Research on Engineering the Saccharomyces uvarum for Constructing a High Efficiency to Degrade Malic Acid and Low Yield of Diacetyl Biosynthesis Pathway

Diacetyl is a flavor compound in wine with a low threshold (1-2 mg/L). It is produced during alcoholic fermentation (AF) <i>Saccharomyces</i> and malolactic fermentation (MLF) initiated by lactic acid bacteria (LAB). Whereas, the environment after AF suppresses the normal metabolism of LAB after AF. Researchs have shown the influence on diacetyl mechanisms of the genes <i>ILV2</i>, <i>ILV6</i>, <i>ILV3</i>, <i>ILV5</i>, <i>BDH1</i>, <i>BDH2</i>, and gene <i>aldB</i> from <i>Lactobacillus plantarum</i> in <i>Saccharomyce uvarum</i> WY1. While we found that the diacetyl contents produced by mutants after MLF (Co-fermentation and Seq-fermentation) were significantly improved compared to AF alone. Moreover, the genes <i>mae1</i> and <i>mae2</i> from <i>S. pombe</i>, and gene <i>mleS</i> from <i>L. lactis</i> exhibited significant effect on deacidification in our previous study, but the diacetyl of the mutants showed obvious improvement in this study. Thus the effects of association mutation of genes (<i>ILV2</i>, <i>ILV6</i>, <i>ILV3</i>, <i>ILV5</i>, <i>BDH1</i>, <i>BDH2</i>, <i>aldB</i>, <i>mae1</i>, and <i>mleS</i>) on deacidification, diacetyl and other flavors (organic acids, higher alcohols and esters) metabolism in <i>S. uvarum</i> after AF were detected in the study. Among all the mutants, strains V6AmS, V635mS, and V6B12mS showed the most favorable results. Specifically, the L-malic acid contents decreased to 1.26 g/L, 1.18 g/L, and 1.19 g/L, respectively. Concurrently, diacetyl levels were reduced by 52.56%, 61.84%, and 65.31%. The production of n-propanol increased by 18.84%, 20.89%, and 28.12%, whereas isobutanol levels decreased by 37.01%, 42.36%, and 44.04%, and isoamyl alcohol levels decreased by 19.28%, 19.79%, and 16.74%, compared to the parental strain WY1. Additionally, the concentration of lactate ester in the wine increased to 13.162 mg/L, 14.729 mg/L, and 14.236 mg/L, respectively.