Construction and optimization of efficient glucose–xylose co-fermenting yeast Yarrowia lipolytica for green and sustainable succinic acid production from lignocellulosic biomass

Abstract

Xylose is the second most abundant carbohydrate present in nature, while its inefficient utilization severely restricts the economic viability of lignocellulosic biomass-based biorefinery. Herein, metabolic engineering strategies involving xylose metabolism and the succinic acid (SA) synthetic pathway were developed in Yarrowia lipolytica for the production of SA from lignocellulosic hydrolysate. First, the Ylsdh5 gene (succinate dehydrogenase subunit 5) was inactivated in Y. lipolytica BZ, which can grow on xylose as the sole carbon source, thereby obtaining a strain capable of synthesizing SA from xylose. Subsequently, the glucose–xylose assimilating rate and SA titers were further optimized by blocking the by-product pathway and enhancing the SA synthetic pathways. Then, with the overexpression of the crucial mitochondrial dicarboxylic acid transporter YlDic, the obtained SA producer Y. lipolytica BDic5 showed excellent xylose assimilation performance, which could utilize all the glucose and xylose in either pure culture or hydrolysate fermentation. Remarkably, BDic5 exhibited robust growth in 30% solid-loading of corn stover hydrolysate without hydrolysate detoxification or dilution, and the fermentation process did not require neutral pH maintenance. Finally, up to 105.42 g L⁻¹ SA was produced from undetoxified lignocellulosic hydrolysate using the fed-batch strategy in a 3 L bioreactor, which was the highest SA titer achieved from lignocellulosic feedstock to date. Following downstream purification of the acidic fermentation broth, 61.75% of the total SA with purity of 92.81% was recovered. These promising results indicated that the recombinant strain exhibited great potential for bioconversion of lignocellulosic biomass into bio-SA, which demonstrated great prospects for industrial production.