Improvement of xylose uptake and ethanol production in recombinant Saccharomyces cerevisiae through an inverse metabolic engineering approach

Abstract

We used an inverse metabolic engineering approach to identify gene targets for improved xylose assimilation in recombinant Saccharomyces cerevisiae. Specifically, we created a genomic fragment library from Pichia stipitis and introduced it into recombinant S. cerevisiae expressing XYL1 and XYL2. Through serial subculturing enrichment of the transformant library, 16 transformants were identified and confirmed to have a higher growth rate on xylose. Sequencing of the 16 plasmids isolated from these transformants revealed that the majority of the inserts (10 of 16) contained the XYL3 gene, thus confirming the previous finding that XYL3 is the consensus target for increasing xylose assimilation. Following a sequential search for gene targets, we repeated the complementation enrichment process in a XYL1 XYL2 XYL3 background and identified 15 fast-growing transformants, all of which harbored the same plasmid. This plasmid contained an open reading frame (ORF) designated PsTAL1 based on a high level of homology with S. cerevisiae TAL1. To further investigate whether the newly identified PsTAL1 ORF is responsible for the enhanced-growth phenotype, we constructed an expression cassette containing the PsTAL1 ORF under the control of a constitutive promoter and transformed it into an S. cerevisiae recombinant expressing XYL1, XYL2, and XYL3. The resulting recombinant strain exhibited a 100% increase in the growth rate and a 70% increase in ethanol production (0.033 versus 0.019 g ethanol/g cells . h) on xylose compared to the parental strain. Interestingly, overexpression of PsTAL1 did not cause growth inhibition when cells were grown on glucose, unlike overexpression of the ScTAL1 gene. These results suggest that PsTAL1 is a better gene target for engineering of the pentose phosphate pathway in recombinant S. cerevisiae.

FIG. 1.

Distributions of colony sizes for the enriched populations after series of serial transfers on YSC agar plates with glucose and xylose. On a glucose plate (A), the colony sizes of the enriched population (total number of colonies counted, 351) showed a uniform distribution, with a mean area of 4.0 mm². However, on a xylose plate (B), the colony sizes of the enriched population (total number of colonies counted, 1,191) showed a distribution that can be described as a mixed population with two different means, 1.5 and 4.0 mm².

FIG. 2.

Growth assay for the 204 colonies screened on a YSC agar plate with xylose. Cells grown on glucose in a 96-well plate were spotted onto an agar plate with xylose. Replicate growth assays were performed (plates B and D are replicates of plates A and C, respectively). For each plate the image was captured and visualized in black and white for easy comparison.

FIG. 3.

Location of PsTAL1 in the isolated plasmid, pTAL1I (A), and phylogenetic tree of transaldoase proteins found in the GenBank database. The homologues of transaldolase were identified by a BLAST search, and the protein sequences found were then aligned by CLUSTALW. The tree was generated from the aligned region with bootstrapping. aa, amino acids; ORF, open reading frame.

FIG. 4.

Xylose uptake and cell growth profile for S. cerevisiae strains YSX3-C (A and C) and YSX3-TAL1M (B and D). Cells were cultured in 50 ml of YSC medium with 20 g/liter of xylose (A and B) and 40 g/liter of xylose (C and D). The data are the averages of two replicate experiments. Symbols: ⧫, cell mass; ▪, xylose; ▴, xylitol.

FIG. 5.

Comparison of ethanol production by S. cerevisiae strains YSX3-C (▴) and YSX4-TAL1M (▪) under oxygen-limited conditions. Cells were grown on glucose, harvested, and inoculated into 20 ml of YSC medium with 40 g/liter of xylose. The initial OD₆₀₀ was about 12 to 15. EtOH, ethanol.