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.

The immunosuppressive effects of low molecular weight chitosan on thymopentin-activated mice bearing H22 solid tumors.

In the present study, the low molecular weight of chitosan (CS) was prepared and its activity on thymopentin-activated mice bearing H22 solid tumors was further researched. The purity and molecular weight of CS were determined by UV and HPGPC spectra, and its immunosuppressive effects on H22 tumor-bearing mice were evaluated through determination on immune organs, cells and cytokines. Results showed that CS contained little impurities with the average molecular weight of 1.20 × 104 Da. The in vivo antitumor experiments demonstrated that CS facilitated to destroy immune organs (thymuses and spleens), suppress immune cells (lymphocytes, macrophages and NK cells) activities and reduce immune-related cytokines (TNF-α, IFN-γ, IL-2 and IL-4) expressions of H22 tumor-bearing mice even with simultaneous TP5 stimulation. Our data suggested that CS could not be applied to improve immune response in cancer-bearing patients, but might be employed for treatments on autoimmune diseases or organ transplant patients.

Enhanced Production of Ethyl Lactate in Saccharomyces cerevisiae by Genetic Modification.

Ethyl lactate is an important flavor substance in baijiu, and it is also one of the common raw materials in the production of flavors and spices. In this study, we first established the ethyl lactate biosynthesis pathway in Saccharomyces cerevisiae α(L) by introducing propionyl coenzyme A transferase (Pct) and alcohol acyltransferase (AAT), and the results showed that strain α(L)-CP-Ae produced the most ethyl lactate 239.53 ± 5.45 mg/L. Subsequently, the copy number of the Pctcp gene and AeAT9 gene was increased, and the modified strain α(L)-tCP-tAe produced 346.39 ± 3.99 mg/L ethyl lactate. Finally, the porin gene (por2) and the mitochondrial pyruvate carrier gene (MPC2) were knocked to impede mitochondrial transport of pyruvate, and the final modified strain α(L)-tCP-tAeΔpor2 produced ethyl lactate 420.48 ± 6.03 mg/L.

Increased Acetate Ester Production of Polyploid Industrial Brewer's Yeast Strains via Precise and Seamless "Self-cloning" Integration Strategy.

BACKGROUND: Enhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand. OBJECTIVES: For increasing the ethyl acetate yield of industrial brewer's yeast strain, all the ATF1 alleles were overexpressed through "self-cloning" integration strategy. MATERIAL AND METHODS: Escherichia coli strain DH5α was utilized for plasmid construction. ATF1 alleles were overexpressed through a precise and seamless insertion of the PGK1 promoter in industrial brewer's yeast strain S6. In addition, growth rates, ATF1 mRNA levels, AATase activity, the fermentation performance of the engineered strains, and gas chromatography (GC) analysis was conducted. RESULTS: The two engineered strains (S6-P-12 and S6-P-30) overexpressed all ATF1 alleles but unaffected normal growth. The ATF1 mRNA levels of the S6-P-12 and S6-P-30 were all 4-fold higher than that of S6. The AATase (Alcohol acetyl transferases, encoded by ATF1 gene) activity of the two engineered strains was all 3-fold higher than that of the parent strain. In the beer fermentation at 10 ℃, the concentrations of ethyl acetate produced by the engineered strains S6-P-12 and S6-P-30 was increased to 23.98 and 24.00 mg L-1, respectively, about 20.44% and 20.54% higher than that of S6. CONCLUSIONS: These results verify that the ethyl acetate yield could be enhanced by the overexpressed of ATF1 in the polyploid industrial brewer's yeast strains via "self-cloning" integration strategy. The present study provides a reference for target gene modification in the diploid or polyploid industrial yeast strains.

Identification by comparative transcriptomics of core regulatory genes for higher alcohol production in a top-fermenting yeast at different temperatures in beer fermentation.

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.

Improved xylose tolerance and 2,3-butanediol production of Klebsiella pneumoniae by directed evolution of rpoD and the mechanisms revealed by transcriptomics.

