Cuiying Zhang

Effects of MIG1, TUP1 and SSN6 deletion on maltose metabolism and leavening ability of baker's yeast in lean dough

BackgroundGlucose repression is a global regulatory system in baker’s yeast. Maltose metabolism in baker’s yeast strains is negatively influenced by glucose, thereby affecting metabolite productivity (leavening ability in lean dough). Even if the general repression system constituted by MIG1, TUP1 and SSN6 factors has already been reported, the functions of these three genes in maltose metabolism remain unclear. In this work, we explored the effects of MIG1 and/or TUP1 and/or SSN6 deletion on the alleviation of glucose-repression to promote maltose metabolism and leavening ability of baker’s yeast.ResultsResults strongly suggest that the deletion of MIG1 and/or TUP1 and/or SSN6 can exert various effects on glucose repression for maltose metabolism. The deletion of TUP1 was negative for glucose derepression to facilitate the maltose metabolism. By contrast, the deletion of MIG1 and/or SSN6, rather than other double-gene or triple-gene mutations could partly relieve glucose repression, thereby promoting maltose metabolism and the leavening ability of baker’s yeast in lean dough.ConclusionsThe mutants of industrial baker’s yeast with enhanced maltose metabolism and leavening ability in lean dough were developed by genetic engineering. These baker’s yeast strains had excellent potential industrial applications.

Optimization of 2,3‐butanediol production by Enterobacter cloacae in simultaneous saccharification and fermentation of corncob residue

Corncob residue, a waste in xylose or xylitol production, was utilized to produce 2,3‐butanediol (2,3‐BD) via simultaneous saccharification and fermentation (SSF). This study developed the optimal conditions for production of 2,3‐BD by using a heat‐resistant strain, Enterobacter cloacae UV4, to perform SSF of the corncob residue. Urea, lactic acid, sodium citrate, and MgSO4, selected by the Plackett–Burman experiment, were determined to be significant independent variables to conduct the response surface experiment. With the optimized medium, a total production of 28.923 g/L for 2,3‐BD and acetoin (BA) was obtained at 60 H. Furthermore, 43.162 g/L of BA production and 0.553 g/L/H of productivity were obtained by fed‐batch SSF, which was 0.424 g diol/g consumed corncob residue. The results suggest that the waste corncob residue could be used as an available substrate for the production of 2,3‐BD by E. cloacae UV4, as well as a potential resource to improve the economics of microbial compound production.

A two-step integration method for seamless gene deletion in baker's yeast.

In this study, we developed a seamless gene deletion method through a two-step integration protocol to construct an industrial bakers yeast with NTH1 deletion. A fusion fragment consisted of the upstream sequence, and the downstream sequence of NTH1 was subcloned into an integrating plasmid containing a URA3 counter-selection marker for excision of unwanted DNA. The plasmid was integrated into the genomic NTH1 locus of recipient bakers yeast, leading to tandem repeats of the upstream flank and the downstream flank. Pop-out of the URA3 marker occurs by integration recombination between either the downstream flank repeats or the upstream flank repeats. Integration recombination between the repeats results in NTH1 deletion without any heterologous DNA and reversion to a wild-type strain. The desired deletion occurred with a frequency of approximately 10(-5). Polymerase chain reaction verification and sequence analysis confirmed the NTH1 disruption and the absence of integrated plasmid sequences in the genome of the selected strain. In addition, the mutant with NTH1 deletion exhibits a higher trehalose accumulation and consequently displays a higher viability of yeast cells after freezing. Thus, this method proposes a protocol to construct mutant yeast without leaving behind any heterologous DNA sequences and will facilitate the genetic engineering of any yeast.

Effects of SNF1 on Maltose Metabolism and Leavening Ability of Baker's Yeast in Lean Dough

Maltose metabolism of bakers yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression, thereby delaying the dough fermentation. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. The Snf1 protein kinase is well known to be essential for the response to glucose repression and required for transcription of glucose-repressed genes including the maltose-utilization genes (MAL). In this study, the SNF1 overexpression and deletion industrial bakers yeast strains were constructed and characterized in terms of maltose utilization, growth and fermentation characteristics, mRNA levels of MAL genes (MAL62 encoding the maltase and MAL61 encoding the maltose permease) and maltase and maltose permease activities. Our results suggest that overexpression of SNF1 was effective to glucose derepression for enhancing MAL expression levels and enzymes (maltase and maltose permease) activities. These enhancements could result in an 18% increase in maltose metabolism of industrial bakers yeast in LSMLD medium (the low sugar model liquid dough fermentation medium) containing glucose and maltose and a 15% increase in leavening ability in lean dough. These findings provide a valuable insight of breeding industrial bakers yeast for rapid fermentation.

