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Background: Human serum albumin (HSA) is the most abundant protein in plasma, playing crucial roles in regulating osmotic pressure and maintaining protein homeostasis. It is widely applied in the clinical treatment of various diseases. HSA can be purified from plasma or produced using recombinant DNA technology. Due to the improved efficiency and reduced costs, a growing body of research has focused on enhancing albumin production through bacterial strain overexpression. However, there have been few studies on the effect of albumin on the characteristics of the overexpressing-strain itself, particularly stress resistance. In this study, we utilized Lactiplantibacillus plantarum (L. plantarum) AR113 as the expression host and successfully constructed the albumin overexpression strain AR113-pLLY01 through gene editing technology. The successful expression of albumin was achieved and subsequently compared with the wild-type strain AR113-pIB184., Results: The results demonstrated that the survival rate of AR113-pLLY01 was also significantly better than that of AR113-pIB184 after lyophilization. In addition, AR113-pLLY01 exhibited a significantly better protective effect than AR113-pIB184 at pH 3, indicating that albumin possesses a certain tolerance to acidic stress. At bile salt concentrations higher than 0.03%, both strains showed limited growth, but at a concentration of 0.02%, AR113-pLLY01 had a significant protective effect., Conclusion: This study suggest that albumin can improve strain tolerance, which has significant implications for future applications. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Streptococcus thermophilus is an important strain widely used in dairy fermentation, with distinct urea metabolism characteristics compared to other lactic acid bacteria. The conversion of urea by S. thermophilus has been shown to affect the flavor and acidification characteristics of milk. Additionally, urea metabolism has been found to significantly increase the number of cells and reduce cell damage under acidic pH conditions, resulting in higher activity. However, the physiological role of urea metabolism in S. thermophilus has not been fully evaluated. A deep understanding of this metabolic feature is of great significance for its production and application. Genome-scale metabolic network models (GEMs) are effective tools for investigating the metabolic network of organisms using computational biology methods. Constructing an organism-specific GEM can assist us in comprehending its characteristic metabolism at a systemic level., Results: In the present study, we reconstructed a high-quality GEM of S. thermophilus S-3 (iCH492), which contains 492 genes, 608 metabolites and 642 reactions. Growth phenotyping experiments were employed to validate the model both qualitatively and quantitatively, yielding satisfactory predictive accuracy (95.83%), sensitivity (93.33%) and specificity (100%). Subsequently, a systematic evaluation of urea metabolism in S. thermophilus was performed using iCH492. The results showed that urea metabolism reduces intracellular hydrogen ions and creates membrane potential by producing and transporting ammonium ions. This activation of glycolytic fluxes and ATP synthase produces more ATP for biomass synthesis. The regulation of fluxes of reactions involving NAD(P)H by urea metabolism improves redox balance., Conclusion: Model iCH492 represents the most comprehensive knowledge-base of S. thermophilus to date, serving as a potent tool. The evaluation of urea metabolism led to novel insights regarding the role of urease. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria according to the demands of the user., Results: We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR - and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results., Conclusion: Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results - a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. Software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: It has been well accepted that lactic acid bacteria (LAB) are the main bacterial genera present during the brewing of Chinese rice wine (CRW). LAB plays a decisive role in the flavor quality of CRW; however, its application in CRW has previously been overlooked. Therefore, effects of different LAB as co-fermenter on the flavor characteristics of CRW were investigated., Results: Co-fermentation of LAB increased the utilization rate of reducing sugar, concentration of lactic acid, amino acid nitrogen and total acidity, as well as the content of volatile flavor compounds. Different LAB doses had little effect on the flavor profiles of CRW, but the species of LAB greatly affected the flavor characteristic. The flavor of CRW co-fermented with Lactococcus lactis was characterized by long-chain fatty acid ethyl esters, while co-fermentation with Weissella confusa highlighted the ethyl esters of low molecular weight and short carbon chains in the resultant CRW. Alcohol compounds were dominant in the CRW co-fermented using Pediococcus pentosaceus., Conclusion: The co-fermentation of LAB increased the number of volatile flavor compounds, especially esters. LAB exhibited great potential in the application of CRW industry to enrich the flavor characteristics and enhance the flavor diversity of the final product. