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

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.

Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains

“…The glucose consumption rate during both aerobic and anaerobic resting state was similar to Zm6 reference in the Zm6 cat mutant ( Table 1). The ethanol yields in Zm6 cat were lower, especially under aerobic conditions, supporting the phenotype of Zm6 cat mutant with higher respiratory rate and accumulation of acetaldehyde [17]. Interestingly, the NADH/ NAD ratio was similar to that of Zm6 under aerobic conditions, whereas its increase was not observed in Zm6 cat under transition to anaerobic conditions; rather a slight decrease took place ( Table 2).…”

“…The Zm6 cat mutant was included in this study due to its interesting phenotype, an elevated aerobic respiration capacity [32], and accordingly, an improved capacity of acetaldehyde synthesis [31]. This strain was shown to be sensitive to hydrogen peroxide due to the lack of catalase activity [32], but the mutation did not cause growth deficiency under aerobic condition, suggesting that the mutant was still able to cope with general oxidative stress [17]. The mechanism by which the mutant upregulates oxygen consumption is not understood, but it is likely that its redox dynamics is altered.…”

“…In Z. mobilis, there are two alcohol dehydrogenase enzymes (AdhI encoded by adhA and AdhII encoded by adhB). The Zm6 adhB strain was chosen for metabolomic study because of its altered redox dynamics [33,34], and also its potential use for industrial acetaldehyde production due to lower ethanol-synthesising activity [17].…”

“…Another recent work [12] monitored the metabolome of exponentially growing cultures during anaerobic to aerobic transition. Kalnenieks et al [17] reported metabolomes of aerobic batch cultures of several Z. mobilis mutants. These studies revealed dynamic shift of the metabolome and some of its consequences in growing Z. mobilis cells.…”

An assessment of serial co-cultivation approach for generating novel Zymomonas mobilis strains

“…In addition to ethanol production, rewiring metabolic pathways to produce other useful compounds has lately been explored in Z. mobilis [3][4][5][6][7]. This is to utilize and exploit its intrinsic capacity of fast catabolism that comes with small biomass accumulation [1].…”