Increase ethyl acetate production in Saccharomyces cerevisiae by genetic engineering of ethyl acetate metabolic pathway

Abstract

Ethyl acetate has attracted much attention as an important chemical raw material and a flavor component of alcoholic beverages. In this study, the biosynthetic pathway for the production of ethyl acetate in Chinese liquor yeast was unblocked. In addition to engineering Saccharomyces cerevisiae to increased intracellular CoA and acetyl-CoA levels, we also increased the combining efficiency of acetyl-CoA to ethanol. The genes encoding phosphopantothenate-cysteine ligase, acetyl-CoA synthetase, and alcohol acetyltransferase were overexpressed by inserting the strong promoter PGK1p and the terminator PGK1t, respectively, and then combine them. Our results finally showed that the ethyl acetate levels of all engineering strains were improved. The final engineering strain CLy12a-ATF1-ACS2-CAB2 had a significant increase in ethyl acetate yield, reaching 610.26 (± 14.28) mg/L, and the yield of higher alcohols was significantly decreased. It is proved that the modification of ethyl acetate metabolic pathway is extremely important for the production of ethyl acetate from Saccharomyces cerevisiae.

Metabolic Engineering of Saccharomyces cerevisiae for Ethyl Acetate Biosynthesis

“…In order to improve the ethyl acetate production, the activity of AATase that catalysts the synthesis of ethyl acetate from acetyl-CoA and ethanol, should be enhanced (Figure ). As is demonstrated in earlier research, those transformants carrying multiple copies of the ATF1 gene are more vibrant in AATase process to generate acetate esters with higher concentration when compared with those strains restricted to bear only one copy of ATF1 gene . Therefore, to address the above-mentioned problem, ATF1 gene was coexpressed in ACS1 and ALD6 genes expression cassettes.…”

“…Yeast cells were transformed using the lithium acetate/single-stranded carrier (LiAc/SS) method . These PCR fragments were inserted into a specific locus in the yeast genome through intracellular homologous recombination . The loci GAL80 gene was involved in transcriptional regulation in galactose metabolism, the knockdown of which would not severely affect target product accumulation or cell growth mainly involving glucose metabolism. , The details of yeast strain construction are summarized in the Supporting Methods.…”

“…The yeast cells were developed in 5 mL of 8°Bx corn hydrolysate medium with extra yeast extract of a concentration of 0.5% at a temperature of 30 °C lasting for 24 h, after which they were transported into a 150 mL conical flask of 45 mL of 12°Bx corn hydrolysate with extra yeast extract of a concentration of 0.5% to be cultured at 30 °C, the process of which lasted for 16 h 3 . Twice in total, 15 mL from the yeast which had been developed in advance was taken out and put into a 250 mL conical flask with 135 mL of 18°Bx corn hydrolysate medium with supplementation of 1 mL nutritive salt . The conical flask (250 mL) was sealed closely with parafilm to create a fermenting environment with a restricted amount of oxygen, and at the same time a narrow exit was left for carbon dioxide by punching a small hole on the parafilm with a 0.5 mm injection needle …”

“…Baijiu produced from China is one of the most famous distilled liquors in the world, together with brandy, gin, rum, vodka, and whiskey . Ethyl acetate has been known as an important flavor compound in Baijiu, which could provide an enjoyable scent of fruit (solvent-like) and affect the sensory quality and flavor traits . Moreover, ethyl acetate is a unique compound that could be used as a clear indicator of Baijiu style and quality in national standards .…”

Formation of basic soybean‐flavor substances using pork fat soaked in rice‐flavored liquor

“…Esterification was enhanced at 50°C, resulting in more ethyl acetate compared to RT. Ethyl acetate is an important flavor substance in all liquors, and in small quantities, it confers a good bouquet and fruity flavor (Dong et al, 2019). Pork fat soaked in rice liquor had a significantly increased ethyl acetate content, which would benefit the formation of soybean flavor.…”