Genetic Strategies and Mechanisms for Improving Ribonucleic Acid Levels of Saccharomyces pastorianus.

Developing microorganisms with a high ribonucleic acid (RNA) content is crucial for the RNA industry. Numerous studies have been conducted to enhance RNA production in yeast cells through genetic engineering, yet precise mechanisms remain elusive. Previously, upregulation of TAL1 or PGM2 and deleting PRS5 or DBP8 individually could increase the RNA content in Saccharomyces pastorianus. In this study, within these genetically modified strains, the intracellular nucleotide levels notably increased following cell fragmentation. Deletion of PRS5 and DBP8 within the strain prompted the upregulation of genes sharing similar functions, consequently augmenting the flow of the gene pathway. Furthermore, the upregulation of genes encoding cell-cycle-dependent protein kinases (CDK) was observed in the G03-△PRS5 strain. The influence of TAL1 and PGM2 on RNA content was attributed to the pentose phosphate pathway (PPP). The RNA content of polygenic recombinant strains, G03-△PRS5+△DBP8 and G03-△PRS5+△DBP8+PGM2, displayed the most significant improvement, increasing by 71.8 and 80.1% when compared to the parental strain. Additionally, the maximum specific growth rate of cells increased in these strains. This study contributes valuable insights into the genetic mechanisms underlying high nucleic acid synthesis in S. pastorianus.

Enhanced expression of a novel trypsin from Streptomyces fradiae in Komagataella phaffii GS115 through combinational strategies of propeptide engineering and self-degredation sites modification.

This study aimed to enhance the expression level of a novel trypsin gene from Streptomyces fradiae ATCC14544 in Komagataella phaffii GS115 through the combinational use of propeptide engineering and self-degradation residues modification strategies. An artificial propeptide consisted of thioredoxin TrxA, the bovine propeptide DDDDK and the hydrophobic peptide FVEF was introduced to replace the original propeptide while the self-degradation residue sites were predicted and analyzed through alanine screening. The results showed that the quantity and enzymatic activity of asft with engineered propeptide reached 47.02 mg/mL and 33.9 U/mL, which were 9.6 % and 59.29 % higher than those of wild-type (42.9 mg/mL and 13.8 U/mL). Moreover, the introduction of R295A/R315A mutation further enhanced the enzymatic activity (58.86 U/mL) and obviously alleviated the phenomena of self-degradation. The tolerance of trypsin towards alkaline environment was also improved since the optimal pH was shifted from pH 9.0 to pH 9.5 and the half-life value at pH 10 was significantly extended. Finally, the fermentation media composition and condition were optimized and trypsin activity in optimal condition reached 160.58 U/mL, which was 2.73-fold and 11.64-fold of that before optimization or before engineering. The results obtained in this study indicated that the combinational use of propeptide engineering and self-degradation sites modification might have great potential application in production of active trypsins.

Characterization of color, metabolites and microbial community dynamics of doubanjiang during constant temperature fermentation.

This study aimed to elaborate the effect of temperature on doubanjiang fermentation. Two batches of constant-temperature groups were prepared and their physicochemical parameters, color formation, metabolites and microbial community dynamics during fermentation were determined and compared with those of natural temperature fermentation group. The results showed that fermentation at 40 °C could accelerate the accumulation of amino nitrogen, reducing sugar, amino acids, organic acids and various volatile metabolites while it was able to inhibit the growth of conditionally pathogenic bacteria, such as Klebsiella and Salmonella. However, high concentrations of total acids and biogenic amines, protrusive burnt flavor and darker color were observed in constant temperature fermentation, which were unfavorable for doubanjiang quality. Higher fermentation temperature lowered the diversity of bacterial community and favored the growth of Bacillus genus. The correlation between key microbial genera and doubanjiang quality indexes were significantly different among different temperatures. This study would deep our understanding of the roles of temperature ondoubanjiangfermentation.

[Manipulation of isocitrate dehydrogenase genes affects the anti-autolytic ability of lager yeast].

