Workshop environment heterogeneity shaped the microbiome and metabolome profiles during Xiasha round of Jiangxiangxing Baijiu.

Workshop with different fermentation years plays an essential role in the yield and quality of Baijiu. In actual production, the quality of base Baijiu in newly built workshop is inferior to the older one. In this study, the microbiota of workshop environment and fermentation process from two workshops namely N (ferment 2 years) and O (ferment 20 years) and flavor compounds were studied during Xiasha round. Results showed workshop O accumulated more environmental microorganisms and fungi including P. kudriavzevii, Wickerhamomyces anomalus and Saccharomyces sp mainly came from ground. Yeasts including Pichia, Cyberlindnera, Wickerhamomyces and Candida were responsible for flavor substances formation in O while Saccharopolyspora was in N. This study for the first time explored the reasons for the brewing differences among N and O workshop from perspectives of workshop environment, microbial community and flavor substances, providing new ideas for guiding production as well as improvement of Baijiu quality.

Atroposelective Synthesis of Aldehydes via Alcohol Dehydrogenase-Catalyzed Stereodivergent Desymmetrization.

Axially chiral aldehydes have emerged recently as a unique class of motifs for drug design. However, few biocatalytic strategies have been reported to construct structurally diverse atropisomeric aldehydes. Herein, we describe the characterization of alcohol dehydrogenases to catalyze atroposelective desymmetrization of the biaryl dialdehydes. Investigations into the interactions between the substrate and key residues of the enzymes revealed the distinct origin of atroposelectivity. A panel of 13 atropisomeric monoaldehydes was synthesized with moderate to high enantioselectivity (up to >99% ee) and yields (up to 99%). Further derivatization allows enhancement of the diversity and application potential of the atropisomeric compounds. This study effectively expands the scope of enzymatic synthesis of atropisomeric aldehydes and provides insights into the binding modes and recognition mechanisms of such molecules.

Engineering extracellular electron transfer to promote simultaneous brewing wastewater treatment and chromium reduction.

Microbial fuel cell (MFC) technology has been developed for wastewater treatment in the anodic chamber, and heavy metal reduction in the cathodic chamber. However, the limited extracellular electron transfer (EET) rate of exoelectrogens remained a constraint for practical applications of MFCs. Here, a MFC system that used the electricity derived from anodic wastewater treatment to drive cathodic Cr6+ reduction was developed, which enabled an energy self-sustained approach to efficiently address Cr6+ contamination. This MFC system was achieved by screening exoelectrogens with a superior EET rate, promoting the exoelectrogenic EET rate, and constructing a conductive bio-anode. Firstly, Shewanella algae-L3 was screened from brewing wastewater acclimatized sludge, which generated power density of 566.83 mW m-2. Secondly, to facilitate EET rate, flavin synthesis gene operon ribADEHC was overexpressed in engineered S. algae-L3F to increase flavins biosynthesis, which promoted the power density to 1233.21 mW m-2. Thirdly, to facilitate interface electron transfer, carbon nanotube (CNT) was employed to construct a S. algae-L3F-CNT bio-anode, which further enhanced power density to 3112.98 mW m-2. Lastly, S. algae-L3F-CNT bio-anode was used to harvest electrical energy from brewing wastewater to drive cathodic Cr6+ reduction in MFC, realizing 71.43% anodic COD removal and 98.14% cathodic Cr6+ reduction. This study demonstrated that enhanced exoelectrogenic EET could facilitate cathodic Cr6+ reduction in MFC.

Effects of Four Strains of Actinomycetes on the Content of Terpenoids in Baijiu.

Terpenoids not only are an important health factor in baijiu but also contribute to the elegance and finesse of baijiu, and actinomycetes act as an important source of terpenoids in baijiu. Four strains of actinomycetes-Streptomyces violascens (SPQ1), S. sampsonii (SPS1), S. thermophilus (SPG1), and S. griseus (SPH1)-obtained from the Daqu, pit mud, fermented grains and air, respectively, in the production of baijiu were used in solid-state and liquid fermentation with five brewing raw materials as the substrates. The terpenoids in the metabolites were analyzed and compared using gas chromatography-mass spectrometry (GC-MS). We found that the four strains of actinomycetes produced 31 terpenoids from the hydrolysates of five fermentation substrates during liquid fermentation, and the total terpenoid content was 989.94 µg/kg in the fermentation products. After 28 days of solid-state fermentation, the four actinomycete strains produced 64 terpenoids using the five fermentation substrates, and the total terpenoid content was 23,651.52 µg/kg in the fermentation products. The different fermentation substrates and fermentation methods have a great influence on the terpenoids produced by actinomycetes.

