Selected acetyl-coa synthase enzymes for reduction of acetate production in yeast
An acetyl coenzyme and acetic acid technology, applied in the direction of enzymes, fermentation, enzymes, etc., can solve the problem of only moderate acetic acid reduction
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[0108] Example 1. Construction of Expression Vectors for Testing Acetyl-CoA Synthase
[0109] Using the chromosomal DNA of the corresponding host as a template, the coding regions of Saccharomyces cerevisiae genes ACS1 and ACS2 and the coding region of Z. rouxii gene ACS2 were amplified by PCR. Based on the known amino acid sequences, the gene encoding the acs mutant L641P of Salmonella typhimurium and the gene encoding M. bristle acsA1 were synthesized. The codon composition of the synthetic genes was optimized to correspond to the codon bias of highly expressed genes in Saccharomyces cerevisiae. The nucleotide and amino acid sequences of all genes encoding the various ACSs of this study are listed in SEQ ID NOs: 1-10. The vector pX(DeltaTDH_ACSSalty) was assembled using conventional genetic engineering methods ( figure 1 ). All yeast-derived elements in this vector (ie, the THD3 promoter and ENO2 transcription terminator, the 3' and 5' sequences of the yeast delta-element...
example 2
[0110] Example 2. Testing the effect of different ACS on acetic acid production in yeast
[0111] To test the ability of different ACSs to help yeast absorb excess acetic acid, test strains of acetic acid-overproducing yeast were used. By transforming a commercially available S. cerevisiae strain with the large SwaI fragment of the vector PATH1 (TDH_A2_ADE2) ura3 (ade2 mutant) to obtain this strain (referred to herein as A2) ( figure 2 ). Strain A2 expresses three enzymes: phosphoketolase, phosphotransacetylase, and acylated acetaldehyde dehydrogenase that make up the "phosphotransacetylase pathway". compared to the wild-type precursor strain Strain A2 produced significantly higher levels of acetate. The reasons for the increased production of acetate in strain A2 are, firstly, acetylphosphate, the product of the phosphoketolase reaction, is chemically unstable and can be hydrolyzed to acetate and phosphate spontaneously; secondly, acetyl-CoA formed in the phosphoketola...
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