Yeast autolysis affects the flavor and quality of beer. The regulation of yeast autolysis is a need for industrial beer production. Previous studies on brewer's yeast autolysis showed that the citric acid cycle-related genes had a great influence on yeast autolysis. To explore the contribution of isocitrate dehydrogenase genes in autolysis, the IDP1 and IDP2 genes were destroyed or overexpressed in typical lager yeast Pilsner. The destruction of IDP1 gene improved the anti-autolytic ability of yeast, and the anti-autolytic index after 96 h autolysis was 8.40, 1.5 times higher than that of the original strain. The destruction of IDP1 gene increased the supply of nicotinamide adenine dinucleotide phosphate (NADPH) and the NADPH/NADP+ ratio was 1.94. After fermentation, intracellular ATP level was 1.8 times higher than that of the original strain, while reactive oxygen species (ROS) was reduced by 10%. The destruction of IDP2 gene resulted in rapid autolysis and a decrease in the supply of NADPH. Anti-autolytic index after 96 h autolysis was 4.03 and the NADPH/NADP+ ratio was 0.89. After fermentation, intracellular ATP level was reduced by 8% compared with original strain, ROS was 1.3 times higher than that of the original strain. The results may help understand the regulation mechanism of citric acid cycle-related genes on yeast autolysis and provide a basis for the selection of excellent yeast with controllable anti-autolytic performance.

[Effect of mitophagy related genes on the antioxidant properties of Saccharomyces cerevisiae].

Mitophagy is a process whereby cells selectively remove mitochondria through the mechanism of autophagy, which plays an important role in maintaining cellular homeostasis. In order to explore the effect of mitophagy genes on the antioxidant activities of Saccharomyces cerevisiae, mutants with deletion or overexpression of mitophagy genes ATG8, ATG11 and ATG32 were constructed respectively. The results indicated that overexpression of ATG8 and ATG11 genes significantly reduced the intracellular reactive oxygen species (ROS) content upon H2O2 stress for 6 h, which were 61.23% and 46.35% of the initial state, respectively. Notable, overexpression of ATG8 and ATG11 genes significantly increased the mitochondrial membrane potential (MMP) and ATP content, which were helpful to improve the antioxidant activities of the strains. On the other hand, deletion of ATG8, ATG11 and ATG32 caused mitochondrial damage and significantly decreased cell vitality, and caused the imbalance of intracellular ROS. The intracellular ROS content significantly increased to 174.27%, 128.68%, 200.92% of the initial state, respectively, upon H2O2 stress for 6 h. The results showed that ATG8, ATG11 and ATG32 might be potential targets for regulating the antioxidant properties of yeast, providing a new clue for further research.

Improving ribonucleic acid production in Saccharomyces pastorianus via in silico genome-scale metabolic network model.

Ribonucleic acid (RNA) and its degradation products are important biomolecules widely used in the food and pharmaceutical industries for their flavoring and nutritional functions. In this study, we used a genome-scale metabolic network model (GSMM) to explore genetic targets for nucleic acid synthesis in a Saccharomyces pastorianus strain (G03). Yeast 8.5.0 was used as the base model, which accurately predicted G03's growth. Using OptForce, we found that overexpression of ARO8 and ATP1 among six different strategies increased the RNA content of G03 by 58.0% and 74.8%, respectively. We also identified new metabolic targets for improved RNA production using a modified GSMM called TissueModel, constructed using the GIMME transcriptome constraint tool to remove low-expressed reactions in the model. After running OptKnock, the RNA content of G03-△BNA1 and G03-△PMA1 increased by 44.6% and 39.8%, respectively, compared to G03. We suggest that ATP1, ARO8, BNA1, and PMA1 regulate cell fitness, which affects RNA content. This study is the first to identify strategies for RNA overproduction using GSMM and to report that regulation of ATP1, ARO8, BNA1, and PMA1 can increase RNA content in S. pastorianus. These findings also provide valuable knowledge on model reconstruction for S. pastorianus.

Revealing the Succession of Spatial Heterogeneity of the Microbial Community during Broad Bean Paste Fermentation.