Intrahepatic osteopontin signaling by CREBZF defines a checkpoint for steatosis-to-NASH progression.

BACKGROUND AND AIMS: NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. APPROACH AND RESULTS: Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. CONCLUSIONS: Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.

The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity.

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

Engineering Shewanella oneidensis to efficiently harvest electricity power by co-utilizing glucose and lactate in thin stillage of liquor industry.

Thin stillage, rich in glucose and lactate, can seriously pollute water resources when directly discharged into the natural environment. Microbial fuel cells (MFC), as a green and sustainable technology, could utilize exoelectrogens to break down organics in wastewater and harvest electricity. Nevertheless, Shewanella oneidensis MR-1, cannot utilize thin stillage for efficient power generation. Here, to enable S. oneidensis to co-utilize glucose and lactate from thin stillage, an engineered S. oneidensis G7∆RSL1 was first created by constructing glucose metabolism pathway, promoting glucose and lactate co-utilization, and enhancing biofilm formation. Then, to enhance biofilm conductivity, we constructed a 3D self-assembled G7∆RSL1-rGO/CNT biohybrid with maximum power density of 560.4 mW m-2 and 373.7 mW m-2 in artificial and actual thin stillage, respectively, the highest among the reported genetically engineered S. oneidensis with thin stillage as carbon source. This study provides a new strategy to facilitate practical applications of MFC in wastewater remediation and efficient power recovery.

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach.

BACKGROUND: Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and ß-amyrin on simple sugar and waste cooking oil. RESULTS: We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L ß-amyrin. CONCLUSIONS: The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

Application Potential of Baijiu Non-Saccharomyces Yeast in Winemaking Through Sequential Fermentation With Saccharomyces cerevisiae.

To explore the potential application of non-Saccharomyces yeasts screened from Baijiu fermentation environment in winemaking, the effect of four Baijiu non-Saccharomyces yeasts (two Zygosaccharomyces bailii and two Pichia kudriavzevii) sequentially fermented with Saccharomyces cerevisiae on the physicochemical parameters and volatile compounds of wine was analyzed. The results indicated that there was no obvious antagonism between S. cerevisiae and Z. bailli or P. kudriavzevii in sequential fermentations, and all strains could be detected at the end of alcoholic fermentation. Compare with S. cerevisiae pure fermentation, Z. bailii/S. cerevisiae sequential fermentations significantly reduced higher alcohols, fatty acids, and ethyl esters and increased acetate esters; P. kudriavzevii/S. cerevisiae sequential fermentations reduced the contents of C6 alcohols, total higher alcohols, fatty acids, and ethyl esters and significantly increased the contents of acetate esters (especially ethyl acetate and 3-methylbutyl acetate). Sequential fermentation of Baijiu non-Saccharomyces yeast and S. cerevisiae improved the flavor and quality of wine due to the higher ester content and lower concentration of higher alcohols and fatty acids, non-Saccharomyces yeasts selected from Baijiu fermentation environment have potential applications in winemaking, which could provide a new strategy to improve wine flavor and quality.

Engineering Yarrowia lipolytica to Produce Itaconic Acid From Waste Cooking Oil.

Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.

[Production of carboxylic acids by metabolically engineered Yarrowia lipolytica: a review].

Yarrowia lipolytica is a non-conventional yeast with unique physiological and metabolic characteristics. It is suitable for production of various products due to its natural ability to utilize a variety of inexpensive carbon sources, excellent tolerance to low pH, and strong ability to secrete metabolites. Currently, Y. lipolytica has been demonstrated to produce a wide range of carboxylic acids with high efficiency. This article summarized the progress in engineering Y. lipolytica to produce various carboxylic acids by using metabolic engineering and synthetic biology approaches. The current bottlenecks and solutions for high-level production of carboxylic acids by engineered Y. lipolytica were also discussed, with the aim to provide useful information for relevant studies in this field.

Metabolic engineering of microbes for monoterpenoid production.

Monoterpenoids are an important class of natural products that are derived from the condensation of two five­carbon isoprene subunits. They are widely used for flavouring, fragrances, colourants, cosmetics, fuels, chemicals, and pharmaceuticals in various industries. They can also serve as precursors for the production of many industrially important products. Currently, monoterpenoids are produced predominantly through extraction from plant sources. However, the small quantity of monoterpenoids in nature renders this method of isolation non-economically viable. Similarly impractical is the chemical synthesis of these compounds as they suffer from high energy consumption and pollutant discharge. Microbial biosynthesis, however, exists as a potential solution to these hindrances, but the transformation of cells into efficient factories remains a major impediment. Here, we critically review the recent advances in engineering microbes for monoterpenoid production, with an emphasis on categorized strategies, and discuss the challenges and perspectives to offer guidance for future engineering.

Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study.

BACKGROUND: In biological cells, promoters drive gene expression by specific binding of RNA polymerase. They determine the starting position, timing and level of gene expression. Therefore, rational fine-tuning of promoters to regulate the expression levels of target genes for optimizing biosynthetic pathways in metabolic engineering has recently become an active area of research. RESULTS: In this study, we systematically detected and characterized the common promoter elements in the unconventional yeast Yarrowia lipolytica, and constructed an artificial hybrid promoter library that covers a wide range of promoter strength. The results indicate that the hybrid promoter strength can be fine-tuned by promoter elements, namely, upstream activation sequences (UAS), TATA box and core promoter. Notably, the UASs of Saccharomyces cerevisiae promoters were reported for the first time to be functionally transferred to Y. lipolytica. Subsequently, using the production of a versatile platform chemical isoamyl alcohol as a test study, the hybrid promoter library was applied to optimize the biosynthesis pathway expression in Y. lipolytica. By expressing the key pathway gene, ScARO10, with the promoter library, 1.1-30.3 folds increase in the isoamyl alcohol titer over that of the control strain Y. lipolytica Po1g KU70∆ was achieved. Interestingly, the highest titer increase was attained with a weak promoter PUAS1B4-EXPm to express ScARO10. These results suggest that our hybrid promoter library can be a powerful toolkit for identifying optimum promoters for expressing metabolic pathways in Y. lipolytica. CONCLUSION: We envision that this promoter engineering strategy and the rationally engineered promoters constructed in this study could also be extended to other non-model fungi for strain improvement.

GAT1 Gene, the GATA Transcription Activator, Regulates the Production of Higher Alcohol during Wheat Beer Fermentation by Saccharomyces cerevisiae.

Uncoordinated carbon-nitrogen ratio in raw materials will lead to excessive contents of higher alcohols in alcoholic beverages. The effect of GAT1 gene, the GATA transcription activator, on higher alcohol biosynthesis was investigated to clarify the mechanism of Saccharomyces cerevisiae regulating higher alcohol metabolism under high concentrations of free amino nitrogen (FAN). The availability of FAN by strain SDT1K with a GAT1 double-copy deletion was 28.31% lower than that of parent strain S17, and the yield of higher alcohols was 33.91% lower. The transcript levels of the downstream target genes of GAT1 and higher alcohol production in the double-copy deletion mutant suggested that a part of the effect of GAT1 deletion on higher alcohol production was the downregulation of GAP1, ARO9, and ARO10. This study shows that GATA factors can effectively regulate the metabolism of higher alcohols in S. cerevisiae and provides valuable insights into higher alcohol biosynthesis, showing great significance for the wheat beer industry.

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae.

OBJECTIVES: Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus. RESULTS: Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower. CONCLUSIONS: Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

Structural characterization and immunomodulatory activity of mycelium polysaccharide from liquid fermentation of Monascus purpureus (Hong Qu).

Alkaline extracted endopolysaccharides (MPS) from Monascus purpureus (Hong Qu) mycelium were successfully separated into four sub-fractions, namely MPS-1 (18.0 %), MPS-2 (27.1 %), MPS-3 (12.6 %) and MPS-4 (14.7 %), by DEAE-Cellulose column chromatography. By combining monosaccharide composition analysis, methylation analysis and 1D & 2D NMR, the structure of sub-fractions was systematically characterized. Both MPS-1 and MPS-2 were comprised of mannose, glucose and galactose in the molar ratio of 1.5:1.6:1.0 and 10.6:1.0:13.8, respectively. The backbone of them both consisted of 2-α-Manp with several different branched chains. However, MPS-1 contained glucose based sugar residues such as 3-Glcp and 4-Glcp which were not shown on MPS-2. The proposed structures of MPS-3 and MPS-4 were not obtained due to the fairly complex molecular structure and relatively low yield. Moreover, based on the RAW 264.7 cells model, MPS-2 could significantly promote cytokines secretion including IL-6, TNF-α, and IL-10 and improve expression levels of the related mRNA.

Analysis of the molecular basis of Saccharomyces cerevisiae mutant with high nucleic acid content by comparative transcriptomics.

