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Determination of WatR binding site in Methylomonas sp. DH-1 after lactate adaptive evolution

ABSTRACT: JHM80 strain was obtained by adaptive evolution of Methylomonas sp. DH-1 strain against 8.0 g/L of lactate. The tolerance against lactate of JHM80 was obtained by the overexpression of watR (weak acid tolerance regulator). In order to discover the target genes of WatR, FLAG epitope tag was added to WatR of JHM80 strain and ChIP-seq was performed.

ORGANISM(S): Methylomonas sp. DH-1

PROVIDER: GSE206216 | GEO | 2023/08/08

REPOSITORIES: GEO

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Project description:Background: Evolutionary engineering is a powerful approach to isolate suppressor mutants and industrially relevant genotypes. Until recently, DNA microarray analysis was the only affordable genome-wide approach to identify the responsible mutations. This situation has changed due to the rapidly decreasing costs of whole genome (re)sequencing. DNA microarray-based mRNA expression analysis and whole genome resequencing were combined in a study on lactate transport in Saccharomyces cerevisiae. Jen1p is the only S. cerevisiae lactate transporter reported in literature. To identify alternative lactate transporters, a jen1Δ strain was evolved for growth on lactate. Results: Two independent evolution experiments yielded Jen1p-independent growth on lactate (μmax 0.14 and 0.18 h-1 for single-cell lines IMW004 and IMW005, respectively). Whereas mRNA expression analysis did not provide leads, whole-genome resequencing showed different single nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Analysis of mRNA levels and depth of coverage of DNA sequencing combined with karyotyping, gene deletions and diagnostic PCR showed that in IMW004 an isochromosome III (~475 kb), which contains two additional copies of ADY2C755G, was formed via crossover between YCLWΔ15 and YCRCΔ6. Introduction of the ADY2 alleles in a jen1 ady2 strain resulted in growth on lactate (μmax 0.14 h-1 for Ady2pLeu219Val and 0.12 h-1 for Ady2pAla252Gly). Conclusions: Whole-genome resequencing of yeast strains obtained from independent evolution experiments enabled rapid identification of a key gene that was not identified by mRNA expression analysis of the same strains. Reverse metabolic engineering showed that mutated alleles of ADY2 (C655G and C755G) encode efficient lactate transporters. The goal of the present study was to investigate whether evolutionary engineering and subsequent elucidation of the underlying mutations can lead to the identification of alternative lactate transporters in S. cerevisiae. Transport of carboxylic acids plays a key role in weak organic acids stress and the responsible membrane transporters are often poorly studied and encoded by multiple redundant genes . Additionally, import of lactate is an essential step in the reutilization of lactate produced during intense exercise in mammals [8]. In S. cerevisiae, Jen1p was previously identified as the only efficient lactate importer by generation of a UV-mutant unable to grow on lactate and subsequent functional complementation with a genomic library . The lactate uptake rate of the resulting jen1 knockout strain was close to the detection limit. Interestingly, export of lactate in engineered lactate producing S. cerevisiae is unaffected by deletion of JEN1 (our unpublished results). These observations suggest the presence of at least one alternative lactate transporter. To test this, a jen1 knockout strain was constructed and evolved for faster growth on lactate as the sole carbon and energy source. The evolved strains were subjected to a combination of mRNA expression analysis and whole genome DNA (re)sequencing to identify the relevant mutations. The resulting leads were tested for lactate transport activity via knockout studies in the evolved strains and reverse engineering of the portable genetic elements into non-evolved strains.

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Determination of WatR binding site in Methylomonas sp. DH-1 after lactate adaptive evolution

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