Improvement of Acetaldehyde Production in Zymomonas mobilis by Engineering of Its Aerobic Metabolism.

Uldis Kalnenieks,Agris Pentjuss,Oliver Sawodny,Inese Strazdina,Robert K Poole,Steffi Strähler,Per Bruheim,Katsuya Fuchino,Katherine M Pappas,Katja Bettenbrock,Julia Rex,Elina Balodite,Reinis Rutkis

doi:10.3389/fmicb.2019.02533

Abstract

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers.

Highlights

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by chemical industry as the entry point for the synthesis of acetic anhydride, acetic acid, butadiene, butanol, crotonaldehyde, and other commodity chemicals (Danner and Braun, 1999; Moore et al, 2017)
Glucose is converted into equimolar amounts of acetaldehyde and carbon dioxide minus the carbon that is used for synthesis of biomass precursors
Since the main product of Z. mobilis is ethanol, a major improvement of the acetaldehyde yield is anticipated by minimizing the flux through the alcohol dehydrogenase (ADH)

Summary

Introduction

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by chemical industry as the entry point for the synthesis of acetic anhydride, acetic acid, butadiene, butanol, crotonaldehyde, and other commodity chemicals (Danner and Braun, 1999; Moore et al, 2017). Acetaldehyde is an important aroma compound in yogurt and other dairy products (Bongers et al, 2005). In microorganisms, this compound is the metabolic precursor of ethanol. In aerobic cultures of ethanologenic microorganisms, it accumulates as a volatile, inhibitory byproduct because of withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. Due to its toxicity, aerobic cultures tend to convert acetaldehyde into less toxic products: reduce it to ethanol or oxidize it to acetate depending on the culture conditions.

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Results

Conclusion