This study aimed to elaborate the assembly processes and metabolic regulation of the microbial community under the conditions of environmental factors and artificial intervention using broad bean paste (BBP) fermentation as a tractable research object. Spatial heterogenicity of amino acid nitrogen, titratable acidity, and volatile metabolites were observed between upper and lower layers after fermentation for 2 weeks. Amino nitrogen contents in the upper fermented mash reached 0.86, 0.93, and 1.06 g/100 g at 2, 4, and 6 weeks, respectively, which were significantly higher than those of mash located at the lower layer (0.61, 0.79, and 0.78 g/100 g). Moreover, higher concentrations of titratable acidity were accumulated in upper layers (2.05, 2.25 and 2.56 g/100g) than those in lower layers, and the differentiation of volatile metabolites was the greatest (R = 0.543) at 36 days, after which the BBP flavor profiles converged with the fermentation progress. The successive heterogenicity of the microbial community in the mid-late stage was also found during fermentation, and Zygosaccharomyces, Staphylococcus, and Bacillus had heterogeneous characteristics driven by sunlight, water activity, and microbial interactions. This study provided new insights into the mechanisms underlying the succession and assembly of the microbial community of BBP fermentation, which also laid new clues for researches of the microbial communities in complex ecosystems. IMPORTANCE Gaining insights into the community assembly processes is essential and valuable for the elaboration of underlying ecological patterns. However, current studies about microbial community succession in multispecies fermented food usually treat the research object as a whole, are focused exclusively on temporal dimensions, and have ignored the changes of community structure in spatial dimensions. Therefore, dissecting the community assembly process from the view of spatiotemporal dimensions will be a more comprehensive and detailed perspective. Here, we found the heterogenicity of the BBP microbial community under the traditional production technology from spatial and temporal scales, systematically analyzed the relationship between the spatiotemporal succession of community and the difference of BBP quality, and elucidated the roles of environmental factors and microbial interactions to drive the heterogeneous succession of the microbial community. Our findings provide a new insight into understanding the association between microbial community assembly and the quality of BBP.

Effect of environmental stresses during fermentation on brewing yeast and exploration on the novel flocculation-associated function of RIM15 gene.

Flocculation of brewer's yeast is an environment-friendly and cost-effective way to separate yeast cells from fermentation broth for subsequent production. Diverse genetic background and complex fermentation environment cause difficulty to explore flocculation mechanism and regulate yeast flocculation. In this study, comparative transcriptome analysis was carried out between an industrial brewing yeast and its flocculation-enhanced mutant strain, unveiling the differentially-expressed genes were enriched in response to stresses. The expression level of Lg-FLO1 was the highest among all FLO genes. Environmental stresses of fermentation were simulated to stimulated yeast cells and it was found that nitrogen and amino acid starvation promoted the process of flocculation. It is the first time to reveal the nutrient-responsive gene RIM15 has a novel genetic function regulating flocculation. The study provides novel direction and strategies to manage yeast flocculation and achieve effective cell utilization in fermentation.

Development of a Rapid and Sensitive Fluorescence Sensing Method for the Detection of Acetaldehyde in Alcoholic Beverages.

Acetaldehyde is regarded as an important flavor compound in alcoholic beverages. With the advantages of rapidity, low cost and high sensitivity, fluorescent probe could be used as a new tool for the detection of acetaldehyde. Here, an effective fluorescence sensing method based on fluorescent probe N1 (FPN1) was established in this study. The function of FPN1 relies on the nucleophile substitution reaction and photoinduced electron transfer (PET), resulting in a fluorescence increase. Remarkably, the pretreatment background removal method (BRM) was successfully applied for removal of the interference of pyruvate and acetal. The linearity range (LR), limit of detection (LOD) and recovery of the fluorescence sensing method with BRM were 0.0053-200 mg/L, 0.0016 mg/L and 94.02-108.12%, respectively, which showed a broader detection range and better performance on sensitivity compared with the traditional quantitation using gas chromatography (GC). Furthermore, successful application of the method in real samples indicated the advantages of low-cost and rapidity for small-scale detection while assuring the accuracy, which provides a new strategy for the detection of acetaldehyde concentration in alcoholic beverages.

Fed-Batch Fermentation of Saccharomyces pastorianus with High Ribonucleic Acid Yield.

