Engineering of Yarrowia lipolytica transporters for high-efficient production of biobased succinic acid from glucose

Abstract

Background: Succinic acid (SA) is a crucial metabolic intermediate and platform chemical. Development of biobased processes to achieve sustainable SA production has attracted more and more attention in biotechnology industry. Yarrowia lipolytica has a strong tricarboxylic acid cycle and tolerates low pH conditions, thus making it a potential platform for SA production. However, its SA titers in glucose media remain low.

Results: In this study, we screened mitochondrial carriers and C4-dicarboxylic acid transporters to enhance SA secretion in Y. lipolytica. PGC62-SYF-Mae strain with efficient growth and SA production was constructed by optimizing SA biosynthetic pathways and expressing the transporter SpMae1. In fed-batch fermentation, this strain produced 101.4 g/L SA with a productivity of 0.70 g/L/h and a yield of 0.37 g/g glucose, which is the highest SA titer achieved using yeast, with glucose as the sole carbon resource.

Conclusion: Our results indicated that transporter engineering is a powerful strategy to achieve the efficient secretion of SA in Y. lipolytica, which will promote the industrial production of bio-based SA.

Keywords: Glucose fermentation; Succinic acid; Transporter engineering; Yarrowia lipolytica.

Fig. 1

Schematic diagram of the SA biosynthetic pathways and its export route in Y. lipolytica. There are three major metabolic pathways involve in the SA biosynthesis of Y. lipolytica: (1) reductive branch of the TCA cycle (red line); (2) oxidative TCA cycle (green line); (3) glyoxylate bypass (blue line). Dotted lines indicated the deleted genes for SA accumulation. Pck phosphoenolpyruvate carboxykinase, Scs2 succinyl-CoA synthase β subunit, Frd fumarate reductase, Mls malate synthase, Icl isocitrate lyase, Yhm2 mitochondrial citrate transporter

Fig. 2

Effect of overexpression and suppression of YlDic1 gene on the cell growth and SA production of Y. lipolytica PGC62 strain. PGC62-YlDic was derived from PGC62 strain with overexpressed YlDic1 gene. PGC62-YlDici was derived from PGC62 strain with down-regulated YlDic1 gene through CRISPRi method. Error bars show the SDs of 3 biological replicates

Fig. 3

Overexpression of C4-dicarboxylic acid transporters from different species to improve the SA production of Y. lipolytica. a Nine potential C4-dicarboxylic acid transporters were selected according to the phylogenetic tree; b effect of the different C4-dicarboxylic acid transporters on the SA production of Y. lipolytica. Error bars show the SDs of 3 biological replicates

Fig. 4

Construction of the Y. lipolytica chassis cell through optimizing SA biosynthetic pathways. a Effect of the different genetic modifications on SA production of Y. lipolytica. To enhance the metabolic flux of reductive TCA, oxidative TCA and glyoxylate pathway, the expression cassettes of TbFrd, YlScs2 and YlYhm2-YlMls-YlIcl were overexpressed in PGC62 strain, respectively; b fermentation profile of the engineered strain PGC62-SYF with optimized SA biosynthetic pathway in shaking flasks. Error bars show the SDs of 3 biological replicates

Fig. 5

Comparison of the fermentation profiles between different SA producing Y. lipolytica strains. a Differences in SA production performance of different engineered strains. b Differences in the cell growth of different engineered strains. Error bars show the SDs of 3 biological replicates

Fig. 6

Optimization of culture conditions for SA over-production in 1 L bioreactor. a Comparison of the SA production performance between different Y. lipolytica engineered strains; b growth curves of different Y. lipolytica engineered strains; c effect of the pH values on the SA production of PGC62-SYF-Mae strain; d effect of the air flows and stirring rates on the SA production of PGC62-SYF-Mae strain. Error bars show the SDs of 3 technical replicates

Fig. 7

Kinetics of cell growth, glucose consumption and SA production during fed-batch culture of Y. lipolytica engineered strain PGC62-SYF-Mae in bioreactor. Error bars show the SDs of 3 biological replicates