a secondary metabolite composed of complex heterogeneous polymers. Fungal melanin is considered to be a sustainable and biodegradable natural pigment and has a variety of functional properties and biological activities. On one hand, due to its own specific properties it can play the role of antioxidant, anti-radiation, adsorption, and photoprotection. On the other hand, it has good biological activities such as hepatoprotective effect, hypolipidemic effect and anti-cancer. Therefore, it is widely used in various fields of daily life, including dyeing, food, biomedical and commercial industry. It is conducive to environmental protection and human health. However, the insolubility of fungal melanin in water, acids and organic solvents has been an obstacle to its commercial applications. Thus, the chemical modification methods of fungal melanin are summarized to increase its solubility and expand the application fields. Although fungal melanin has been used in many industries, as the structure and function of fungal melanin and modified melanin are further studied, more functional properties and bioactivities are expected to be discovered for a wide range of applications in the future.

Enhancement of S-adenosylmethionine production by deleting thrB gene and overexpressing SAM2 gene in Bacillus amyloliquefaciens.

OBJECTIVES: To improve the S-adenosylmethionine (SAM) production in methionine-free medium, effects of deleting genes of SAM decarboxylase (speD) and homoserine kinase (thrB) on SAM titers were investigated, and the SAM synthetase gene (SAM2) was also overexpressed. RESULTS: In B. amyloliquefaciens HSAM2, deleting speD to block the SAM utilization pathway significantly reduced the SAM titer. After knockout of thrB to block the branched pathway, the resulted mutant HSAM4 produced 143.93 mg/L SAM, increasing by 42% than HSAM2. Further plasmid-based expression of SAM2 improved the SAM titer to 226.92 mg/L, and final optimization of key fermentation parameters resulted in the maximum SAM titer of 412.01 mg/L in flasks batch fermentation. CONCLUSIONS: Deleting thrB and overexpressing SAM2 gene were efficient for enhanced SAM production in B. amyloliquefaciens. The maximum SAM titer in flasks batch fermentation was much higher than that of previous reports.

Metabolomics analysis reveals global acetoin stress response of Bacillus licheniformis.

INTRODUCTION: Acetoin serves as a high value-added platform with a broad range of applications, and can be effectively produced by Bacillus licheniformis. However, its toxicity to the producing strain hinders the higher acetoin production, and current knowledge about the acetoin resistance mechanisms of B. licheniformis is quite limited. OBJECTIVES: To comprehensively investigate the metabolic changes in B. licheniformis under acetoin stress. METHODS: We used gas chromatography-mass spectrometry based untargeted metabolomics approach to measure the metabolic profiles of B. licheniformis under 20, 40 and 80 g/L acetoin stress. Transcriptional analysis was conducted to verify the metabolomics results. RESULTS: A total of 119 metabolites were identified in our experiment. The metabolic responses of B. licheniformis to acetoin stress were as follows: (i) pentose phosphate pathway and tricarboxylic acid (TCA) cycle were negatively affected by acetoin stress. In turn, glyoxylate cycle was activated to supply malic acid. (ii) Acetoin stress induced the accumulation of serine, valine, leucine and protective osmolytes (glycine and proline). (iii) Acetoin stress induced a higher saturated fatty acid ratio, which indicated a lower fluidity of cell membrane that could inhibit the entry of acetoin into cytoplasm. (iv) Synthesis of phosphatidylserine was enhanced, and phosphatidylethanolamine content was probably increased under acetoin stress. CONCLUSIONS: This study revealed the metabolic perturbations of B. licheniformis to acetoin stress. In response to acetoin stress, glyoxylate cycle was activated, protective osmolytes were accumulated, saturated fatty acid ratio was elevated and synthesis of phosphatidylserine was enhanced in B. licheniformis.

Balancing the carbon flux distributions between the TCA cycle and glyoxylate shunt to produce glycolate at high yield and titer in Escherichia coli.

The glyoxylate shunt is a branch of the tricarboxylic acid (TCA) cycle which directly determines the synthesis of glycolate, and the balance between the glyoxylate shunt and TCA cycle is very important for the growth of Escherichia coli. In order to accumulate glycolate at high yield and titer, strategies for over-expressing glycolate pathway enzymes including isocitrate lyase (AceA), isocitrate dehydrogenase kinase/phosphatase (AceK) and glyoxylate reductase (YcdW) were analyzed. The genes encoding these three enzymes were transcribed under the control of promoter pTrc on pTrc99A, to form pJNU-3, which was harbored by strain Mgly1, resulting in strain Mgly13. Strain Mgly13 produced glycolate with 0.385 g/g-glucose yield (45.2% of the theoretical yield). Citrate synthase (GltA) converted excess acetyl-CoA and oxaloacetate to citrate and was over-expressed by pJNU-4 (pCDFDuet-1 backbone). Thus, the resulting strain Mgly134 produced glycolate with a 0.504 g/g-glucose yield (59.3% of the theoretical yield). We then eliminated the pathways involved in the degradation of glycolate, resulting in strain Mgly434, which produced glycolate with 92.9% of the theoretical yield. Following optimization of fermentation, the maximum glycolate titer from strain Mgly434 was 65.5 g/L.

