Reconstruction Of Scheffersomyces Stipitis Synthetic Strain For Fumaric Acid Production From Xylose

Fumaric acid, a C4-dicarboxylic acid, is widely used in food, pharmaceutical and chemical industries. The U.S. Department of Energy has identified fumaric acid as one of the top 12 building block chemicals that can be served as an important precursor for the synthesis of high-value products. Rhizopus species were the main fumaric acid production strains. However, due to the complex morphological and growth characteristics, the process controllability and scales of industrial production were strongly limited. Moreover, with the low frequency of genomic integration and lacking effective gene manipulation tools, it is still very difficult to further improve fumaric acid production of filamentous fungi by metabolic engineering. As the most abundant renewable bioresource on the earth, lignocellulose has attracted more and more attentions for its conversion into energy and chemicals. Xylose is the major component in the lignocellulose hydrolysate and is regarded as the second abundant sugar in lignocellulose after glucose. Efficient utilization of xylose is one of the biggest obstacles for commercial conversion of lignocellulose to useful compounds.In this work, Scheffersomyces stipitis, the yeast with excellent xylose-utilizing ability, was firstly engineered for fumaric acid production from xylose with the heterologous fumaric acid synthetic reductive pathway from Rhizopus oryzae FM19, including pyruvate carboxylase, malate dehydrogenase and fumarase. 1.86 g/L fumaric acid was produced by the initial fumaric acid synthetic strain PSRPMF under the oxygen-limited condition.In order to improve the fumaric acid production, the fermentation behaviors of the initial fumaric acid synthesis strain were rationally analyzed and three strategies were implemented, including increasing the reductive pathway activity by codon optimization, blocking the fumaric acid conversion in tricarboxylic acid cycle by knocking out the native fumarases, and improving the fumaric acid transportation by overexpressing heterologous transporter. Firstly, codon optimization was performed according to the S. stipitis codon preference. The activities of pyruvate carboxylase, malate dehydrogenase and fumarate were increased by 28.12%, 17.35% and 16.13% respectively and relative expression levels of three enzymes were increased by 35.39%, 20.2% and 18.75% respectively. The fumaric acid titer was up to 2.65 g/L. Then,the deletion of native fumarase was implemented to regulate carbon metabolic flux and reduce the fumaric acid consumption. The fumaric acid final concentration reached to 3.21g/L. Moreover, the effective fumaric acid transporter YMAE1 was overexpressed to improve the fumaric acid capacity, so that the intracellular fumaric acid titer declined to 0.36 mg/g DCW, a 43.75% lower, which promoting the fumaric acid production. Finally, the final fumaric acid synthetic strain PSYPMFfS was obtained and the fumaric acid titer reached to 4.67 g/L, significantly increased by 37.92-fold than that of the control strain PSPYSS. It was indicated that the S. stipitis was a promising platform for fumaric acid production from xylose.