Dongguang Xiao

Discovering the role of the apolipoprotein gene and the genes in the putative pullulan biosynthesis pathway on the synthesis of pullulan, heavy oil and melanin in Aureobasidium pullulans

Pullulan produced by Aureobasidium pullulans presents various applications in food manufacturing and pharmaceutical industry. However, the pullulan biosynthesis mechanism remains unclear. This work proposed a pathway suggesting that heavy oil and melanin may correlate with pullulan production. The effects of overexpression or deletion of genes encoding apolipoprotein, UDPG-pyrophosphorylase, glucosyltransferase, and α-phosphoglucose mutase on the production of pullulan, heavy oil, and melanin were examined. Pullulan production increased by 16.93 and 8.52% with the overexpression of UDPG-pyrophosphorylase and apolipoprotein genes, respectively. Nevertheless, the overexpression or deletion of other genes exerted little effect on pullulan biosynthesis. Heavy oil production increased by 146.30, 64.81, and 33.33% with the overexpression of UDPG-pyrophosphorylase, α-phosphoglucose mutase, and apolipoprotein genes, respectively. Furthermore, the syntheses of pullulan, heavy oil, and melanin can compete with one another. This work may provide new guidance to improve the production of pullulan, heavy oil, and melanin through genetic approach.

Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans

BackgroundAureobasidium pullulans is a yeast-like fungus that can ferment xylose to generate high-value-added products, such as pullulan, heavy oil, and melanin. The combinatorial expression of two xylose reductase (XR) genes and two xylitol dehydrogenase (XDH) genes from Spathaspora passalidarum and the heterologous expression of the Piromyces sp. xylose isomerase (XI) gene were induced in A. pullulans to increase the consumption capability of A. pullulans on xylose.ResultsThe overexpression of XYL1.2 (encoding XR) and XYL2.2 (encoding XDH) was the most beneficial for xylose utilization, resulting in a 17.76% increase in consumed xylose compared with the parent strain, whereas the introduction of the Piromyces sp. XI pathway failed to enhance xylose utilization efficiency. Mutants with superior xylose fermentation performance exhibited increased intracellular reducing equivalents. The fermentation performance of all recombinant strains was not affected when glucose or sucrose was utilized as the carbon source. The strain with overexpression of XYL1.2 and XYL2.2 exhibited excellent fermentation performance with mimicked hydrolysate, and pullulan production increased by 97.72% compared with that of the parent strain.ConclusionsThe present work indicates that the P4 mutant (using the XR/XDH pathway) with overexpressed XYL1.2 and XYL2.2 exhibited the best xylose fermentation performance. The P4 strain showed the highest intracellular reducing equivalents and XR and XDH activity, with consequently improved pullulan productivity and reduced melanin production. This valuable development in aerobic fermentation by the P4 strain may provide guidance for the biotransformation of xylose to high-value products by A. pullulans through genetic approach.

Overexpression of SNF4 and deletions of REG1- and REG2-enhanced maltose metabolism and leavening ability of baker’s yeast in lean dough

Maltose metabolism of baker’s yeast (Saccharomyces cerevisiae) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker’s yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1/REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant ΔREG1ΔREG2 + SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker’s yeast strains had excellent potential applications.

Effect of ILV6 Deletion and Expression of aldB from Lactobacillus plantarum in Saccharomyces uvarum on Diacetyl Production and Wine Flavor

Diacetyl generates an aromatic off-flavor in wine at a high level. The present study expressed α-acetolactate decarboxylase (ALDB) from Lactobacillus plantarum and/or inactivated acetohydroxyacid synthase (Ilv6) in Saccharomyces uvarum, and the effects on diacetyl production and wine flavor in mutants were investigated through sequential fermentation and cofermentation in mixed cultures of S. uvarum and L. plantarum. The diacetyl content of WYDΔ6 (disrupted one ILV6 allele), WYSΔ6 ( ILV6 complete deletion), WYADΔ6 (disrupted one ILV6 allele with aldB expression), and WYASΔ6 ( ILV6 complete deletion with aldB expression) decreased by 25.71%, 41.30%, 47.77%, and 50.00%, respectively, after sequential fermentation and decreased by 15.15%, 26.72%, 35.26%, and 43.80%, respectively, after cofermentation, compared with that of the parental strain. In addition, Ilv6 inactivation not only decreased the acetic acid content but also balanced the flavor profile in wine effectively. This work provided a valuable insight into the metabolic pathway of diacetyl and wine flavor in S. uvarum.

