Translocation of Zymomonas mobilis pyruvate decarboxylase to periplasmic compartment for production of acetaldehyde outside the cytosol.

Acetaldehyde, a valuable commodity chemical, is a volatile inhibitory byproduct of aerobic fermentation in Zymomonas mobilis and in several other microorganisms. Attempting to improve acetaldehyde production by minimizing its contact with the cell interior and facilitating its removal from the culture, we engineered a Z. mobilis strain with acetaldehyde synthesis reaction localized in periplasm. For that, the pyruvate decarboxylase (PDC) was transferred from the cell interior to the periplasmic compartment. This was achieved by the construction of a Z. mobilis Zm6 PDC-deficient mutant, fusion of PDC with the periplasmic signal sequence of Z. mobilis gluconolactonase, and the following expression of this fusion protein in the PDC-deficient mutant. The obtained recombinant strain PeriAc, with most of its PDC localized in periplasm, showed a twofold higher acetaldehyde yield, than the parent strain, and will be used for further improvement by directed evolution.

Effects of acetate on Kluyveromyces marxianus DSM 5422 growth and metabolism.

Metabolically active cells produce a wide array of metabolites that can inhibit their growth. Acetate is a widely known preservative, and it is also produced by yeast cells during their growth. Kluyveromyces marxianus DSM 5422 is a promising yeast strain that could be employed in biotechnological processes, but the knowledge of its stress physiology is scarce. Here, we investigate the effects of acetate on growth and changes in cell population structure during adaptation to elevated concentrations of acetate in K. marxianus DSM 5422. Our results indicate that acetate inhibits growth in a pH-dependent manner and has pronounced effects if yeast is grown on lactose or galactose. When challenged with acetate, culture extends lag phase, during which cells adapt to elevated acetate concentrations, and growth reoccurs, albeit at a slower rate, when majority of the population is acetate resistant. Acetate resistance is maintained only if acetate is present in the media or if the culture has reached end of active growth phase. This study shows possible caveats in lactose fermentation with K. marxianus and gives a further perspective in non-conventional yeast applications in biotechnology.