A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis.

BACKGROUND: Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. RESULTS: In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis. We observed dramatic decreases in the concentrations of both target proteins (45 and 49%, respectively) in a sppA deficient strain, while the extracellular protein yields of nattokinase and α-amylase were increased by 30 and 67% respectively in a strain overexpressing SppA. In addition, biomass, specific enzyme activities and the relative gene transcriptional levels were also enhanced due to the overexpression of sppA, while altering the expression levels of tepA had no effect on the concentrations of the secreted target proteins. CONCLUSIONS: Our results confirm that SppA, but not TepA, plays an important functional role for protein secretion in B. licheniformis. Our results indicate that the sppA overexpression strain, B. licheniformis BL10GS, could be used as a promising host strain for the industrial production of heterologous secreted proteins.

Decreased formation of branched-chain short fatty acids in Bacillus amyloliquefaciens by metabolic engineering.

OBJECTIVES: To reduce the unpleasant odor during 1-deoxynojirimycin (DNJ) production, the genes of leucine dehydrogenase (bcd) and phosphate butryltransferase (ptb) were deleted from Bacillus amyloliquefaciens HZ-12, and the concentrations of branched-chain short fatty acids (BCFAs) and DNJ were compared. RESULTS: By knockout of the ptb gene, 1.01 g BCFAs kg-1 was produced from fermented soybean by HZ-12Δptb. This was a 56% decrease compared with that of HZ-12 (2.27 g BCFAs kg-1). Moreover, no significant difference was found in the DNJ concentration (0.7 g kg-1). After further deletion of the bcd gene from HZ-12Δptb, no BCFAs was detected in fermented soybeans with HZ-12ΔptbΔbcd, while the DNJ yield decreased by 26% compared with HZ-12. CONCLUSIONS: HZ-12Δptb had decreased BCFAs formation but also maintained the stable DNJ yield, which contributed to producing DNJ-rich products with decreased unpleasant smell.

High-level production of α-amylase by manipulating the expression of alanine racamase in Bacillus licheniformis.

OBJECTIVES: To improve target protein production by manipulating expression levels of alanine racemase in Bacillus licheniformis. RESULTS: The gene of dal was identified to be responsible for alanine racemase function. Based on the selection marker of dal, a food-grade expression system was constructed in B. licheniformis, and effects of different dal expression levels mediated by promoters on α-amylase production were investigated. The highest α-amylase activity (155 U/ml) was obtained in BL10D/pP43SAT-PtetDal, increased by 27% compared with that of the control strain BL10/pP43SAT in tetracycline-based system (123 U/ml). Moreover, the dal transcriptional level was not correlated positively with that of amyL. CONCLUSIONS: A food-grade system for high-level production of α-amylase was constructed in B. licheniformis, revealing that expression levels of selection marker significantly affected target protein production.

Efficient production of free fatty acids from ionic liquid-based acid- or enzyme-catalyzed bamboo hydrolysate.

Two engineered Escherichia coli strains, DQ101 (MG1655 fadD -)/pDQTES and DQ101 (MG1655 fadD -)/pDQTESZ were constructed to investigate the free fatty acid production using ionic liquid-based acid- or enzyme-catalyzed bamboo hydrolysate as carbon source in this study. The plasmid, pDQTES, carrying an acyl-ACP thioesterase 'TesA of E. coli in pTrc99A was constructed firstly, and then (3R)-hydroxyacyl-ACP dehydratase was ligated after the TesA to give the plasmid pDQTESZ. These two strains exhibited efficient fatty acid production when glucose was used as the sole carbon source, with a final concentration of 2.45 and 3.32 g/L, respectively. The free fatty acid production of the two strains on xylose is not as efficient as that on glucose, which was 2.32 and 2.96 g/L, respectively. For mixed sugars, DQ101 (MG1655 fadD -)-based strains utilized glucose and pentose sequentially under the carbon catabolite repression (CCR) regulation. The highest total FFAs concentration from the mixed sugar culture reached 2.81 g/L by DQ101 (MG1655 fadD -)/pDQTESZ. Furthermore, when ionic liquid-based enzyme-catalyzed bamboo hydrolysate was used as the carbon source, the strain DQ101 (MG1655 fadD -)/pDQTESZ could produce 1.23 g/L FFAs with a yield of 0.13 g/g, and while it just produced 0.65 g/L free fatty acid with the ionic liquid-based acid-catalyzed bamboo hydrolysate as the feedstock. The results suggested that enzymatic catalyzed bamboo hydrolysate with ionic liquid pretreatment could serve as an efficient feedstock for free fatty acid production.