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: Probiotic lactic acid bacterium Lactiplantibacillus plantarum is widely used in the dairy and other fermented food industries. L. plantarum AR113 harbors a C 30 carotenoid operon crtNM based on genomic analysis, but the yield of C 30 carotenoid is only 8.1 μg g -1 DCW., Results: To improve the productivity of C 30 carotenoid, crtNM from L. plantarum AR113 was cloned and reconstructed in Escherichia coli BL21(DE3). The proteins crtN and crtM were successfully expressed based on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the carotenoid was detected using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). In comparison with the constitutive promoter P 44 , the use of the inducible T7 promoter significantly increased the carotenoid content in E. coli. The fermentation conditions were also optimized with induction by 0.5 mmol/L IPTG at 20 °C for 7 h. The yield of C 30 carotenoid reached 154.5 μg g -1 DCW, which was 18-fold higher than that of L. plantarum AR113. The 2,2-diphenyl-1-picryl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6sulfonic acid (ABTS) radical scavenging capacity of C 30 carotenoids synthesized by heterologous expression in E. coli was also higher than that of the antioxidant food additive butylated hydroxytoluene., Conclusions: Our findings suggest that E. coli has strong potential as a basic chassis for the production of C 30 carotenoids from Lactiplantibacillus with high antioxidant activity. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: Streptococcus thermophilus is a major starter used in the dairy industry and it could improve the flavor of fermented products. It is necessary to improve biomass of S. thermophilus for its application and industrialization. The utilization of carbon sources directly affects the biomass of S. thermophilus. Therefore, the carbohydrate metabolism of S. thermophilus should be investigated., Results: In the present study, metabolic parameters and gene expression of S. thermophilus S-3 with different carbon sources were investigated. The physicochemical results showed that S. thermophilus S-3 had high lactose utilization. Transcriptome analysis found that approximately 104 genes were annotated onto 15 carbohydrate metabolic pathways, of which 15 unigenes were involved in the phosphotransferase system and 75 were involved in the ATP-binding cassette transporter system. In addition, 171 differentially expressed genes related to carbohydrate metabolism were identified. Expression of the galactose metabolism genes lacSZ and galKTEM increased significantly from the lag phase to the mid-exponential growth phase as a result of the global regulator protein, catabolite control protein A (CcpA). The high expression of galK in the mid- to late- phases indicated that the metabolite galactose is re-transported for intracellular utilization. CcpA regulation may also induce high expressions of glycolytic pathway regulated-genes related to lactose utilization, including ldh, fba, eno, pfkA, bglA, pgi, pgm and pyk, producing optimal glycolytic flux and S. thermophilus S-3 growth., Conclusion: The present study provides new insights into the carbon metabolism regulation and provide theoretical support for high-density fermentation of S. thermophilus S-3. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: Adhesion is considered important for Lactiplantibacillus to persist in the human gut and for it to exert probiotic effects. Lactiplantibacillus plantarum contains a considerable number and variety of genes encoding bile salt hydrolases (bsh), but their effects on microbial adhesion remain poorly understood. To clarify the effects of four bsh on adhesion, we tried to knock out bsh (Δbsh) of L. plantarum AR113 using the CRISPR-Cas9 method, and compared the growth, auto-aggregation (R AA ), co-aggregation (R CA ), surface hydrophobicity (A HC ) of AR113 wild-type and Δbsh strains and their adhesion abilities to HT29 cells., Results: We first obtained the AR113 Δbsh1,3,2,4 strain with four bsh knocked out. Their growth was significantly slower than the wild-type strain cultured in De Man, Rogosa, and Sharpe medium (MRS) with 3.0 g L -1 glyco- or tauro-conjugated bile acid. Bsh had no significant effect on the growth of ten strains cultured in MRS, but Δbsh1 inhibited their growth when cultured in MRS containing 3.0 g L -1 sodium glycocholate, whereas Δbsh4 instead promoted their growth in MRS with 3.0 g L -1 sodium glycocholate and sodium taurocholate. R CA and R AA were linearly positive for all strains except AR113 Δbsh2,4, and A HC and R AA were negatively correlated for most strains excluding AR113 Δbsh2, with R AA  = 6.38-25.05%, R CA  = 5.17-9.22%, and A CH  = 3.22-47.71%. The adhesion ability of ten strains cultured in MRS was higher than that of strains cultured in MRS with 3.0 g L -1 bovine bile, and it was related to bsh2., Conclusion: Bsh differentially affected the adhesion of AR113 series strains. This adds to the available information about substrate-gene-performance, and provides new information to enable engineering to regulate the colonization of Lactiplantibacillus. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Background: As a natural food additive, exopolysaccharide (EPS) produced by Streptococcus thermophilus can improve product viscosity and texture. The protein EpsA is a putative pathway-specific transcriptional regulator for EPS biosynthesis in S. thermophilus., Results: According to comparative analysis of EPS biosynthetic gene clusters, a conserved region of epsA (609 bp) was employed to design primer pair epsA-F/R as a molecular marker for the isolation of EPS-producing (EPS + ) S. thermophilus. Two EPS + S. thermophiles strains, AR333 and S-3, were band-positive, whereas Lactococcus lactis NZ9000 (non-EPS-producing, EPS - ), Lactobacillus casei LC2W (EPS + ) and L. plantarum AR113 (EPS + ) were negative by polymerase chain reaction (PCR) amplicon bands using the epsA probe. This indicated good specificity of the epsA probe to EPS + S. thermophilus. Moreover, based on PCR screening with the epsA probe, 23 positive strains were isolated and identified as S. thermophilus from our microbial library and natural fermented milk with 141.3-309.2 mg L -1 of EPS production, demonstrating the validity of our molecular marker screening method., Conclusion: The designed molecular marker of epsA can rapidly screen EPS + S. thermophilus, which has potential application in the dairy and other food industries. