Improvement of L-arabinose fermentation by modifying the metabolic pathway and transport in Saccharomyces cerevisiae

Abstract

The L-arabinose utilization pathway was established in Saccharomyces cerevisiae, by expressing the codon-optimized araA, araB, and araD genes of Lactobacillus plantarum. After overexpressing the TAL1, TKL1, RPE1, RKI1, and GAL2 genes and adaptive evolution, the L-arabinose utilization of the recombinant strain became efficient. The resulting strain displayed a maximum specific growth rate of 0.075 h(-1), a maximum specific L-arabinose consumption rate of 0.61 g h(-1) g(-1) dry cell weight, and a promising ethanol yield of 0.43 g g(-1) from L-arabinose fermentation.

Figure 1

The physical maps of the plasmids (a) YIp5-ara, (b) pYX2422-TEF1araA/HXT7araA, and (c) pJFE318-GAL2.

Figure 2

The expression of araA (black bars), araB (gray bars), and araD (blank bars) of strains BSW2AP and BSW3AP compared to strain BSW1AT. The fold-changes of mRNA levels of these genes are normalized to the expression of ACT1. The tested strains were cultivated on 20 g L⁻¹ glucose. The values given are obtained from three independent measurements.

Figure 3

The L-arabinose fermentation of strains in shaker flasks. Growth capacity (a), L-arabinose consumption (b), arabitol formation (c), and ethanol formation (d) by BSW1AT (▲), BSW2AP (■), and BSW3AP (⚫). The strains were cultured in 40 mL SC medium with 20 g L⁻¹ L-arabinose at 30°C, 200 r min⁻¹ with an initial OD₆₀₀ of 0.5. The data are the averages of three independent experiments.

Figure 4

The anaerobic batch fermentation of BSW3AP (a) and BSW3AG (b) on 20 g L⁻¹ arabinose. Levels of OD₆₀₀ (■), arabinose (◆), ethanol (▲), Glycerol (⚫), and acetate (×). The fermentation was performed in 1.4 L fermentors with a working volume of 900 mL. Anaerobic conditions were maintained by sparging nitrogen (0.1 L min⁻¹); the agitation rate was 500 r min⁻¹. The pH was maintained at 5.0 by automatically pumping in 1 mol L⁻¹ NaOH and 1 mol L⁻¹ H₃PO₄. The initial biomass was 0. 2 g DCW L⁻¹. The 20 g L⁻¹ L-arabinose was used as the carbon source in SC plus CSM-LEU-URA medium, and 200 μg mL⁻¹ G418 was supplied in the fermentation of strain BSW3AG. The data are the average of duplicate determinations.