Enhancement of L-valine production in Bacillus licheniformis by blocking three branched pathways.

OBJECTIVES: Bacillus licheniformis WX-02 is used for the production of many valuable chemicals. Here, we have sought to improve L-valine production by blocking the metabolic pathways related to branched-chain amino acids. RESULTS: The synthesis genes of L-leucine (leuA) and L-isoleucine (ilvA) were deleted to obtain mutant strains. L-Valine yields of WX-02ΔleuA and WX-02ΔilvA reached 33.2 and 21.1 mmol/l, respectively, which are 22 and 14 times higher than the wild-type WX-02 (1.53 mmol/l). After further deletion of L-lactate dehydrogenase gene (ldh) from WX-02ΔleuA, the productivity reached 0.47 mmol/l h, an increase of 19 %. CONCLUSION: We provide a possibility to over-produce L-valine using genetically-modified B. licheniformis using remodeling of the biosynthetic pathway to L-valine.

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

Improvement of lichenysin production in Bacillus licheniformis by replacement of native promoter of lichenysin biosynthesis operon and medium optimization.

Lichenysin is a biodegradable surfactant with huge potential for recovering crude oil from the oil reservoir. The current production of lichenysin is made through fermentation from wild strain of Bacillus licheniformis, which is limited by low yield. The aim of this work was to improve lichenysin-producing capability of a wide strain B. licheniformis WX-02. Lichenysin produced from WX-02 was first extracted, purified, and identified. Through the substitution of the promoter of lichenysin biosynthesis operon, the mutants B. licheniformis WX02-P43lch, WX02-Pxyllch, and WX02-Psrflch were constructed with the constitutive promoter (P43), the xylose-inducible promoter (P xyl ), and the surfactin operon promoter (P srf ), respectively. A consistent change trend was observed between lichenysin production and lchAA gene transcription, confirming the strength of the promoters as an important factor for lichenysin synthesis. Among the three mutants, WX02-Psrflch produced the highest lichenysin yield. The production by the mutant WX02-Psrflch was further improved with the optimization of the major medium components including glucose, NH4NO3, and Na2HPO4/KH2PO4. Under 30 g/L glucose, 5 g/L NH4NO3, and 80 mM/60 mM Na2HPO4/KH2PO4, the strain WX02-Psrflch produced 2,149 mg/L lichenysin, a 16.8-fold improvement compared to that of wild strain WX-02.

Hepatoprotective Effect of Annulohypoxylon stygium Melanin on Acute Alcoholic Liver Injury in Mice.

Annulohypoxylon stygium melanin (AsM) has various functional properties such as antioxidant and anti-radiation, but its biological activity in vivo has not been fully investigated. In this study, we researched the effects of AsM on the protection against acute liver injury in mice and its mechanism. The results showed that AsM had no significant effect on body weight in mice but reduced the liver index. It was able to significantly decrease the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the contents of triglyceride (TG) and total cholesterol (TC) in mice. Simultaneously, it raised the levels of superoxide dismutase (SOD), peroxidase (CAT), and glutathione peroxidase (GSH-Px), which obviously exceeded those of the EtOH group. AsM could significantly lower the levels of inflammatory factors, with inhibition rates of 68.30%, 29.0%, and 19.50% for IL-1ß, IL-6, and TNF-α, respectively. H&E and Oil red O staining also showed that AsM ameliorated liver damage and lipid accumulation in mice. The protective mechanism of AsM may be associated to the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signaling pathway, which could activate the downstream antioxidant enzymes heme oxygenase-1 (HO-1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC). These findings confirmed that AsM had an alleviating effect on alcoholic liver injury and provided new thoughts for the development of natural product.

Phellinus linteus polysaccharides: A review on their preparation, structure-activity relationships, and drug delivery systems.

