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.

Conversion of CO 2 into organic acids by engineered autotrophic yeast

“…However, considering a well-known bioprocess like citric acid production which yields more than 200 g L −1 , the organic acid titers we reached in a shake flask are still far from a feasible industrial process ( 23 ). So far, the highest itaconic acid titers in bioreactors reached by a native producer and an engineered yeast is 160 and 55 g L −1 , respectively ( 34 , 35 ). We could produce up to 600 mg L −1 of lactic acid using our autotropic K. phaffii strain which is already close to the titers reached in cyanobacteria of ∼1 g L −1 ( 36 , 37 ) but still far away from industrially relevant titers of far over 100 g L −1 ( 38 ).…”

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach

“…In this study, we applied a combinatorial engineering strategy, consisting of both protein and metabolic engineering to achieve substantial amyrin biosynthesis in Y. lipolytica . We also leveraged on Y. lipolytica ’s intrinsic ability to utilize WCO as feedstock and tested a small scale bioprocess for circular and cost-effective production of triterpenoids [ 18 ]. At the optimal temperature of ~ 30 °C and k L a of ~ 84 h −1 , amyrin titers of at least 125 mg/L (103 mg/L α-amyrin and 22 mg/L β-amyrin) and 110 mg/L (85 mg/L α-amyrin and 25 mg/L β-amyrin) were attained in glucose and WCO shake flask cultures, respectively (Additional file 1 : Table S2).…”

“…This is not the case for S. cerevisiae in which the pyruvate decarboxylation pathway supplying cytosolic acetyl-CoA is strongly competed by the ethanol fermentation [ 17 ]. More importantly, Y. lipolytica can metabolize a variety of carbon substrates for growth, including waste cooking oil (WCO) and mixed food waste hydrolysate [ 18 , 19 ]. This permits the valorization of waste streams and reduces the overall cost of production.…”

Mitochondrial Engineering of Yarrowia lipolytica for Sustainable Production of α-Bisabolene from Waste Cooking Oil

“…This result suggests that acetyl-CoA from fatty acid degradation in the mitochondria and peroxisomes accumulates more in the cytoplasm than in the mitochondria, which supplies the cytoplasmic MVA pathway more efficiently with the substrate. Since the β-oxidation of long-chain fatty acids occurs mainly in peroxisomes, 35 the peroxisomal supply of acetyl-CoA from WCO may play an important role in the generation of acetyl-CoA-derived products, thus suggesting the potential benefit of the peroxisome as a subcellular factory for α-bisabolene production from WCO. Moreover, the straight substitution of WCO for glucose contributes to higher biomasses for all the engineered strains, demonstrating WCO as a preferred substrate for cell growth, as this yeast was originally isolated from lipid-rich materials.…”