Tolerance to high product concentrations is a major barrier to achieving economically viable processes for bio-based chemical production. Chemical tolerance mechanisms are often unknown, thus their rational design is not achievable. To reveal unknown tolerance mechanisms we used an automated platform to evolve Escherichia coli to grow in previously toxic concentrations of 11 chemicals that have applications as polymer precursors, chemical intermediates, or biofuels. Re-sequencing of isolates from 88 independently evolved 2 populations, reconstruction of mutations, and cross-compound tolerance profiling was employed to uncover general and specific tolerance mechanisms. We found that: 1) the broad tolerance of strains towards chemicals varied significantly depending on the chemical stress condition under which the strain was evolved; 2) the strains that acquired high levels of NaCl tolerance also became broadly tolerant to most chemicals; 3) genetic tolerance mechanisms included alterations in regulatory, cell wall, transcriptional and translational functions, as well as more chemical-specific mechanisms related to transport and metabolism; 4) using pre-tolerized starting strains can significantly enhance subsequent production of chemicals when a production pathway is inserted; and 5) only a subset of the evolved isolates showed improved production indicating that this approach is especially useful when a large number of independently evolved isolates are screened for production.We provide a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 224 chemical tolerant strains.
The yeast Saccharomyces cerevisiae is widely used in industrial biotechnology for the production of fuels, chemicals, food ingredients, food and beverages, and pharmaceuticals. To obtain high‐performing strains for such bioprocesses, it is often necessary to test tens or even hundreds of metabolic engineering targets, preferably in combinations, to account for synergistic and antagonistic effects. Here, we present a method that allows simultaneous perturbation of multiple selected genetic targets by combining the advantage of CRISPR/Cas9, in vivo recombination, USER assembly and RNA interference. CRISPR/Cas9 introduces a double‐strand break in a specific genomic region, where multiexpression constructs combined with the knockdown constructs are simultaneously integrated by homologous recombination. We show the applicability of the method by improving cis,cis‐muconic acid production in S. cerevisiae through simultaneous manipulation of several metabolic engineering targets. The method can accelerate metabolic engineering efforts for the construction of future cell factories.
Background Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars—in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. Results The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. Conclusions The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.
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