High methionine intake alters gut microbiota and lipid profile and leads to liver steatosis in mice.

Methionine is an important sulfur-containing amino acid. Health effects of both methionine restriction (MR) and methionine supplementation (MS) have been studied. This study aimed to investigate the impact of a high-methionine diet (HMD) (1.64% methionine) on both the gut and liver functions in mice through multi-omic analyses. Hepatic steatosis and compromised gut barrier function were observed in mice fed the HMD. RNA-sequencing (RNA-seq) analysis of liver gene expression patterns revealed the upregulation of lipid synthesis and degradation pathways, cholesterol metabolism and inflammation-related nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. Metagenomic sequencing of cecal content demonstrated a shift in gut microbial composition with an increased abundance of opportunistic pathogens and gut microbial functions with up-regulated lipopolysaccharide (LPS) biosynthesis in mice fed HMD. Metabolomic study of cecal content showed an altered gut lipid profile and the level of bioactive lipids, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitoylethanolamide (PEA), linoleoyl ethanolamide (LEA) and arachidonoyl ethanolamide (AEA), that carry anti-inflammatory effects significantly reduced in the gut of mice fed the HMD. Correlation analysis demonstrated that gut microbiota was highly associated with liver and gut functions and gut bioactive lipid content. In conclusion, this study suggested that the HMD exerted negative impacts on both the gut and liver, and an adequate amount of methionine intake should be carefully determined to ensure normal physiological function without causing adverse effects.

Decoding the Qu-aroma of medium-temperature Daqu starter by volatilomics, aroma recombination, omission studies and sensory analysis.

Qu-aroma is of great significance for evaluation the quality of Daqu starter. This study aimed to decode the Qu-aroma of medium-temperature Daqu (MT-Daqu) via "top-down" and "bottom-up" approaches. Firstly, 52 aroma descriptors were defined to describe the MT-Daqu aroma by quantitative descriptive analysis. Secondly, 193 volatile organic compounds (VOCs) were identified from 42 MT-Daqu samples by HS-SPME-GC-MS, and 43 dominant VOCs were screened out by frequence of occurrence or abundance. By Thin Film (TF)-SPME-GC-O-MS, 27 odors and 90 VOCs were detected in MT-Daqu mixture, and 14 odor-active VOCs were screened out by odor intensity. Thirdly, a five-level MT-Daqu aroma wheel was constructed by matching 52 aroma descriptors and 37 aroma-active VOCs. Finally, Qu-aroma of MT-Daqu was reconstructed with 37 aroma-active VOCs and evaluated by omission experiments. Hereinto, 26 key aroma-active VOCs were determined by OAV value ≥1, including isovaleric acid, 1-hexanol, isovaleraldehyde, 2-octanone, trimethylpyrazine, γ-nonalactone, 4-vinylguaiacol, etc.

Restricted intake of sulfur-containing amino acids reversed the hepatic injury induced by excess Desulfovibrio through gut-liver axis.

Diet is a key player in gut-liver axis. However, the effect of different dietary patterns on gut microbiota and liver functions remains unclear. Here, we used rodent standard chow and purified diet to mimic two common human dietary patterns: grain and plant-based diet and refined-food-based diet, respectively and explored their impacts on gut microbiota and liver. Gut microbiota experienced a great shift with notable increase in Desulfovibrio, gut bile acid (BA) levels elevated significantly, and liver inflammation was observed in mice fed with the purified diet. Liver inflammation and elevated gut BA levels also occurred in mice fed with the chow diet after receiving Desulfovibrio desulfuricans ATCC 29,577 (DSV). Restriction of sulfur-containing amino acids (SAAs) prevented liver injury mainly through higher hepatic antioxidant and detoxifying ability and reversed the elevated BA levels due to excess Desulfovibrio. Ex vivo fermentation of human fecal microbiota with primary BAs demonstrated that DSV enhanced production of secondary BAs. Higher concentration of both primary and secondary BAs were found in the gut of germ-free mice after receiving DSV. In conclusion, Restriction of SAAs in diet may become an effective dietary intervention to prevent liver injury associated with excess Desulfovibrio in the gut.

Differences in the Ability of Lactic Acid Bacteria To Prevent Acute Alcohol-Induced Liver Injury via the Gut Microbiota-Bile Acid-Liver Axis.

