Impact of yeast lipid pathway engineering and bioprocess strategy on cellular physiology and lipid content

Microbially-produced lipids have attracted attention due to their positive environmental benefits and commercial value. We have undertaken lipid pathway engineering in Saccharomyces cerevisiae yeast to improve productivity and explore barriers to enhanced lipid production. Research to date has targeted enhanced fatty acid (FA) biosynthesis or lipid accumulation pathways for engineering, however, we have taken stepwise manipulation of up to 6 genes that impact FA synthesis, lipid accumulation and sequestration of stored triacylglycerol (TAG). Initially, expression of individual genes tested effects on yeast growth and lipid production; expression of diacylglycerol acyltransferase DGAT1 and a knockout of the triglyceride lipase 3 (?Tgl3) increased lipid significantly, and an aldehyde dehydrogenase/acetyl-CoA synthetase fusion Ald6-ACS was found to reduce growth. Then, base strains were prepared for enhanced lipid accumulation and sequestration steps by combining DGAT1 and ?Tgl3 for base strain 1 (B1) and to this was added the lipid droplet stabilization protein, caleosin (Atclo1) to form B2. No impact on growth rate was observed with B1 and B2 compared with control but cell viability, measured by propidium iodide exclusion and flow cytometry, was reduced. Total lipid was increased ~1.8-fold in base strains over control. Next, FA biosynthesis genes Ald6-ACS alone or in combination with acetyl-CoA carboxylase (ACC) were coexpressed within B1 and B2. Expression of these additional genes significantly improved cellular lipid content (up to 7.97% DCW basis, 2.6-fold over control) but severely reduced yeast growth and cell viability. To address this drawback, a two-stage process was designed where FA biosynthesis and lipid accumulation genes were differentially regulated by glucose/galactose feeding, which convincingly ameliorated the negative effects resulting in normal cell growth, very high lipid productivity
(307 mg/L, 4.6-fold than control) and improved cell viability. The work has demonstrated that cell viability and other physiological measures are key indicators to guide successful metabolic engineering strategies.