Methods for producing designer esters and assessing alcohol acyltransferase specificity for ester biosynthesis

A method of selectively synthesizing a butyryl-coenzyme A (CoA) derived ester via microbial biosynthesis, the method comprising:

• selecting a target ester to be synthesized by microbial biosynthesis from the group consisting of butyl acetate, butyl butyrate, and ethyl butyrate;
• providing a first expression vector, the first expression vector encoding a first plurality of enzymes that convert acetyl-CoA to butyryl-CoA selected from Escherichia coli atoB (atoBEc), Clostridium acetobutylicum Hbd (hbdCa), Clostridium acetobutylicum crt (crtCa), and Treponema denticola ter (terra);
• providing a second expression vector that encodes a second plurality of enzymes selected from Zymomonas mobilis pdc (pdcZm) and Zymomonas mobilis adhB (adhBZm) or Clostridium acetobutylicum adhE2 (adhE2Ca) for alcohol synthesis and Candida boidinii fdh (fdhCb) for facilitating the NADH production, wherein (i) at least one of the second plurality of enzymes facilitates production of nicotinamide adenine dinucleotide and hydrogen (NADH), (ii) at least one of the enzymes is an alcohol acetyltransferase selected from Saccharomyces cerevisiae ATF1 (ATF1Sc) when an acetate ester is the target ester or Fragaria ananassa (cultivated strawberry) SAAT (SAATFa) when an acylate ester is the target ester, and wherein (iii) at least one of the second plurality of enzymes selectively determines a pathway for either butanol or ethanol synthesis;
• and
• inserting the first expression vector and the second expression vector into a microbial host cell selected from the group consisting of EcJWBA2, EcJWEB2, EcJWBB2, EcJWBA15, EcJWEB7, and EcJWBB8,
• contacting the microbial cells with carbon source in culture media;
• and
• culturing the microbial host to produce butyryl-coenzyme A derived esters;
• wherein expression of the first and second expression vectors in the microbial host cell produces the target ester.