Probiotics can regulate gut microbiota and protect against acute alcohol-induced liver injury through the gut-liver axis. However, efficacy is strain-dependent, and their mechanism remains unclear. This study investigated the effect of lactic acid bacteria (LAB), including Lacticaseibacillus paracasei E10 (E10), Lactiplantibacillus plantarum M (M), Lacticaseibacillus rhamnosus LGG (LGG), Lacticaseibacillus paracasei JN-1 (JN-1), and Lacticaseibacillus paracasei JN-8 (JN-8), on the prevention of acute alcoholic liver injury in mice. We found that LAB pretreatment reduced serum alanine transaminase (ALT) and aspartate transaminase (AST) and reduced hepatic total cholesterol (TC) and triglyceride (TG). JN-8 pretreatment exhibited superior efficacy in improving hepatic antioxidation. LGG and JN-8 pretreatment significantly attenuated hepatic and colonic inflammation by decreasing the expression of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) and increasing the expression of interleukin 10 (IL-10). JN-1 and JN-8 pretreatments have better preventive effects than other LAB pretreatment on intestinal barrier dysfunction. In addition, the LAB pretreatment improved gut microbial dysbiosis and bile acid (BA) metabolic abnormality. All of the strains were confirmed to have bile salt deconjugation capacities in vitro, where M and JN-8 displayed higher activities. This study provides new insights into the prevention and mechanism of LAB strains in preventing acute alcoholic liver injury.

A Free Amino Acid Diet Alleviates Colorectal Tumorigenesis through Modulating Gut Microbiota and Metabolites.

Colorectal cancer (CRC), a major global health concern, may be influenced by dietary protein digestibility impacting gut microbiota and metabolites, which is crucial for cancer therapy effectiveness. This study explored the effects of a casein protein diet (CTL) versus a free amino acid (FAA)-based diet on CRC progression, gut microbiota, and metabolites using carcinogen-induced (AOM/DSS) and spontaneous genetically induced (ApcMin/+ mice) CRC mouse models. Comprehensive approaches including 16s rRNA gene sequencing, transcriptomics, metabolomics, and immunohistochemistry were utilized. We found that the FAA significantly attenuated CRC progression, evidenced by reduced colonic shortening and histopathological alterations compared to the CTL diet. Notably, the FAA enriched beneficial gut bacteria like Akkermansia and Bacteroides and reversed CRC-associated dysbiosis. Metabolomic analysis highlighted an increase in ornithine cycle metabolites and specific fatty acids, such as Docosapentaenoic acid (DPA), in FAA-fed mice. Transcriptomic analysis revealed that FAA up-regulated Egl-9 family hypoxia inducible factor 3 (Egln 3) and downregulated several cancer-associated pathways including Hippo, mTOR, and Wnt signaling. Additionally, DPA was found to significantly induce EGLN 3 expression in CRC cell lines. These results suggest that FAA modulate gut microbial composition, enhance protective metabolites, improve gut barrier functions, and inhibit carcinogenic pathways.

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Efficient production and characterization of a newly identified trehalase for inhibiting the formation of bacterial biofilms.

Trehalase has attracted widespread attention in medicine, agriculture, food, and ethanol industry due to its ability to specifically degrade trehalose. Efficient expression of trehalase remains a challenge. In this study, a putative trehalase-encoding gene (Tre-zm) from Zunongwangia mangrovi was explored using gene-mining strategy and heterologously expressed in E. coli. Trehalase activity reached 3374 U·mL-1 after fermentation optimization. The scale-up fermentation in a 15 L fermenter was achieved with a trehalase production of 15,068 U·mL-1. The recombinant trehalase TreZM was purified and characterized. It displayed optimal activity at 35 °C and pH 8.5, with Mn2+, Sn2+, Na+, and Fe2+ promoting the activity. Notably, TreZM showed significant inhibition effect on biofilm forming of Staphylococcus epidermidis. The combination of TreZM with a low concentration of antibiotics could inhibit 70 % biofilm formation of Staphylococcus epidermidis and 28 % of Pseudomonas aeruginosa. Hence, this study provides a promising candidate for industrial production of trehalase and highlights its potential application to control harmful biofilms.

High-Level Extracellular Expression of Hyaluronate Lyase HylP in Bacillus subtilis for Hyaluronan Degradation.

