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Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

Qi X, Zha J, Liu GG, Zhang W, Li BZ, Yuan YJ - Front Microbiol (2015)

Bottom Line: Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1.Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold.Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University Tianjin, China ; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University Tianjin, China.

ABSTRACT
Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003.

No MeSH data available.


Related in: MedlinePlus

Xylose consumption (A), xylitol production (B), and cell growth (C) of strains expressing different modules of XylA-XKS1. Promoters TDH1, PGK1, and TDH3 were applied to control the expression of XKS1. Promoters PGK1, TDH3, and HXK2 were used to express XylA. Set the expression strength of promoter HXK2p as 1 unit, the expression strengths of TDH1p, TDH3p and PGK1p are calculated to be ∼2.5, ∼9, and ∼6 units based on a previous study, respectively (Lu and Jeffries, 2007). In total, 9 (3 × 3) strains were constructed and evaluated for the oxygen-limited fermentation on xylose, conducted in 50 mL YPX in 250 mL flasks with the initial cell density at 0.5 of OD600. The samples were harvested at 72 h and analyzed of the OD600 values and components. The dry cell weight (DCW) was calculated from OD600 with the coefficient of 0.526 g DCW/L ⋅ OD600.
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Figure 1: Xylose consumption (A), xylitol production (B), and cell growth (C) of strains expressing different modules of XylA-XKS1. Promoters TDH1, PGK1, and TDH3 were applied to control the expression of XKS1. Promoters PGK1, TDH3, and HXK2 were used to express XylA. Set the expression strength of promoter HXK2p as 1 unit, the expression strengths of TDH1p, TDH3p and PGK1p are calculated to be ∼2.5, ∼9, and ∼6 units based on a previous study, respectively (Lu and Jeffries, 2007). In total, 9 (3 × 3) strains were constructed and evaluated for the oxygen-limited fermentation on xylose, conducted in 50 mL YPX in 250 mL flasks with the initial cell density at 0.5 of OD600. The samples were harvested at 72 h and analyzed of the OD600 values and components. The dry cell weight (DCW) was calculated from OD600 with the coefficient of 0.526 g DCW/L ⋅ OD600.

Mentions: The enzyme products of XylA and XKS1 catalyze the formation of 5-phosphate-xylulose that was metabolized by the following non-oxidative PPP (Jin et al., 2003; Lonn et al., 2003). Balancing the expression of XylA and XKS1 can be a way to optimize xylose metabolic pathway and improve xylose utilization. We evaluated the effects of different promoters on balancing the expression of XylA and XKS1. The promoters with different transcriptional strengths were applied to tune the expression of XylA and XKS1. Promoters TDH3p, HXK2p, and PGK1p were applied to express XylA in a multicopy plasmid pRS425 (Lu and Jeffries, 2007). Promoters TDH1p, TDH3p, and PGK1p were used to control the expression of XKS1. This strategy generated nine recombinant strains with different expression levels of XylA and XKS1 (Figure 1).


Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

Qi X, Zha J, Liu GG, Zhang W, Li BZ, Yuan YJ - Front Microbiol (2015)

Xylose consumption (A), xylitol production (B), and cell growth (C) of strains expressing different modules of XylA-XKS1. Promoters TDH1, PGK1, and TDH3 were applied to control the expression of XKS1. Promoters PGK1, TDH3, and HXK2 were used to express XylA. Set the expression strength of promoter HXK2p as 1 unit, the expression strengths of TDH1p, TDH3p and PGK1p are calculated to be ∼2.5, ∼9, and ∼6 units based on a previous study, respectively (Lu and Jeffries, 2007). In total, 9 (3 × 3) strains were constructed and evaluated for the oxygen-limited fermentation on xylose, conducted in 50 mL YPX in 250 mL flasks with the initial cell density at 0.5 of OD600. The samples were harvested at 72 h and analyzed of the OD600 values and components. The dry cell weight (DCW) was calculated from OD600 with the coefficient of 0.526 g DCW/L ⋅ OD600.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4612707&req=5

Figure 1: Xylose consumption (A), xylitol production (B), and cell growth (C) of strains expressing different modules of XylA-XKS1. Promoters TDH1, PGK1, and TDH3 were applied to control the expression of XKS1. Promoters PGK1, TDH3, and HXK2 were used to express XylA. Set the expression strength of promoter HXK2p as 1 unit, the expression strengths of TDH1p, TDH3p and PGK1p are calculated to be ∼2.5, ∼9, and ∼6 units based on a previous study, respectively (Lu and Jeffries, 2007). In total, 9 (3 × 3) strains were constructed and evaluated for the oxygen-limited fermentation on xylose, conducted in 50 mL YPX in 250 mL flasks with the initial cell density at 0.5 of OD600. The samples were harvested at 72 h and analyzed of the OD600 values and components. The dry cell weight (DCW) was calculated from OD600 with the coefficient of 0.526 g DCW/L ⋅ OD600.
Mentions: The enzyme products of XylA and XKS1 catalyze the formation of 5-phosphate-xylulose that was metabolized by the following non-oxidative PPP (Jin et al., 2003; Lonn et al., 2003). Balancing the expression of XylA and XKS1 can be a way to optimize xylose metabolic pathway and improve xylose utilization. We evaluated the effects of different promoters on balancing the expression of XylA and XKS1. The promoters with different transcriptional strengths were applied to tune the expression of XylA and XKS1. Promoters TDH3p, HXK2p, and PGK1p were applied to express XylA in a multicopy plasmid pRS425 (Lu and Jeffries, 2007). Promoters TDH1p, TDH3p, and PGK1p were used to control the expression of XKS1. This strategy generated nine recombinant strains with different expression levels of XylA and XKS1 (Figure 1).

Bottom Line: Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1.Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold.Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University Tianjin, China ; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University Tianjin, China.

ABSTRACT
Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ∼10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003.

No MeSH data available.


Related in: MedlinePlus