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Designing of Lactococcus lactis platform for isobutanol production using multiple rounds of adaptive laboratory evolution

ABSTRACT: This work was primarily focused on removing the main bottleneck for isobutanol production which is the toxicity of isobutanol towards its host and the major reason for its low-level production. Recently, there have been many reports where people have tried various strategies including continuous in-situ product removal from the bioreactor which led to a significant increase in the final product titers. Clearly, it is the intrinsic tolerance levels of the host which decides the end product titers. Importantly, if the host microbe can originally tolerate higher concentrations of toxin (here isobutanol) then it is likely to have a better potential for further improvement of tolerance levels. In our work, we tried to enhance the isobutanol tolerance of wild type NZ9000 using continuous culture. We used the principle of adaptive laboratory evolution to enhance the natural tolerance ability (0.8% isobutanol) of the wild type strain. The strain was cultivated for more than 60 days (>250 generations), while increasing the selection pressure (here isobutanol) gradually in the feed. This finally led to the strain that showed exceptionally higher tolerance (4%) for isobutanol. Transcriptomic analysis also showed fluctuations in gene expression levels from a wide range of categories, precluding any attempt to reverse engineer this phenotype.

ORGANISM(S): Lactococcus cremoris

PROVIDER: GSE107996 | GEO | 2020/07/08

REPOSITORIES: GEO

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Project description:BACKGROUND:Isobutanol is a promising next generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titers. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases on adaptation to exogenous isobutanol stress. RESULTS:The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced rpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodelling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS:We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for futurue global phenotype engineering. Two strains (WT strain and G3.2 mutant strain), each with two culture conditions (with and without isobutanol in medium). Three biological replicates for each strain/culture condition. Twelve samples in total.

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Designing of Lactococcus lactis platform for isobutanol production using multiple rounds of adaptive laboratory evolution

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