Hyaluronate lyase (HA lyase) has potential in the industrial processing of hyaluronan. In this study, HylP, an HA lyase from Streptococcus pyogenes phage (SPB) was successfully expressed in Bacillus subtilis. To improve the extracellular enzyme activity of HylP in B. subtilis, signal peptide engineering systematic optimization was carried out, and cultured it from shake flasks and fermenters, followed by purification, characterization, and analysis of degradation products. The results showed that the replacement of the signal peptide increased the extracellular enzyme activity of HylP from 1.0 × 104 U/mL to 1.86 × 104 U/mL in the shake flask assay, and using a 20 L fermenter in a batch fermentation process, the extracellular enzyme activity achieved the level of 1.07 × 105 U/mL. HylP exhibited significant thermal and pH stability in the temperature range of 40 °C and pH range of 4-8, respectively. The enzyme showed optimum activity at 40 °C and pH 6, with significant activity in the presence of Na+, Mg2+, and Co2+ ions. Degradation analysis showed that HylP efficiently degraded hyaluronan as an endonuclease, releasing unsaturated disaccharides. These comprehensive findings underscore the substantial industrial potential of HylP for hyaluronan processing applications, offering valuable insights into enzyme characterization and optimization of expression for potential industrial utilization.

Rapid detection of six Oceanobacillus species in Daqu starter using single-cell Raman spectroscopy combined with machine learning.

Many traditional fermented foods and beverages industries around the world request the addition of multi-species starter cultures. However, the microbial community in starter cultures is subject to fluctuations due to their exposure to an open environment during fermentation. A rapid detection approach to identify the microbial composition of starter culture is essential to ensure the quality of the final products. Here, we applied single-cell Raman spectroscopy (SCRS) combined with machine learning to monitor Oceanobacillus species in Daqu starter, which plays crucial roles in the process of Chinese baijiu. First, a total of six Oceanobacillus species (O. caeni, O. kimchii, O. iheyensis, O. sojae, O. oncorhynchi subsp. Oncorhynchi and O. profundus) were detected in 44 Daqu samples by amplicon sequencing and isolated by pure culture. Then, we created a reference database of these Oceanobacillus strains which correlated their taxonomic data and single-cell Raman spectra (SCRS). Based on the SCRS dataset, five machine-learning algorithms were used to classify Oceanobacillus strains, among which support vector machine (SVM) showed the highest rate of accuracy. For validation of SVM-based model, we employed a synthetic microbial community composed of varying proportions of Oceanobacillus species and demonstrated a remarkable accuracy, with a mean error was less than 1% between the predicted result and the expected value. The relative abundance of six different Oceanobacillus species during Daqu fermentation was predicted within 60 min using this method, and the reliability of the method was proved by correlating the Raman spectrum with the amplicon sequencing profiles by partial least squares regression. Our study provides a rapid, non-destructive and label-free approach for rapid identification of Oceanobacillus species in Daqu starter culture, contributing to real-time monitoring of fermentation process and ensuring high-quality products.

Lactic acid fermentation of goji berries (Lycium barbarum) prevents acute alcohol liver injury and modulates gut microbiota and metabolites in mice.

Juice fermented with lactic acid bacteria (LAB) has received attention due to its health benefits, such as antioxidant and anti-inflammatory. Previous research on LAB-fermented goji juice mainly focused on exploring the changes in the metabolite profile and antioxidant activity in vitro, whereas the liver protection properties of LAB-fermented goji juice in vivo are still unknown. This study aimed to investigate the effects of Lacticaseibacillus paracasei E10-fermented goji juice (E10F), Lactiplantibacillus plantarum M-fermented goji juice (MF), Lacticaseibacillus rhamnosus LGG-fermented goji juice (LGGF) on preventing acute alcoholic liver injury with physiology, gut microbial, and metabolic profiles in mice. Compared with goji juice, E10F, MF, and LGGF enhanced the protective effect against liver injury by reducing serum alanine transaminase (ALT) levels, improving the hepatic glutathione (GSH) antioxidant system, and attenuating inflammation by decreasing the levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-ß. Furthermore, E10F, MF, and LGGF increased intestinal integrity, restructured the gut microbiota including Bacteroides and Lactobacillus, and altered gut microbial metabolites including kyotorphin, indolelactic acid, and N-methylserotonin. Pretreatment of different LAB-fermented goji juice in mice showed significant differences in gut microbiota and metabolism. The correlation analysis demonstrated that the increase of Lactobacillus, indolelactic acid, and N-methylserotonin by E10F, MF, and LGGF was positively correlated with reduced inflammation and improved liver and gut function. Taken together, E10F, MF, and LGGF all have the potential to be converted into dietary interventions to combat acute alcoholic liver injury. It provided a reference for the study of the hepatoprotective effect of LAB-fermented goji juice.

