Engineering Yarrowia lipolytica to Produce Itaconic Acid From Waste Cooking Oil

Abstract

Itaconic acid (IA) is a high-value organic acid with a plethora of industrial applications. In this study, we seek to develop a microbial cell factory that could utilize waste cooking oil (WCO) as raw material for circular and cost-effective production of the abovementioned biochemical. Specifically, we expressed cis-aconitic acid decarboxylase (CAD) gene from Aspergillus terreus in either the cytosol or peroxisome of Yarrowia lipolytica and assayed for production of IA on WCO. To further improve production yield, the 10 genes involved in the production pathway of acetyl-CoA, an intermediate metabolite necessary for the synthesis of cis-aconitic acid, were individually overexpressed and investigated for their impact on IA production. To minimize off-target flux channeling, we had also knocked out genes related to competing pathways in the peroxisome. Impressively, IA titer up to 54.55 g/L was achieved in our engineered Y. lipolytica in a 5 L bioreactor using WCO as the sole carbon source.

Keywords: Y. lipolytica; itaconic acid; peroxisome; subcellular engineering; waste cooking oil.

FIGURE 1

Simplified schematic of IA biosynthetic pathway in Y. lipolytica. Engineered Y. lipolytica uptakes and converts extracellular carbon sources such as glucose and waste cooking oil into IA products. Genes and metabolites of the native TCA and glyoxylate cycle pathway are identified in black, while heterologously introduced genes are shown in green and the endogenous genes used in this paper are shown in red. LIP2, lipases; POX1-6, six difffferent acyl-CoA oxidases; MFE1, multifunctional enzyme; POT1, peroxisomal thiolase; PEX10, a proteins required for peroxisome assembly; CAT, carnitine acetyltransferases; ICL, isocitrate lyase; CAD, iso-aconitic acid decarboxylase; ACO, aconitase; MLS, malate synthase; MDH, malate dehydrogenase; CIT, citrate synthase.

FIGURE 2

IA production in Y. lipolytica strains expressing the CAD gene. The titer of IA and biomass of Y. lipolytica were determined by shaking flask fermentation of Po1g-CAD strain and control strain Po1g in YPO culture. All values presented are the mean of three biological replicates ± standard deviation.

FIGURE 3

Investigation of the localization of peroxisomes. (A) Schematic diagram of experimental design. hrGFPO-ePTS1 is used to specififically mark peroxisomes in Y. lipolytica. Nile red is used to show intracellular peroxisomes regions. (B) Localization observation of peroxisomes use the Nile red and strain Po1g-hrGFPO-ePTS1 through LSCM.

FIGURE 4

IA production in Y. lipolytica strains expressing the CAD-ePTS1 gene. The titer of IA and biomass were determined by shaking flask fermentation of Po1g-CAD-ePTS1 strain and control strain Po1g in YPO culture. All values presented are the mean of three biological replicates ± SD.

FIGURE 5

Effects of overexpressing genes involved in the acetyl-CoA production pathway on IA production. The genes involved in the acetyl-CoA production pathway, consisting of LIP2, POX1-6, MFE1, POT1 and PEX10, were overexpressed individually. Titers of IA produced by the strains were quantified after 6 days of cultivation in shake flasks with YPO medium. All values presented are the mean of three biological replicates ± standard deviation. *p<0.05, significantly different from control by ANOVA.

FIGURE 6

IA production in bioreactor of Y. lipolytica strain expressing the 2G-ICLΔ gene. The titer of IA and biomass were determined by bioreactor fermentation of Po1g-2G-ICLΔ strain in YPO culture. All values presented are the mean of three biological replicates ± SD.