Phellinus linteus polysaccharides exhibit antitumor, immunomodulatory, anti-inflammatory, and antioxidant properties, mitigate insulin resistance, and enhance the diversity and abundance of gut microbiota. However, the bioactivities of P. linteus polysaccharides vary owing to the complex structure, thereby, limiting their application. Various processing strategies have been employed to modify them for improving the functional properties and yield. Herein, we compare the primary modes of extraction and purification employed to improve the yield and purity, review the structure-activity relationships, and discuss the application of P. linteus polysaccharides using nano-carriers for the encapsulation and delivery of various drugs to improve bioactivity. The limitations and future perspectives are also discussed. Exploring the bioactivity, structure-activity relationship, processing methods, and delivery routes of P. linteus polysaccharides will facilitate the development of functional foods and dietary supplements rich in P. linteus polysaccharides.

Preparation and characterization of metal-polyphenol networks encapsulated in sodium alginate microbead hydrogels for catechin and vitamin C delivery.

A novel encapsulation system was designed, utilizing sodium alginate (SA) polysaccharide as the matrix and easily absorbed Fe2+ as the metal-organic framework, to construct microbead scaffolds with both high catechins (CA) and vitamin C (Vc) loading and antioxidant properties. The structure of microbead hydrocolloids was investigated using SEM, XPS, FTIR, XRD and thermogravimetry, and the antioxidant activity, in vitro digestion and the release of CA and Vc were evaluated. These results revealed that the microbead hydrocolloids SA-CA-Fe and SA-CA-Vc-Fe exhibited denser and stronger cross-linking structures, and the formation of inter- and intramolecular hydrogen and coordination bonds improved thermal stability. Moreover, SA-CA-Fe (44.9 % DPPH and 47.8 % ABTS) and SA-CA-Vc-Fe (89.9 % DPPH and 89.3 % ABTS) displayed strong antioxidant activity. Importantly, they were non-toxic in Caco2 cells. The SA-CA-Fe and SA-CA-Vc-Fe achieved significantly higher CA (56.9 and 62.7 %, respectively) and Vc (42.2 %) encapsulation efficiency while maintaining higher CA and Vc release in small intestinal environment. These results suggested that SA polysaccharide-based encapsulation system using Fe2+ framework as scaffold had greater potential for delivery and controlled release of CA and Vc than conventional hydrocolloids, which could provide new insights into the construction of high loading, safe, targeted polyphenol delivery system.

Production, physico-chemical properties and antioxidant activity of melanin from Annulohypoxylon stygium (Lév.) Y.M. Ju, J.D. Rogers and H.M. Hsieh.

To obtain higher melanin production in liquid culture, culture conditions of Annulohypoxylon stygium (Lév.) Y.M. Ju, J.D. Rogers and H.M. Hsieh were optimised. The results showed that using single factor experiment and orthogonal test, the optimised production of melanin reached 2.20 g/L, which was 2.06 times higher than that of the control group. In addition, it was speculated that A. stygium melanin (AsM) was 3,4-dihydroxyphenylalanine (DOPA) melanin and showed an amorphous irregular structure. Moreover, it had good solubility in alkaline solution. AsM showed good antioxidant activity at a concentration of 500 mg/L, with DPPH, ABTS and OH radicals scavenging activities of 90.83%, 75.36% and 70.90%, respectively. AsM prevented alcohol-induced oxidative damage and oxidative stress in HepG2 cells by inhibiting the decrease of antioxidant key enzyme activity under alcohol stimulation. It was proved to have a great potential for application as a natural antioxidant and a substitute for synthetic pigments.

Enhancement of Green Production of Heme by Deleting Odor-Related Genes from Bacillus amyloliquefaciens Based on CRISPR/Cas9n.

Heme is a crucial component in endowing plant-based meat analogs with flavor and color. This study aimed to develop a green strategy for heme production by reducing fermentation off-odor and accelerating heme synthesis. First, an efficient CRISPR/Cas9n system was constructed in Bacillus amyloliquefaciens to construct the odor-reducing chassis cell HZC9nΔGPSU, and the odor substances including the branched-chain short fatty acids, putrescine, and ammonia were reduced by 62, 70, and 88%, respectively. Meanwhile, the hemA gene was confirmed to be the key gene for enhanced heme synthesis. Various hemA genes were compared to obtain the best gene dhemA, and the catalysis mechanism was explained by molecular docking simulation. After further expression of dhemA in HZC9nΔGPSU, the heme titer of HZC9nΔGPSU/pHY-dhemA reached 11.31 ± 0.51 mg/L, 1.70-fold higher than that of HZC9n/pHY-dhemA. The knockout of off-odor-related genes reduced the odor substances and enhanced the heme synthesis, which is promising for the green production of high-quality heme.