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris.

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum-related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. KEY POINTS: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation.

Solibacillus daqui sp. nov., isolated from high-temperature Daqu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS111008T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu, and was characterized by polyphasic taxonomy. This novel isolate grew in the presence of 0-5 % (w/v) NaCl, at pH 6.0-9.0 and 25-45 °C; optimum growth was observed with 1 % (w/v) NaCl, at pH 8.0 and 30 °C. A comparative analysis of the 16S rRNA gene sequence (1461 bp) of strain ZS111008T showed highest similarity to Solibacillus silvestris DSM12223T (96.7%), followed by Solibacillus cecembensis PN5T (96.6%) and Solibacillus isronensis AMCK01000046 (96.5%). The DNA G+C content of strain ZS111008T was 37.21 mol%. The respiratory quinone was identified as menaquinone-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and one unknown phospholipid. Lys was detected as the diagnostic diamino acid in the cell wall. Based on morphological characteristics, chemotaxonomic characteristics and physiological properties, strain ZS111008T represents a novel species of the genus Solibacillus, for which the name Solibacillus daqui sp. nov. is proposed. The type strain for this proposed species is ZS111008T (=CGMCC 1.19455T=JCM 35214T).

Microbial biogeography of pit mud from an artificial brewing ecosystem on a large time scale: all roads lead to Rome.

IMPORTANCE: Baijiu is a typical example of how humans employ microorganisms to convert grains into new flavors. Mud cellars are used as the fermentation vessel for strong-flavor Baijiu (SFB) to complete the decomposition process of grains. The typical flavor of SFB is mainly attributed to the metabolites of the pit mud microbiome. China has a large number of SFB-producing regions. Previous research revealed the temporal profiles of the pit mud microbiome in different geographical regions. However, each single independent study rarely yields a thorough understanding of the pit mud ecosystem. Will the pit mud microbial communities in different production regions exhibit similar succession patterns and structures under the impact of the brewing environment? Hence, we conducted research in pit mud microbial biogeography to uncover the impact of specific environment on the microbial community over a long time scale.

Lentibacillus daqui sp. nov., isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS110521T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu and was characterised by polyphasic taxonomy. This novel isolate grew in the presence of 0-20 % (w/v) NaCl, at pH 6.0-9.0 and 20-50 °C; optimum growth was observed with 8-10 % (w/v) NaCl, at pH 7.0 and 37 °C. A comparative analysis of the 16S rRNA gene sequence (1460 bp) of ZS110521T revealed that it displayed the highest similarity to Lentibacillus populi WD4L-1T (95.5 %), followed by Lentibacillus garicola SL-MJ1T (95.4 %) and Lentibacillus lacisalsi BH260T (95.2 %). ANI and dDDH values between ZS110521T and other strains of species of the genus Lentibacillus were less than 78 and 28 %, respectively. The predominant cellular fatty acids (> 10 %) of ZS110521T were anteiso-C17 : 0 (37.8 %), anteiso-C15 : 0 (28.1 %) and iso-C16 : 0 (15.5 %). The respiratory quinone was identified as menaquinone-7 (MK-7) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The polyphasic taxonomic data and the results of chemotaxonomic analysis confirmed that ZS110521T represents a novel species, for which the name Lentibacillus daqui sp. nov. is proposed. The type strain of this proposed species is ZS110521T (=CGMCC 1.19456T =JCM 35213T).

Technology and functional insights into the nicotinamide mononucleotide for human health.

Nicotinamide mononucleotide (NMN), a naturally occurring biologically active nucleotide, mainly functions via mediating the biosynthesis of NAD+. In recent years, its excellent pharmacological activities including anti-aging, treating neurodegenerative diseases, and protecting the heart have attracted increasing attention from scholars and entrepreneurs for production of a wide range of formulations, including functional food ingredients, health care products, active pharmaceuticals, and pharmaceutical intermediates. Presently, the synthesis methods of NMN mainly include two categories: chemical synthesis and biosynthesis. With the development of biocatalyst engineering and synthetic biology strategies, bio-preparation has proven to be efficient, economical, and sustainable methods. This review summarizes the chemical synthesis and biosynthetic pathways of NMN and provides an in-depth investigation on the mining and modification of enzyme resources during NMN biosynthesis, as well as the screening of hosts and optimization of chassis cells via metabolic engineering, which provide effective strategies for efficient production of NMN. In addition, an overview of the significant physiological functions and activities of NMN is elaborated. Finally, future research on technical approaches to further enhance NMN synthesis and strengthen clinical studies of NMN are prospected, which would lay the foundation for further promoting the application of NMN in nutrition, healthy food, and medicine in the future. KEY POINTS: • NMN supplementation effectively increases the level of NAD+. • The chemical and biological synthesis of NMN are comprehensively reviewed. • The impact of NMN on the treatment of various diseases is summarized.

