Thermal and Solvent Stress Cross-Tolerance Conferred to Corynebacterium glutamicum by Adaptive Laboratory Evolution

Abstract

bReinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.A high-GϩC, Gram-positive bacterium, Corynebacterium glutamicum, was first discovered in Japan as a natural producer of glutamic acid (1) and has been exploited for industrial production of amino acids for over 50 years. Microbes employed in industrial-scale fermentation encounter a variety of stresses (2, 3), including thermal stress, which stems from heat generated by metabolic activities of microbial catalysts. Careful control of temperature during fermentation processes is crucial to protect microbes from thermal stress and to achieve optimal productivity. Engineering microbes with improved tolerance for thermal stress enables fermentation with more flexible temperature requirements and thereby leads to reductions in cooling costs. Different approaches have been developed to improve bacterial stress tolerance (4-6). Among them, adaptive laboratory evolution (ALE) makes use of naturally occurring mutations to generate individuals better adapted to certain environments (7-9). ALE has been successfully applied to metabolic engineering, as well as evolutionary studies on different organisms (10-15). Studies on Escherichia coli provide evidence that ALE can be a powerful tool to ameliorate bacterial stress tolerance (16)(17)(18)(19)(20). Independent groups have reported on ALE of the bacter...

Evolutionary engineering of Corynebacterium glutamicum

“…In an economically optimized bioprocess, microorganisms should be able to grow on low‐purity feedstock (lower substrate costs) and at favorable process conditions (e.g., at a higher temperature, to reduce cooling costs). An improved heat tolerance was reported for C. glutamicum after a repetitive batch ALE (rbALE) of 65 days, in which the temperature was gradually increased from 38 °C to 41.5 °C . Sequencing of three evolved isolates revealed a surprisingly high amount of 295 point mutations and two genomic deletions in total.…”

“…Interestingly, both the reverse engineered strain and the evolved strain lost the ability to grow on ethanol. This showed that, in contrast to an observed beneficial cross‐tolerance for isobutanol after ALE for increased temperatures, a single‐target ALE approach is typically associated with significant negative trade‐off effects.…”

Trehalose acts as a uridine 5′‐diphosphoglucose‐competitive inhibitor of trehalose 6‐phosphate synthase in Corynebacterium glutamicum

“…E. coli strains were grown at 37°C in LB medium [49]. Growth at high temperatures was evaluated as described previously [9]. Growth at high temperatures was evaluated as described previously [9].…”

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum

“…Here, industrial strain development strongly benefits from adaptive evolution approaches, in which strains typically feature only a few mutations and which enable the enrichment of nonintuitive beneficial mutations by improving growth at the same time (Abatemarco et al, 2013;Portnoy et al, 2011). Up to now, laboratory evolution experiments of mainly fitness-linked phenotypes have been performed by exposing microorganisms to sequentially increasing levels of environmental stress (Eckdahl et al, 2015;Lee et al, 2013;Marietou et al, 2014;Oide et al, 2015;Reyes et al, 2014). Especially in the case of the yeast Saccharomyces cerevisiae, adaptation to an improved ethanol tolerance has been proven useful for increasing product formation (Alper et al, The bottom line of almost all reported adaptive evolution approaches is selection for improved growth and survival, which usually coincides with increased product formation -especially in the case of growth-coupled processes (Feist et al, 2010).…”

“…Short generation times and a natural mutation frequency of 10 À 10 to 10 À 9 mutations per base pair per replication cycle enable the selection of beneficial phenotypical traits from high genetic diversity (Barrick and Lenski, 2013). During the last few years, laboratory evolution strategies went more and more into the focus to adapt industrial producer strains to detrimental growth conditions such as oxidative and thermal stress (Lee et al, 2013;Oide et al, 2015;Sandberg et al, 2014;Tenaillon et al, 2012), to improve product formation (Raman et al, 2014;Reyes et al, 2014;Xie et al, 2015) or solvent tolerance (Atsumi et al, 2010;Lee et al, 2011;Oide et al, 2015) (for reviews discussing the use of adaptive evolution approaches in metabolic engineering, see Abatemarco et al, 2013;Portnoy et al, 2011).…”