PGK1 Promoter Library for the Regulation of Acetate Ester Production in Saccharomyces cerevisiae during Chinese Baijiu Fermentation

Appropriate concentrations and proportion of acetate esters and higher alcohols improve the quality of Chinese Baijiu. To regulate the concentrations of acetate esters in Chinese Baijiu, we constructed a PGK1 promoter library through error-prone PCR. Then, we used an enhanced green fluorescent protein as a reporter to characterize the activities of PGK1p mutants. The PGK1p library contained 28 PGK1p mutants and spanned an activity that ranged between 0.1% and 141% of wild-type PGK1p. Seven PGK1p mutants were characterized by an additional reporter β-galactosidase and then used for the overexpression of ATF1 with BAT2 deletion in Saccharomyces cerevisiae a45. The production of ethyl acetate in strains A8, A17, A18, A27, A22, A25, A28, and AWT were 1.66-, 3.09-, 10.59-, 13.07-, 15.99-, 22.67-, 24.06-, and 27.22-fold higher than that of the parental strain. The results on alcohol acetyltransferase (AATase) activity showed that the PGK1p library precisely controlled ATF1 expression and regulated the acetate esters production.

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 β-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.

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae : different roles of the functional domains

BackgroundTup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker’s yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker’s yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated.ResultsThe results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker’s yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker’s yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker’s yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression.ConclusionsThese findings lay a foundation for the optimization of industrial baker’s yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Effects of ATF2 Overexpression with BAT2 Deletion on the Higher Alcohols and Esters in Beer Yeast

During the process of beer fermentation, higher alcohols and esters are two important substances influencing beer flavor. Deletion of BAT2 gene coding amino acid transaminase can effectively reduce production of higher alcohols. On the other hand, alcohol acetyltransferase (AATase) encoded by the ATF2 gene is one of the most important enzymes for acetate ester synthesis. The objective of this study is to construct engineered brewer’s yeast strains for moderate production of acetate esters and less yield of higher alcohols. The industrial beer yeast strain S5 was selected as the parental strain, by means of overexpressing ATF2 and knocking out BAT2 to appropriately increase the concentration of acetate and reduce the concentration of higher alcohols. The engineered strain S5-L, featuring partial BAT2 allelic genes replaced by the constructed ATF2 overexpression cassette, was obtained. The fermentation results indicated that the engineered strain S5-L had a moderate accrete of ethyl acetate compared with the parental strain. The concentration of ethyl acetate produced by the engineered strains S5-L increased to 7.6 mg L−1, about 1.28-fold higher than that produced by the parental S5 cells. The concentration of higher alcohols produced by the engineered strains S5-L reduced to 51.49 mg L−1, about 84.77% of the parental S5 cells. Isobutanol and propanol reduced to 8.30 and 9.72 mg L−1, which was 73.13% and 79.47% of that of the parental strain S5, respectively. Research on the beer yeast genes ATF2 and BAT2 influencing the generation amount of higher alcohol and volatile esters lays a foundation to improve the flavor and quality of beer.

Expression, Purification and Characterization of Maltase from “Quick” Baker’s Yeast

The “quick” baker’s yeasts are capable to rapidly metabolize maltose, thereby improving leavening ability. Given that maltase is the determining factor in maltose fermentation for “quick” baker’s yeast, it is hence necessary to research the properties of the maltase independently for the “quick” baker’s yeast. In this study, the heterogeneous expression of MAL62 encoding for maltase and purification of maltase were well completed. Furthermore, the enzymatic properties of maltase concentrated on the effects of substrate (maltose) and end product (glucose) were investigated as well as the physic-chemical properties and transglycosylation activity. The substrate maltose did not affect the activity of maltase, while the maltase activity was inhibited by the end product. This study provides guidance for the research of maltose metabolism for “quick” baker’s yeast.

Improved Lactose Utilization by Overexpression β-Galactosidase and Lactose Permease in Klebsiella pneumoniae

As an important bio-based chemical product, 2,3-butanediol has a wide range of applications in many fields, such as chemical, fuel, food, and aerospace. Cheese whey powder (CWP), an inexpensive, available, and abundant material, is considered to be an ideal substrate for 2,3-BD fermentation. To improve 2,3-butanediol production, the previous studies mainly focus on the metabolic pathway from pyruvate to 2,3-butanediol or the metabolic pathway of by-products, but studies about improving lactose utilization rate are rarely reported. In the present study, adding exogenous β-galactosidase was proved to favor the lactose utilization and lactose utilization might be the limiting step of lactose fermentation to 2,3-butanediol. ElacY (encoding lactose permease of Escherichia coli) and bgaB (encoding β-galactosidase of K. pneumonia) were overexpressed in K. pneumonia CICC10781. Of the two genes, only overexpression of ElacY promoted lactose utilization of CICC10781, and meanwhile the 2,3-butanediol generation capacity was not affected.