A plant malonyl-CoA synthetase enhances lipid content and polyketide yield in yeast cells

Abstract

Malonyl-CoA is the essential building block of natural products such as fatty acids, polyketides, and flavonoids. Engineering the biosynthesis of fatty acids is important for biofuel production while that of polyketides provides precursors of medicines and nutritional supplements. However, microorganisms maintain a small amount of cellular malonyl-CoA, which could limit production of lipid and polyketides under certain conditions. Malonyl-CoA concentration is regulated by multiple pathways and signals, and changes in intracellular malonyl-CoA often lead to complex alterations in metabolism. In the present work, overexpression of a plant malonyl-CoA synthetase gene (AAE13) in Saccharomyces cerevisiae resulted in 1.6- and 2.4-fold increases in lipid and resveratrol accumulation simultaneously. We also demonstrated that AAE13 partially complemented the temperature-sensitive acc1 mutant, replacing this key enzyme in central metabolism. Mechanistic analysis by CoA quantification and transcriptomic measurement suggested that increases in malonyl-CoA concentration were coupled with drastic reductions in other major CoA compounds and clear suppression of tricarboxylic acid cycle-related genes. These results suggest that malonyl-CoA is a critical target for fatty acid and polyketide engineering and that overexpression of malonyl-CoA synthetic enzymes needs to be combined with upregulation of CoA synthesis to maintain metastasis of central metabolism.

Recent advances in biosynthesis of fatty acids derived products in Saccharomyces cerevisiae via enhanced supply of precursor metabolites

“…Specifically, less than 2-fold improvement was achieved in most cases simply by ACC1 overexpression [21,53,56,61,75,76]. In some studies, ACC1 overexpression did not improve the production at all [42].…”

“…Thus, upstream genes including ethanol dehydrogenase (ADH2) and/or aldehyde dehydrogenase (ALD6) were co-expressed with SeAcs L641P to direct the metabolic fluxes to acetate and then acetyl-CoA biosynthesis. By introducing the engineered PDH-bypass pathway into wild-type yeast strains, the production of α-santalene was increased by 1.75-fold [6], amorphadiene by 1.22-fold [59], 3-HP by about 1.50-fold [75], PHB by 18-fold [33], and n-butanol by 3.10-fold [36], respectively. In another study, the production of FALs could be increased from 140 mg/L to 236 mg/L by introducing such an acetyl-CoA boosting pathway into the engineered yeast strain with increased fluxes to FAB.…”

“…The glyoxylate shunt could be disrupted by knocking out CIT2 or MLS1, which encodes peroxisomal citrate synthase and cytosolic malate synthase, respectively [7]. Compared with the reference strain with an intact glyoxylate shunt, the inactivation of CIT2 or MLS1 increased the production of α-santalene by 1.36-and 2.27-fold [6], 3-hydroxypropionic acid (3-HP) by 1.19-and 1.20-fold [75], and n-butanol by 1.58-and 1.36-fold [36], respectively. Interestingly, it was found that the production of polyhydroxybutyrate (PHB) was impaired in the ∆cit2 or ∆mls1 yeast strain [33].…”

“…3) were introduced to boost the availability of acetyl-CoA in yeast [34,67]. Notably, improved production of amorphadiene [59], α-santalene [6], PHB [33,34], n-butanol [36,43], 3-HP [75], and fatty acids [67] were achieved by engineering the acetyl-CoA pool in S. cerevisiae. Considering the significance of acetyl-CoA in cellular metabolism, nature has evolved various routes to synthesize acetyl-CoA under different conditions.…”

“…Indeed, the overexpression of ACC1 could increase the malonyl-CoA level and the corresponding production of a wide range of fuels and chemicals, including FFAs [53], FALs [21,53], FAEEs [56], 3-HP [56,75], resveratrol [61], and polyketides [76].…”