(1) Background: The degradation products of ribonucleic acid (RNA)are widely used in the food and pharmaceutical industry for their flavoring and nutritional enhancement functions. Yeast is the main source for commercial RNA production, and an efficient strain is the key to reducing production costs; (2) Methods: A mutant Saccharomyces pastorianus G03H8 with a high RNA yield was developed via ARTP mutagenesis and fed-batch fermentation was applied to optimize production capacity. Genome sequencing analysis was used to reveal the underlying mechanism of higher RNA production genetic differences in the preferred mutant; (3) Results: Compared with the highest RNA content of the mutant strain, G03H8 increased by 40% compared with the parental strain G03 after response surface model optimization. Meanwhile, in fed-batch fermentation, G03H8's dry cell weight (DCW) reached 60.58 g/L in 5 L fermenter by molasses flowing and RNA production reached up to 3.58 g/L. Genome sequencing showed that the ribosome biogenesis, yeast meiosis, RNA transport, and longevity regulating pathway were closely related to the metabolism of high RNA production; (4) Conclusion: S. pastorianus G03H8 was developed for RNA production and had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle. This work lays the foundation for efficient RNA content using S. pastorianus.

Transcriptomic and metabolomic analysis reveals genes related to stress tolerance in high gravity brewing.

The fermentation performance of yeast is the key of beer production. High gravity brewing is a commonly used technique in industrial lager beer production and it is environmentally friendly. Therefore, there has been extensive effort toward improving high gravity brewing. In this study, through transcriptomic and metabolomic analysis of two homologous lager yeasts, genes that relate to stress tolerance in high gravity brewing were screened. The results showed EMP pathway and multiple amino acid metabolism pathway were the most enriched pathways, and pyruvate might be the core metabolite. Overexpression and knockdown strains were constructed to verify the genes' functions. The overexpression of MAN2, PCL1 and PFK26 genes were beneficial to fermentation without significantly changes in flavor profiles. The relative intracellular ATP levels can help us understand the change of metabolic flux such as enhancement of sugar consumption. This work is helpful to reveal the stress tolerance mechanism of high gravity brewing and breed yeast strains with improved performance.

Roles of sunlight exposure on chemosensory characteristic of broad bean paste by untargeted profiling of volatile flavors and multivariate statistical analysis.

This study aimed to elaborate the role of sunlight on chemosensory characteristics of broad bean paste (BBP). Two groups of BBP fermentation with sunlight treated (ST) or darkness treated (DT) were prepared and the volatile compounds were revealed and compared via GC-MS and GC-IMS and multivariate statistical analysis. A total of 125 compounds were identified and 24 markers with potential contribution to the BBP flavor characteristics were screened. Sunlight exposure was proposed to favor the accumulation of volatile compounds providing fruity, floral, buttery and roasted aroma during fermentation while volatile flavor compounds with unfavorable aroma were enriched in the darkness-treated group. Through KEGG-enrichment analysis, phenylalanine metabolism was found to play critical roles in regulating volatile flavor compound formation and the predicted synthesis network for 24 marker volatile flavor compounds was constructed. This study may unravel the scientific meaning of the role of sunlight on BBP chemosensory quality.

A Novel Approach to Develop Lager Yeast with Higher NADH Availability to Improve the Flavor Stability of Industrial Beer.

Flavor stability is important for beer quality and extensive efforts have been undertaken to improve this. In our previous work, we proved a concept whereby metabolic engineering lager yeast with increased cellular nicotinamide adenine dinucleotide hydride (NADH) availability could enhance the flavor stability of beer. However, the method for breeding non-genetically modified strains with higher NADH levels remains unsolved. In the current study, we reported a novel approach to develop such strains based on atmospheric and room temperature plasma (ARTP) mutagenesis coupled with 2,4-dinitrophenol (DNP) selection. As a result, we obtained a serial of strains with higher NADH levels as well as improved flavor stability. For screening an optimal strain with industrial application potential, we examined the other fermentation characteristics of the mutants and ultimately obtained the optimal strain, YDR-63. The overall fermentation performance of the strain YDR-63 in pilot-scale fermentation was similar to that of the parental strain YJ-002, but the acetaldehyde production was decreased by 53.7% and the resistance staling value of beer was improved by 99.8%. The forced beer aging assay further demonstrated that the favor stability was indeed improved as the contents of 5-hydroxymethylfurfural in YDR-63 was less than that in YJ-002 and the sensory notes of staling was weaker in YDR-63. We also employed this novel approach to another industrial strain, M14, and succeeded in improving its flavor stability. All the findings demonstrated the efficiency and versatility of this new approach in developing strains with improved flavor stability for the beer industry.

