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

Elina Balodite,Reinis Rutkis,Inese Strazdina,Jekaterina Martynova,Uldis Kalnenieks,Nina Galinina,Zane Lasa

doi:10.1002/mbo3.809

Abstract

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.

Highlights

Acetaldehyde is a highly desirable product of microbial biosynthe‐ sis, because it can be further used as the entry point for the syn‐ thesis of acetic acid, acetic anhydride, butadiene, crotonaldehyde, and other higher value chemicals (Danner & Braun, 1999; Moore et al, 2017)
Like Zymomonas mobilis, ge‐ netically engineered Lactococcus lactis, or yeast, have been inves‐ tigated for the production of acetaldehyde from sugary substrates (Bongers, Hoefnagel, & Kleerebezem, 2005; Wecker & Zall, 1987)
The advantage of Z. mobilis as an acetaldehyde producer is the best rate of its Entner–Doudoroff glycolytic pathway, a very active pyruvate decarboxylase, and, at the same time, an active respiratory chain with low energy‐coupling efficiency, ideally suited for regeneration of NAD+ under condition when ethanol is not the major catabolic product (Kalnenieks, 2006; Rogers, Jeon, Lee, & Lawford, 2007)

Summary

Introduction

Acetaldehyde is a highly desirable product of microbial biosynthe‐ sis, because it can be further used as the entry point for the syn‐ thesis of acetic acid, acetic anhydride, butadiene, crotonaldehyde, and other higher value chemicals (Danner & Braun, 1999; Moore et al, 2017). KEYWORDS acetaldehyde production, periplasm, pyruvate decarboxylase, Zymomonas mobilis 2.1 | Construction of a PDC‐deficient strain and expression of PDC with a periplasmic signal sequence

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