Combinatorial design of a highly efficient xylose-utilizing pathway in Saccharomyces cerevisiae for the production of cellulosic biofuels

Abstract

Balancing the flux of a heterologous metabolic pathway by tuning the expression and properties of the pathway enzymes is difficult, but it is critical to realizing the full potential of microbial biotechnology. One prominent example is the metabolic engineering of a Saccharomyces cerevisiae strain harboring a heterologous xylose-utilizing pathway for cellulosic-biofuel production, which remains a challenge even after decades of research. Here, we developed a combinatorial pathway-engineering approach to rapidly create a highly efficient xylose-utilizing pathway for ethanol production by exploring various combinations of enzyme homologues with different properties. A library of more than 8,000 xylose utilization pathways was generated using DNA assembler, followed by multitiered screening, which led to the identification of a number of strain-specific combinations of the enzymes for efficient conversion of xylose to ethanol. The balancing of metabolic flux through the xylose utilization pathway was demonstrated by a complete reversal of the major product from xylitol to ethanol with a similar yield and total by-product formation as low as 0.06 g/g xylose without compromising cell growth. The results also suggested that an optimal enzyme combination depends on not only the genotype/phenotype of the host strain, but also the sugar composition of the fermentation medium. This combinatorial approach should be applicable to any heterologous pathway and will be instrumental in the optimization of industrial production of value-added products.

Fig 1

Overview of the xylose utilization pathway.

Fig 2

Overall scheme of the combinatorial pathway engineering approach. In step 1, genes encoding enzyme homologues from various organisms are cloned into gene expression cassette plasmids containing a promoter and terminator pair. During PCR amplification, flanking sequences (30 to 40 nt each) homologous to the end and beginning of the promoter and terminator sequences in the expression cassette plasmids are added to each gene. In steps 2 and 3, the entire expression cassette of each gene is amplified with flanking sequences homologous to the neighboring regions of the recipient plasmids or gene cassettes, assembled, and directed into the host S. cerevisiae strains. In steps 1 and 3, assembly is achieved by endogenous homologous recombination, and the recombination efficiency is controlled by the flanking sequences (80 to 100 nt), identical for each homologue. In steps 4 and 5, the libraries containing all possible combinations of the genes are subjected to screening/selection, and the pathway with balanced enzyme properties is identified for each strain.

Fig 3

Library screening for fast growth and high product yield. (a, c, and e) Specific growth rates of 80 (INVSc1; ATCC 4124) and 50 (Classic Turbo Yeast) recombinants selected from xylose plates. (b, d, and f) Fermentation product profiles of the 10 recombinants (second-round screening) of INVSc1, Classic, and ATCC 4124 strains. The 10 recombinants were selected based on the growth rates determined in the 1st-round screening.

Fig 4

Xylose fermentation and product distribution of the selected fast and slow growers of INVSc1 and Classic strains. (a to c) Xylose fermentation profiles of one fast grower (INVSc1-F2 [a]) and two slower growers (INVSc1-S5 [b] and INVSc1-S10 [c]). (d) Volumetric xylose consumption rates (g/liter/h) and product yields (g/g consumed xylose) in the fermentation of INVSc1-F2, -S5, and -S10. (e to g) Xylose fermentation profiles of one fast grower (Classic-F3 [e]) and two slower growers (Classic-S1 [f] and Classic-S10 [g]). (h) Volumetric xylose consumption rates (g/liter/h) and product yields (g/g consumed xylose) in the fermentations of Classic-F3, -S1, and -S10. The error bars represent standard deviations (n = 3).

Fig 5

Xylose fermentation and cofermentation with glucose of the screened recombinants. (a and b) Xylose (4%) fermentation profile of ATCC 4124-F2 and comparison between selected recombinants of the three strains. (c and d) Glucose (4%) and xylose (4%) cofermentation profiles of Classic-F3 and comparison between selected recombinants of the three strains. In panels b and d, the unit for the consumption and production rate was g/liter/h and the unit for the ethanol yield was g/g sugar consumed. The error bars represent standard deviations (n = 3). Statistical significance: *, n = 3, P < 0.05; **, n = 3, P < 0.005.

Fig 6

Effects of glucose cofermentation on the xylose utilization rate. (a and b) Xylose fermentation (a) and cofermentation (b) profiles of INVSc1-F2 and INVSc1-F5. (c) Consumption rate and ethanol yield comparison. The unit for the consumption and production rates was g/liter/h, and the unit for the ethanol yield was g/g sugar consumed. The error bars represent standard deviations (n = 3). Statistical significance: *, n = 3, P < 0.05; **, n = 3, P < 0.005.

Fig 7

Xylose utilization efficiencies of the pathways in different strain backgrounds. Shown are the xylose consumption rates and ethanol yields of the INVSc1 (a), ATCC 4124 (b), and CTY (c) recombinants transformed with 5 pathways identified by screening of the CTY library. The error bars indicate standard deviations (n = 3).

Fig 8

Enzyme activities of XR, XDH, and XKS used for library generation and in the screened xylose pathways. (a) Enzyme activity distributions of XR, XDH, and XKS homologues used for library generation. (b) Measured enzyme activities of five fast growers (INVSc1-F1 to -F5) and five slow growers (INVSc1-S1 to -S5). (c) Average enzyme activities of the 10 fast growers of each strain. The error bars represent standard deviations (n = 2).