Characterization of Cinnamomum kanehirae Extract-Stimulated Triterpenoids Synthesis in Submerged Fermentation of Antrodia camphorata via Untargeted Metabolomics.

The underlying mechanisms of Cinnamomum kanehirae-stimulated growth and metabolism of Antrodia camphorata remain unknown. Herein, we first observed that the methanol extract of C. kanehirae trunk (MECK) (2 g/L) showed a potent stimulatory effect on A. camphorata triterpenoids production (115.6 mg/L). Second, MECK treatment considerably increased the category and abundance of many secondary metabolites in the mycelia. We identified 93 terpenoids (8 newly formed and 49 upregulated) in the MECK-treated mycelia, wherein 21 terpenoids were the same as those in the fruiting bodies. Third, 42 out of the 93 terpenoids were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, mainly involving monoterpenoids and diterpenoids syntheses. Finally, 27 monoterpenes and 16 sesquiterpenes were detected in the MECK, and the two terpenoids with the highest abundance (linalool and α-pinene) were selected for verification and found to considerably increase the terpenoids production of A. camphorata and demonstrate the regulation of mRNA expression levels of nine key genes in the mevalonate pathway via RT-qPCR. This study is beneficial for elucidating the terpenoids synthesis mechanism in A. camphorata.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.

Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins. Accounting for the accumulated knowledge of the pivotal factors in the solubility and folding stability of proteins, this review focuses on advanced technologies and tools of protein engineering, protein quality control systems, and the redesign of expression platforms in prokaryotic expression systems, as well as advances of the cell-free expression technologies for membrane proteins production.

The differences between broad bean koji fermented in laboratory and factory conditions by an efficient Aspergillus oryzae.

Broad bean paste-meju was fermented by a mixture of broad bean koji and saline; koji fermentation is an essential process for the production of broad bean paste-meju. Aspergillus oryzae was the most widely used in sauce fermentation. The purpose of this study was to research the factory adaptability of the highly efficient A. oryzae PNM003 and further evaluate the effect of fermentation conditions and fermentation strains on koji. A. oryzae PNM003 was compared with the widely used strain HN 3.042 not only in the laboratory but also in factory conditions (large scale). Results showed that the koji made with the same starter in the factory had a greater amount of fungi than that in the laboratory. Bacteria and yeast levels in HN_L koji were higher than in PN_L koji. As for fungi constitution, almost only Aspergillus survived in the end through the microorganism self-purification process during koji fermentation. As for the bacterial constitution, koji was grouped by fermentation conditions instead of fermentation starter. PN koji had higher protease activity and a higher content of total acids, amino acid nitrogen, amino acids, and organic acids in the laboratory conditions. Nevertheless, in factory conditions, PN koji and HN koji had similar indexes. As for volatile flavor compounds, koji made with the two starters in the same condition was grouped together. As for the same starter, there were more flavor compounds metabolized in the factory condition than in the laboratory condition, especially esters and alcohols. The results showed PN was a highly efficient strain to ferment koji, but the advantages were expressed more remarkably in laboratory conditions. In brief, the fermented condition had a greater influence than the fermentation starter for broad bean koji.

Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin.

Phospholipase D (PLD) is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification. However, the efficient heterologous expression of PLD is limited by its cell toxicity. In this study, a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL. A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was established, and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation, 191.30-fold higher than that of the control. Under optimum reaction conditions, a 61.61% conversion ratio and 21.07 g/L of phosphatidylserine production were achieved. Finally, the synthesis system of PL derivates was established, which could efficiently synthesis novel PL derivates. The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application, and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates. As far as we know, this work reports the highest level of extracellular PLD expression to date and the enzymatic production of several PL derivates for the first time.