Enhanced acidic resistance ability and catalytic properties of Bacillus 1,3-1,4-ß-glucanases by sequence alignment and surface charge engineering.

High stability at acidic environment is required for 1,3-1,4-ß-glucanase to function in biofuel, brewing and animal feed industries. In this study, a mesophilic ß-glucanase from Bacillus terquilensis CGX 5-1 was rationally engineered through sequence alignment and surface charge engineering to improve its acidic resistance ability. Nineteen singly-site variants were constructed and Q1E, I133L and V134A variants showed better acidic stability without the compromise of catalytic property and thermostability. Furthermore, four multi-site variants were constructed and one double-site variant Q1E/I133L with better stability at acidic environment and higher catalytic property was obtained. The fluorescence spectroscopy and structural analysis showed that more surface negative charge, decreased exposure degree of residue No.1, shifted side chain direction of residue No.133 and the lower total and folding free energy might be the reason for the improvement of acidic stability of Q1E/I133L variant. The obtained Q1E/I133L variant has potential applications in industries.

Metabolic potential of microbial community and distribution mechanism of Staphylococcus species during broad bean paste fermentation.

Although the microbial diversity and structure in bean-based fermented foods have been widely studied, systematic studies on functional microbiota and mechanism of community forms in multi-microbial fermentation systems were still lacking. In this work, the metabolic pathway and functional potential of microbial community in broad bean paste (BBP) were investigated by metagenomics approach, and Staphylococcus, Bacillus, Weissella, Aspergillus and Zygosaccharomyces were found to be the potential predominant populations responsible for substrate alteration and flavor biosynthesis. Among them, Staphylococcus was the most abundant and widespread functional microbe, and closely related Staphylococcus species were diverse and ubiquitously distributed, with the opportunistic pathogen S. gallinarum being the most abundant Staphylococcus specie isolated from BBP. To explain the dominance status of S. gallinarum and species distributions of Staphylococcus genus, we tested the effects of abiotic and biotic factors on three Staphylococcus species using a tractable BBP model, demonstrating that adaptation to environmental conditions (environmental parameters and other functional microbes) led to the dominant position and species coexistence of Staphylococcus, and congeneric competition among Staphylococcus species further shaped ecological distributions of closely related Staphylococcus species. In general, this work revealed the metabolic potential of microbial community and distribution mechanism of Staphylococcus species during BBP fermentation, which could help traditional factories to more precisely control the safety and quality of bean-based fermented foods.

Evaluation and prediction of the biogenic amines in Chinese traditional broad bean paste.

Biogenic amines (BAs) are a threat to the safety of broad bean paste, and biosynthetic mechanism of BA and its regulation are unknown. This study aimed to assess microbial BA synthesis in Chinese traditional broad bean paste and determine favorable fermentation conditions for BA regulation. The BAs content in 27 pastes was within the safe range. 64 strains with potential decarboxylation were screened in Luria-Bertani Glycerol medium and identified as Bacillus spp. Although Bacillus amyloliquefaciens produced highest levels of BAs (70.14 ± 2.69 mg/L) in LBAA, Bacillus subtilis produced 6% more BAs than B. amyloliquefaciens. Meanwhile, temperature was the most remarkable factor affecting BAs production by B. amyloliquefaciens 1-13. Furthermore, the fermented broad bean paste model revealed that BA content increased by 61.2 mg/kg every 10 days at 45 °C, which was approximately threefold of that at 25 °C. An ARIMA prediction model of BAs content was constructed, and the total BAs content of 40 mg/100 g was set as the critical value. This study not only contributed to understanding the BAs formation mechanism, but also provided potential measures to control the BAs in fermented soybean products.

