Efficient production of citric acid from lignocellulose hydrolysate by metabolically engineered Yarrowia lipolytica

Abstract

Lignocellulosic biomass is a reliable renewable feedstock for citric acid fermentation. Low product titer is the bottleneck in the large scale production of cellulosic citric acid by Yarrowia lipolytica. Herein, multiple genetic engineering strategies were explored to construct an engineered Y. lipolytica strain that can efficiently produce citric acid with a high titer and yield. Genes related to TCA cycles were overexpressed to increase citric acid production. Subsequently, genes in the downstream lipid synthesis pathway were deleted to decrease citric acid consumption. The mitochondrial transporter of isocitric acid was also deleted to minimize by-product secretion. Next, six glucose transporter genes, a hexose kinase gene, and a heterologous 6-phosphofructo-1-kinase gene were tested to enhance the efficiency of citric acid production. Consequently, the optimized engineered strain produced 88.2 g L⁻¹ and 73.6 g L⁻¹ citric acid from a pure sugar medium and 30% solid loading hydrolysate, respectively. Finally, in a 3 L bioreactor, 83.6 g L⁻¹ citric acid was produced from 35% solid loading of corn stover hydrolysate via fed-batch fermentation. In this work, an efficient robust yeast cell method was developed for the production of citric acid in a sustainable manner.