Ribonucleic acid (RNA) and its degradation products are important functional components widely used in the food industry. Transcription analysis was used to explore the genetic mechanism underlying nucleic acid synthesis in the chemical mutant Saccharomyces cerevisiae strain BY23-195 with high nucleic acid content. Results showed that ribosome biogenesis, meiosis, RNA transport, mitogen-activated protein kinase (MAPK) signaling pathway, tryptophan metabolism, carbon metabolism, and longevity regulating pathway are closely related to the high nucleic acid metabolism of S. cerevisiae. Fourteen most promising genes were selected to evaluate the effect of single-gene deletion or overexpression on the RNA synthesis of S. cerevisiae. Compared with the RNA content of the parent strain BY23, that of mutant strains BY23-HXT1, BY23-ΔGSP2 and BY23-ΔCTT1 increased by 8.19%, 11.60% and 14.00%, respectively. The possible reason why HXT1, GSP2, and CTT1 affect RNA content is by regulating cell fitness. This work was the first to report that regulating the transcription of HXT1, GSP2, and CTT1 could increase the RNA content of S. cerevisiae. This work also provides valuable knowledge on the genetic mechanism of high nucleic acid synthesis in S. cerevisiae and new strategies for increasing its RNA content.

[Higher alcohols metabolism by Saccharomyces cerevisiae: a mini review].

Higher alcohols are one of the main by-products of Saccharomyces cerevisiae in brewing. High concentration of higher alcohols in alcoholic beverages easily causes headache, thirst and other symptoms after drinking. It is also the main reason for chronic drunkenness and difficulty in sobering up after intoxication. The main objective of this review is to present an overview of the flavor characteristics and metabolic pathways of higher alcohols as well as the application of mutagenesis breeding techniques in the regulation of higher alcohol metabolism in S. cerevisiae. In particular, we review the application of metabolic engineering technology in genetic modification of amino transferase, α-keto acid metabolism, acetate metabolism and carbon-nitrogen metabolism. Moreover, key challenges and future perspectives of realizing optimization of higher alcohols metabolism are discussed. This review is intended to provide a comprehensive understanding of metabolic regulation system of higher alcohols in S. cerevisiae and to provide insights into the rational development of the excellent industrial S. cerevisiae strains producing higher alcohols.

Simultaneous Improvement of Limonene Production and Tolerance in Yarrowia lipolytica through Tolerance Engineering and Evolutionary Engineering.

Limonene is an important plant natural product widely used in food and cosmetics production as well as in the pharmaceutical and chemical industries. However, low efficiency of plant extraction and high energy consumption in chemical synthesis limit the sustainability of industrial limonene production. Recently, the advancement of metabolic engineering and synthetic biology has facilitated the engineering of microbes into microbial cell factories for producing limonene. However, the deleterious effects on cellular activity by the toxicity of limonene is the major obstacle in achieving high-titer production of limonene in engineered microbes. In this study, by using transcriptomics, we identified 82 genes from the nonconventional yeast Yarrowia lipolytica that were up-regulated when exposed to limonene. When overexpressed, 8 of the gene candidates improved tolerance of this yeast to exogenously added limonene. To determine whether overexpression of these genes could also improve limonene production, we individually coexpressed the tolerance-enhancing genes with a limonene synthase gene. Indeed, expression of 5 of the 8 candidate genes enhanced limonene production in Y. lipolytica. Particularly, overexpressing YALI0F19492p led to an 8-fold improvement in product titer. Furthermore, through short-term adaptive laboratory evolution strategy, in combination with morphological and cytoplasmic membrane integrity analysis, we shed light on the underlying mechanism of limonene cytotoxicity to Y. lipolytica. This study demonstrated an effective strategy for improving limonene tolerance of Y. lipolytica and limonene titer in the host strain through the combinatorial use of tolerance engineering and evolutionary engineering.

High-efficiency production of bisabolene from waste cooking oil by metabolically engineered Yarrowia lipolytica.

The natural plant product bisabolene serves as a precursor for the production of a wide range of industrially relevant chemicals. However, the low abundance of bisabolene in plants renders its isolation from plant sources non-economically viable. Therefore, creation of microbial cell factories for bisabolene production supported by synthetic biology and metabolic engineering strategies presents a more competitive and environmentally sustainable method for industrial production of bisabolene. In this proof-of-principle study, for the first time, we engineered the oleaginous yeast Yarrowia lipolytica to produce α-bisabolene, ß-bisabolene and γ-bisabolene through heterologous expression of the α-bisabolene synthase from Abies grandis, the ß-bisabolene synthase gene from Zingiber officinale and the γ-bisabolene synthase gene from Helianthus annuus respectively. Subsequently, two metabolic engineering approaches, including overexpression of the endogenous mevalonate pathway genes and introduction of heterologous multidrug efflux transporters, were employed in order to improve bisabolene production. Furthermore, the fermentation conditions were optimized to maximize bisabolene production by the engineered Y. lipolytica strains from glucose. Finally, we explored the potential of the engineered Y. lipolytica strains for bisabolene production from the waste cooking oil. To our knowledge, this is the first report of bisabolene production in Y. lipolytica using metabolic engineering strategies. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of bisabolene and its utilization in converting waste cooking oil into various industrially valuable products.