BACKGROUND: The biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. RpoD, an encoding gene of the sigma factor, is the key element in global transcription machinery engineering and has been successfully used to improve the fermentation with Escherichia coli. However, whether it can regulate the tolerance in K. pneumoniae remains unclear. RESULTS: In this study, the kpC mutant strain was constructed by altering the expression quantity and genotype of the rpoD gene, and this exhibited high xylose tolerance and 2,3-butanediol production. The xylose tolerance of kpC strain was increased from 75 to 125 g/L, and the yield of 2,3-butanediol increased by 228.5% compared with the parent strain kpG, reaching 38.6 g/L at 62 h. The RNA sequencing results showed an upregulated expression level of 500 genes and downregulated expression level of 174 genes in the kpC mutant strain. The pathway analysis further showed that the differentially expressed genes were mainly related to signal transduction, membrane transport, carbohydrate metabolism, and energy metabolism. The nine most-promising genes were selected based on transcriptome sequencing, and were evaluated for their effects on xylose tolerance. The overexpression of the tktA encoding transketolase, pntA encoding NAD(P) transhydrogenase subunit alpha, and nuoF encoding NADH dehydrogenase subunit F conferred increased xylose consumption and increased 2,3-butanediol production to K. pneumoniae. CONCLUSIONS: These results suggest that the xylose tolerance and 2,3-butanediol production of K. pneumoniae can be greatly improved by the directed evolution of rpoD. By applying transcriptomic analysis, the upregulation of tktA, pntA, and nuoF that were coded are essential for the xylose consumption and 2,3-butanediol production. This study will provide reference for further research on improving the fermentation abilities by means of other organisms.

Genetic engineering to alter carbon flux for various higher alcohol productions by Saccharomyces cerevisiae for Chinese Baijiu fermentation.

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded ß-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.

Reduced production of diacetyl by overexpressing BDH2 gene and ILV5 gene in yeast of the lager brewers with one ILV2 allelic gene deleted.

Diacetyl causes an unwanted buttery off-flavor in lager beer. The production of diacetyl is reduced by modifying the metabolic pathway of yeast in the beer fermentation process. In this study, BDH2 and ILV5 genes, coding diacetyl reductase and acetohydroxy acid reductoisomerase, respectively, were expressed using a PGK1 promoter in Saccharomyces cerevisiae, which deleted one ILV2 allelic gene. Diacetyl contents and fermentation performances were examined and compared. Results showed that the diacetyl content in beer was remarkably reduced by 16.52% in QI2-KP (one ILV2 allelic gene deleted), 55.65% in QI2-B2Y (overexpressed BDH2 gene and one ILV2 allelic gene deleted), and 69.13% in QI2-I5Y (overexpressed ILV5 gene and one ILV2 allelic gene deleted) compared with the host strain S2. The fermentation ability of mutant strains was similar to that of S2. Results of the present study can lead to further advances in this technology and its broad application in scientific investigations and industrial beer production.

Reduced production of ethyl carbamate for wine fermentation by deleting CAR1 in Saccharomyces cerevisiae.

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.

Enhanced production of 2,3-butanediol by overexpressing acetolactate synthase and acetoin reductase in Klebsiella pneumoniae.

Mutants with overexpression of α-acetolactate synthase (ALS), α-acetolactate decarboxylase, and acetoin reductase (AR), either individually or in combination, were constructed to improve 2,3-butanediol (2,3-BD) production in Klebsiella pneumoniae. The recombinant strains were characterized in terms of the enzyme activity, 2,3-BD yield, and expression levels. The recombinant K. pneumoniae strain (KG-rs) that overexpressed both ALS and AR showed an improved 2,3-BD yield. When cultured in the media with five different carbon sources (glucose, galactose, fructose, sucrose, and lactose), the mutant exhibited higher 2,3-BD productivity and production than the parental strain in all the tested carbon sources except for lactose. The 2,3-BD production of KG-rs in a batch fermentation with glucose as the carbon source was 12% higher than that of the parental strain.

Simultaneous determination of furfural, acetic acid, and 5-hydroxymethylfurfural in corncob hydrolysates using liquid chromatography with ultraviolet detection.

A single-laboratory validation study was conducted using HPLC for detecting and quantifying acetic acid, furfural, and 5-hydroxymethylfurfural (HMF) in corncob hydrolysates. A pretreatment procedure using dilute sulfuric acid was optimized for corncob hydrolysis. The final hydrolysates were analyzed by HPLC using a C18 RP column with aqueous 0.01% (v/v) H2SO4-CH3OH (95 + 5) as the mobile phase at a flow rate of 1 mL/min. The wavelengths for detecting the three compounds were changed to their optimal UV detection wavelengths at the time of elution. The wavelength detection adjustments were as follow: 205 nm (0 to 4 min); 284 nm (4 to 7 min); and 276 nm (7 to 10 min). Separation was achieved with a chromatographic run time of 10 min. The calibration curves for the three compounds had correlation coefficients (r2) > or = 99.8%. The analytical range, as defined by the calibration curves, was 0.5-10 mg/L for acetic acid, 0.4-22 mg/L for furfural, and 0.1-18 mg/L for HMF. The LODs for acetic acid, furfural, and HMF were estimated to be 0.05, 0.03, and 0.02 mg/L, respectively; the LOQs were 0.196, 0.135, and 0.074 mg/L, respectively. The RSD values for the intraday precision study ranged from 0.31 to 2.22%, and from 0.57 to 2.43% for the interday study. The mean recovery rates in all compounds were between 100.08 and 101.49%.