Production of triacetic acid lactone from oleic acid by engineering the yeast Candida viswanathii

Abstract

Triacetic acid lactone (TAL) is a promising platform chemical to produce valuable compounds. The development of engineered microbial hosts to efficiently produce TAL from lipid-containing waste streams could be a cost-effective, sustainable and environmentally friendly approach to meet the industrial demand. In this study, we engineered the yeast Candida viswanathii, possessing robust fatty acid conversion capabilities, to develop an alternative route for TAL production from fatty acids that aims to maximize conversion of the acetyl-CoA pool generated by β-oxidation in the peroxisome. To do so, we inactivated the carnitine acetyltransferase gene to block the transport of acetyl-CoA out of the peroxisome and overexpressed the enzymes methylmalonyl-CoA carboxyltransferase, 2-pyrone synthase and pyruvate carboxylase in the peroxisome to convert acetyl-CoA into TAL. We also performed an adaptive laboratory evolution experiment to obtain mutants with higher growth rate in medium with oleic acid and observed marked differences in central carbon metabolism and organic acid production pathways between the evolved and parental strains. These strains were further engineered by integrating additional copies of TAL biosynthetic genes while reducing competing reactions like ω-oxidation and Lipid biosynthesis, resulting in up to 50-fold increase in titers relative to the initial strain, reaching 280 mg/L. This study contributes to the development of bioprocesses that valorize fatty acids as microbial conversion substrates for the production of valuable compounds.

Fig. 1

Genetic modifications applied to C. viswanathii. Deletion of the CAT2 gene retained the acetyl-CoA pool within the peroxisome. Peroxisomal expression of methylmalonyl-CoA carboxyltransferase (MMC) and pyrone synthase (PS) enzymes promoted TAL production from acetyl-CoA while pyruvate carboxylase (PC) was expressed to replenish oxaloacetate. The overexpressed enzymes are indicated in red

Fig. 2

Comparison of a CAT2-deleted C. viswanathii strain expressing a peroxisomal TAL pathway (423B) and the wild type (WT) by cultivating them in media with different concentrations of glycerol (Gly) and oleic acid (OA). a Dry cell weight; b TAL production; c Stability of TAL spiked into the culture broth after 3 days of incubation. Error bars represent the standard deviation of n = 3

Fig. 3

Screening of colonies isolated from mixed population samples collected after adaptive laboratory evolution. Growth profiles for colony 19 (ALE), WT and 423B strains are indicated in red, green and blue color, respectively. All other tested colonies are displayed in different shades of gray

Fig. 4

Comparison of extracellular metabolite profiles between the WT, 423B and ALE strains in a z-score heatmap. Red and blue colors indicate higher or lower relative concentrations of each metabolite, respectively. P-values were calculated from ANOVA performed on the log2(abundance) data and were corrected for multiple hypothesis tests using the Benjamini-Hochberg method. *** p < 0.001, ** p < 0.01, * p < 0.05. Three replicates are shown for each strain

Fig. 5

Comparison of the TAL concentrations produced by the parental and evolved strains before and after knocking out competing pathways. The strains 423B_AA and ALE_BB derive from 423B and ALE, respectively, and were obtained by inactivation of three ω-oxidation genes and two lipid biosynthesis genes. Error bars represent the standard deviation of n = 3

Fig. 6

Comparison of TAL production in 423B_AA-derived strains after transposon mediated pathway integration. a Strains transformed with the original TAL pathway; b strains transformed with the alternative ACC-PS pathway

Fig. 7

TAL production in the original 423B strain expressing only the peroxisomal pathway or a derivative expressing both peroxisomal and cytosolic TAL pathways. Error bars represent the standard deviation of n = 3