Aerobic catabolism and respiratory lactate bypass in Ndh-negative Zymomonas mobilis

Abstract

Ability to ferment in the presence of oxygen increases the robustness of bioprocesses and opens opportunity for novel industrial setups. The ethanologenic bacterium Zymomonas mobilis performs rapid and efficient anaerobic ethanol fermentation, yet its respiratory NADH dehydrogenase (Ndh)-deficient strain (ndh-) is known to produce ethanol with high yield also under oxic conditions. Compared to the wild type, it has a lower rate of oxygen consumption, and an increased expression of the respiratory lactate dehydrogenase (Ldh). Here we present a quantitative study of the product spectrum and carbon balance for aerobically growing ndh-. Ldh-deficient and Ldh-overexpressing ndh- strains were constructed and used to examine the putative role of the respiratory lactate bypass for aerobic growth and production. We show that aerobically growing ndh- strains perform fermentative metabolism with a near-maximum ethanol yield, irrespective of their Ldh expression background. Yet, Ldh activity strongly affects the aerobic product spectrum in glucose-consuming non-growing cells. Also, Ldh-deficiency hampers growth at elevated temperature (42 °C) and delays the restart of growth after 10–15 h of aerobic starvation.

Genome Wide Phosphoproteome Analysis of Zymomonas mobilis Under Anaerobic, Aerobic, and N2-Fixing Conditions

“…In Z. mobilis , we observed a generalized increase, compared to baseline anaerobic growth, in the phosphorylation of most glycolytic enzymes during aerobic and N 2 -fixing growth conditions (Figure 3). The specific rate of glucose consumption changes substantially in these two growth conditions (Kremer et al, 2015; Strazdina et al, 2018), and it is possible that some of the phosphorylation events that we identified may regulate glycolytic enzyme activity leading to overall changes in flux.…”

“…Recent studies have indicated that during aerobic growth, oxidative damage to iron-sulfur (FeS) clusters constitutes a major factor influencing Z. mobilis metabolism and that respiratory enzymes and the ability to form multicellular aggregates are important for its survival (Jones-Burrage et al, 2019; Martien et al, 2019). Despite these and other recent advances (Yang et al, 2009; Rutkis et al, 2016; Strazdina et al, 2018), much remains to be learned about the regulation of Z. mobilis physiology during aerobic growth.…”

A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4

“…As a proof of principle, we tested whether we could efficiently delete an average size gene, choosing the 1.7 kb ldh gene encoding lactate dehydrogenase ( ldh , ZMO0256), since its function should be dispensable in Z. mobilis (Zou et al, 2012; Strazdina et al, 2018; Jones-Burrage et al, 2019). Our suicide plasmid (pPK15534) was engineered to contain two 500 bp sequences, immediately flanking but not including the target gene (pPK15535) (Figure 2).…”

“…Existing genetic tools for Z. mobilis are not optimal for creation of strains with multiple engineered loci. A common approach to insertionally inactivate genes relies on host homologous recombination of an antibiotic resistance gene flanked by Z. mobilis DNA contained on a suicide plasmid, which, once integrated into the genome, can be screened for events leading to loss of delivery plasmid and gene of interest (Senthilkumar et al, 2004; Strazdina et al, 2018). This approach was enhanced by use of lambda red recombinase to increase the frequency of recombination events (Senthilkumar et al, 2004; Bo et al, 2013).…”

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

“…Another distinctive physiological characteristics of Z. mobilis is its high capacity of aerobic respiration, proceeding almost without concomitant energy production [3,[6][7][8]. The primary electron donor in Z. mobilis electron transport chain is NADH [9,10], therefore aerobic respiration results in acetaldehyde production by withdrawal of NADH from ethanol synthesis [3,11]. Yet the full scale of physiological consequences of aeration in Z. mobilis remains to be elucidated.…”