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Background: In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms., Results: To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S-29 were analysed. The addition of n-tetradecane as an extractant during ISEF showed more pronounced up-regulation of ubiquinone and other terpenoid-quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up-regulated. The CoQ2 genes encode puru-hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine-type sesquiterpene antrodin C., Conclusion: This investigation advances our understanding of how two different extractants of n-tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Background: Antroquinonol, a ubiquinone derivative that shows anticancer and anti-inflammatory activities, is produced during solid-state fermentation of Antrodia camphorata; however, it cannot be biosynthesized via conventional submerged fermentation., Results: A method for enhancing the biosynthesis of antroquinonol by controlling pH and adding an oxygen vector in a 7 L bioreactor was studied. In shake-flask experiments, a maximum antroquinonol production of 31.39 ± 0.78 mg L -1 was obtained by fermentation with adding 0.2 g L -1 coenzyme Q 0 (CoQ 0 ), at the 96th hour. Following kinetic analysis of the fermentation process, pH control strategies were investigated. A maximum antroquinonol production of 86.47 ± 3.65 mg L -1 was achieved when the pH was maintained at 5.0, which exhibited an increase of 348.03% higher than the batch without pH regulation (19.30 ± 0.88 mg L -1 ). The conversion rate of CoQ 0 improved from 1.51% to 20.20%. Further research revealed that the addition of n-tetradecane could increase the production of antroquinonol to 115.62 ± 4.87 mg L -1 by increasing the dissolved oxygen in the fermentation broth., Conclusion: The results demonstrated that pH played an important role in antroquinonol synthesis in the presence of the effective precursor CoQ 0 . It was a very effective strategy to increase the yield of antroquinonol by controlling pH and adding oxygen vector. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Background: Antroquinonol have significantly anti-tumour effects on various cancer cells. There is still lack of reports on regulation of environmental factors on antroquinonol production by Antrodia camphorata., Results: An effective submerged fermentation method was employed to induce antroquinonol with adding H 2 O 2 . The production of antroquinonol was 57.81 mg L -1 after fermentation for 10 days when adding 25 mmol L -1 H 2 O 2 at day 4 of the fermentation process. Then, antroquinonol was further increased to 80.10 mg L -1 with cell productivity of 14.94 mg g -1 dry mycelium when the feeding rate of H 2 O 2 was adjusted to 0.2 mmol L -1 h -1 in the 7 L fermentation bioreactor. After inhibiting the generation of reactive oxygen species with the inhibitor diphenyleneiodoium, the synthesis of antroquinonol from A. camphorata was significantly reduced, and the yield was only 3.3 mg L -1 ., Conclusion: The results demonstrated that addition of H 2 O 2 was a very effective strategy to induce and regulate the synthesis of antroquinonol in submerged fermentation. Reactive oxygen species generated by H 2 O 2 during fermentation caused oxidative stress, which induced the synthesis of antroquinonol and other chemical compounds. Moreover, it is very beneficial process to improve production and diversity of the active compounds during liquid fermentation of A. camphorata mycelium. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Background: Although the volatiles present in mushrooms and fungi have been investigated by many researchers, including Antrodia camphorata in submerged fermentation, there are few data available regarding changes in volatile compounds during fermentation. Our research has revealed that solid state fermentation of A. camphorata is highly odiferous compared with submerged cultures and the odor changed with increasing culture time. Therefore the aim of this study was to investigate the changes in volatile compound composition of A. camphorata during solid state fermentation., Results: Altogether, 124 major volatile compounds were identified. The volatile compounds produced by A. camphorata during growth in solid state fermentation were quite different. Oct-1-en-3-ol, octan-3-one and methyl 2-phenylacetate were predominant in exponential growth phase production, while the dominant volatiles produced in stationary phase were octan-3-one and methyl 2-phenylacetate. In stationary phase, lactone compounds in A. camphorata, such as 5-butyloxolan-2-one, 5-heptyloxolan-2-one, 6-heptyloxan-2-one, contributed greatly to peach and fruit-like flavor. Terpene and terpene alcohol compounds, such as 1-terpineol, L-linalool, T-cadinol, (E, E)-farnesol, β-elemene, cis-α-bisabolene and α-muurolene, made different contributions to herbal fresh aroma in A. camphorata. Nineteen volatile sesquiterpenes were detected from solid state fermentation of A. camphorata. The compounds 5-n-butyl-5H-furan-2-one, β-ionone, (-)-caryophyllene oxide, aromadendrene oxide, diepi-α-cedrene epoxide, β-elemene, α-selinene, α-muurolene, azulene, germacrene D, γ-cadinene and 2-methylpyrazine have not hitherto been reported in A. camphorata., Conclusion: The preliminary results suggest that the aroma-active compounds produced by A camphorata in solid state fermentation might serve as an important source of natural aroma compounds for the food and cosmetic industries or antibiotic activity compounds. The sesquiterpenes could be identified as possible taxonomic markers for A. camphorata., (Copyright © 2011 Society of Chemical Industry.)