Development Of Robust Strains Research Articles

Transcriptome analysis of Kluyveromyces marxianus under succinic acid stress and development of robust strains.

Kluyveromyces marxianus has become an attractive non-conventional yeast cell factory due to its advantageous properties such as high thermal tolerance and rapid growth. Succinic acid (SA) is an important platform molecule that has been applied in various industries such as food, material, cosmetics, and pharmaceuticals. SA bioproduction may be compromised by its toxicity. Besides, metabolite-responsive promoters are known to be important for dynamic control of gene transcription. Therefore, studies on global gene transcription under various SA concentrations are of great importance. Here, comparative transcriptome changes of K. marxianus exposed to various concentrations of SA were analyzed. Enrichment and analysis of gene clusters revealed repression of the tricarboxylic acid cycle and glyoxylate cycle, also activation of the glycolysis pathway and genes related to ergosterol synthesis. Based on the analyses, potential SA-responsive promoters were investigated, among which the promoter strength of IMTCP2 and KLMA_50231 increased 43.4% and 154.7% in response to 15 g/L SA. In addition, overexpression of the transcription factors Gcr1, Upc2, and Ndt80 significantly increased growth under SA stress. Our results benefit understanding SA toxicity mechanisms and the development of robust yeast for organic acid production. KEY POINTS: • Global gene transcription of K. marxianus is changed by succinic acid (SA) • Promoter activities of IMTCP2 and KLMA_50123 are regulated by SA • Overexpression of Gcr1, Upc2, and Ndt80 enhanced SA tolerance.

Copper homeostasis as a target to improve Saccharomyces cerevisiae tolerance to oxidative stress

The yeast Saccharomyces cerevisiae is widely used as a cell factory for the biotechnological production of various industrial products. During these processes, yeasts meet different kinds of stressors that often cause oxidative stress and thus impair cell growth. Therefore, the development of robust strains is indispensable to improve production, yield and productivity of fermentative processes.Copper plays a key role in the response to oxidative stress, as cofactor of the cytosolic superoxide dismutase (Sod1) and being contained in metallochaperone and metallothioneines with antioxidant properties. In this work, we observed a higher naturally copper internalization in a robust S. cerevisiae strain engineered to produce the antioxidant l-ascorbic acid (L-AA), compared with the wild type strain. Therefore, we investigated the effect of the alteration of copper homeostasis on cellular stress tolerance. CTR1 and FRE1 genes, codifying for a plasma membrane high-affinity copper transporter and for a cell-surface ferric/cupric reductase, respectively, were overexpressed in both wild type and L-AA cells. Remarkably, we found that the sole FRE1 overexpression was sufficient to increase copper internalization leading to an enhanced stress tolerance toward H2O2 exposure, in both strains under investigation. These findings reveal copper homeostasis as a target for the development of robust cell factories.

The Saccharomyces cerevisiae poly(A) binding protein Pab1 as a target for eliciting stress tolerant phenotypes.

When exploited as cell factories, Saccharomyces cerevisiae cells are exposed to harsh environmental stresses impairing titer, yield and productivity of the fermentative processes. The development of robust strains therefore represents a pivotal challenge for the implementation of cost-effective bioprocesses. Altering master regulators of general cellular rewiring represents a possible strategy to evoke shaded potential that may accomplish the desirable features. The poly(A) binding protein Pab1, as stress granules component, was here selected as the target for obtaining widespread alterations in mRNA metabolism, resulting in stress tolerant phenotypes. Firstly, we demonstrated that the modulation of Pab1 levels improves robustness against different stressors. Secondly, the mutagenesis of PAB1 and the application of a specific screening protocol on acetic acid enriched medium allowed the isolation of the further ameliorated mutant pab1 A60-9. These findings pave the way for a novel approach to unlock industrially promising phenotypes through the modulation of a post-transcriptional regulatory element.