Screening lager yeast with higher ethyl-acetate production by adaptive laboratory evolution in high concentration of acetic acid.

Ethyl-acetate is important for the flavor and aroma of the alcoholic beverages, therefore, there have been extensive efforts toward increasing its production by engineering yeast strains. In this study, we reported a new approach to breed non-genetic modified producing yeast strain with higher ethyl-acetate production for beer brewing. First, we demonstrated the positive effect of higher acetic acid concentration on inducing the expression of acetyl-CoA synthetase (ACS). Then, we applied adaptive laboratory evolution method to evolve strain with higher expression level of ACS. As a result, we obtained several evolved strains with increased ACS expression level as well as ethyl-acetate production. In 3 L scale fermentation, the optimal strain EA60 synthesized more ethyl-acetate than M14 at the same time point. At the end of fermentation, the ethyl-acetate production in EA60 was 21.4% higher than M14, while the other flavor components except for acetic acid were changed in a moderate degree, indicating this strain had a bright prospect in industrial application. Moreover, this study also indicated that ACS1 played a more important role in increasing the acetic acid tolerance of yeast, while ACS2 contributed to the synthesis of cytosol acetyl-CoA, thereby facilitating the production of ethyl-acetate during fermentation.

Genome comparison of three lager yeasts reveals key genes affecting yeast flocculation during beer fermentation.

Yeast flocculation plays an essential role in industrial application. Appropriate flocculation of yeast cells at the end of fermentation benefits the cell separation in production, which is an important characteristic of lager yeast for beer production. Due to the complex fermentation environment and diverse genetic background of yeast strains, it is difficult to explain the flocculation mechanism and find key genes that affect yeast flocculation during beer brewing. By analyzing the genomic mutation of two natural mutant yeasts with stronger flocculation ability compared to the parental strain, it was found that the mutated genes common in both mutants were enriched in protein processing in endoplasmic reticulum, membrane lipid metabolism and other pathways or biological processes involved in stress responses. Further functional verification of genes revealed that regulation of RIM101 and VPS36 played a role in lager yeast flocculation under the brewing condition. This work provided new clues for improving yeast flocculation in beer brewing.

Development of a defined autochthonous starter through dissecting the seasonal microbiome of broad bean paste.

Bean-based fermentation foods are usually ripened in open environment, which would lead to inconsistencies in flavor and quality between batches. The physicochemical metabolism and microbial community of seasonal broad bean paste (BBP) were compared to distinguish discriminant metabolites and unique taxa, as well as their specific reasons for different flavor and quality in this study. Here, we found that environmental variables led to the seasonal distribution of microbiota, and differential microorganisms further contributed to the inconsistency of flavor quality, in which Lactobacillales was responsible for the higher titratable acid and amino acid nitrogen concentration in winter pei, while Saccharomycetales benefited the formation of volatile flavor substances in autumn pei. Additionally, we compared the effect of different combinations of Lactobacillales with Zygosaccharomyces rouxii on the quality of BBP, and found that W. confusa was more suitable for BBP fermentation rather than T. halophilus in terms of sensory characteristics and physicochemical metabolites.

Physicochemical, flavor and microbial dynamic changes during low-salt doubanjiang (broad bean paste) fermentation.

This study aimed to elaborate the roles of salt concentration on doubanjiang (broad bean paste) fermentation. Three sets of doubanjiang samples which had lower salt concentration than commercial doubanjiang were prepared and the physicochemical parameters, biogenic amines, flavor, microbial dynamics were analyzed during fermentation. The salt reduction showed significant effect on the dynamics of bacteria and fungi, thus leading to doubanjiang samples with different properties. Salt reduction during fermentation relieved the osmotic pressure towards microbes, which favored the accumulation of amino acid nitrogen, amino acids, and volatile flavor compounds. However, higher concentrations of total acids and biogenic amines and the existence of conditional pathogens, such as Klebsiella, Cronobacter and Acinetobacter genera, were observed in salt reduced doubanjiang samples, which was undesirable for doubanjiang quality. This study would deep our understanding of the roles of salt on doubanjiang fermentation.