Regulation of Saccharomyces cerevisiae genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.

Improvement of stress tolerance and leavening ability under multiple baking-associated stress conditions by overexpression of the SNR84 gene in baker's yeast.

During the bread-making process, industrial bakers yeast cells are exposed to multiple baking-associated stresses, such as elevated high-temperature, high-sucrose and freeze-thaw stresses. There is a high demand for bakers yeast strains that could withstand these stresses with high leavening ability. The SNR84 gene encodes H/ACA snoRNA (small nucleolar RNA), which is known to be involved in pseudouridylation of the large subunit rRNA. However, the function of the SNR84 gene in bakers yeast coping with baking-associated stresses remains unclear. In this study, we explored the effect of SNR84 overexpression on bakers yeast which was exposed to high-temperature, high-sucrose and freeze-thaw stresses. These results suggest that overexpression of the SNR84 gene conferred tolerance of bakers yeast cells to high-temperature, high-sucrose and freeze-thaw stresses and enhanced their leavening ability in high-sucrose and freeze-thaw dough. These findings could provide a valuable insight for breeding of novel stress-resistant bakers yeast strains that are useful for baking.

MAL62 overexpression and NTH1 deletion enhance the freezing tolerance and fermentation capacity of the baker's yeast in lean dough.

BackgroundTrehalose is related to several types of stress responses, especially freezing response in baker’s yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry.ResultsThe strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress.ConclusionsThe mutants of the industrial baker’s yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.

Effects of GLC7 and REG1 deletion on maltose metabolism and leavening ability of baker's yeast in lean dough.

Maltose metabolism and leavening ability of bakers yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. In this study, we focus on the effects of regulators (GLC7 encoding the catalytic and REG1 encoding the regulatory subunits of protein phosphatase type 1) of glucose repression on maltose metabolism and leavening ability of bakers yeast in lean dough. To this end, GLC7 and/or REG1 deletions were constructed and characterized in terms of the growth characteristics, maltose metabolism, leavening ability, and enzyme activities. The results suggest that GLC7 and/or REG1 deletions increased maltose metabolism and leavening ability at different level with glucose derepression and increased enzymes (maltase and maltose permease) activities. In a medium containing glucose and maltose, at the point of glucose exhaustion the maltose metabolized and the leavening ability were increased 59.3% and 23.1%, respectively, in the case of a REG1 single gene deletion.

Improving freeze-tolerance of baker's yeast through seamless gene deletion of NTH1 and PUT1.

Baker’s yeast strains with freeze-tolerance are highly desirable to maintain high leavening ability after freezing. Enhanced intracellular concentration of trehalose and proline in yeast is linked with freeze-tolerance. In this study, we constructed baker’s yeast with enhanced freeze-tolerance by simultaneous deletion of the neutral trehalase-encoded gene NTH1 and the proline oxidase-encoded gene PUT1. We first used the two-step integration-based seamless gene deletion method to separately delete NTH1 and PUT1 in haploid yeast. Subsequently, through two rounds of hybridization and sporulation-based allelic exchange and colony PCR-mediated tetrad analysis, we obtained strains with restored URA3 and deletion of NTH1 and/or PUT1. The resulting strain showed higher cell survival and dough-leavening ability after freezing compared to the wild-type strain due to enhanced accumulation of trehalose and/or proline. Moreover, mutant with simultaneous deletion of NTH1 and PUT1 exhibits the highest relative dough-leavening ability after freezing compared to mutants with single-gene deletion perhaps due to elevated levels of both trehalose and proline. These results verified that it is applicable to construct frozen dough baker’s yeast using the method proposed in this paper.

Effect of GPD1 and GPD2 Deletion on the Production of Glycerol and Ethanol in the Yeast Saccharomyces cerevisiae

Glycerol is the main by-product in ethanol production during the very high gravity (VHG) fermentation process by Saccharomyces cerevisiae. This study investigates the effect of GPD1 or GPD2 (encoding 3-phosphate dehydrogenase) deletion on the production of glycerol and ethanol through the VHG fermentation. We observed that deletion of GPD1 resulted in 45.30 % reduction in glycerol production compared with the parent strain, and ethanol production reached the levels of 15.8 ± 0.03 (v/v), while we failed to observe such a significant decrease in glycerol production for the GPD2 deletion mutants whose ethanol production was 15.7 ± 0.03 (v/v). It can be concluded that deletion of either GPD1 or GPD2 can elevate ethanol production, and that GPD1 deletion can significantly reduce glycerol production, suggesting that GPD1 plays a dominant role in regulating glycerol synthesis during the process of VHG fermentation.