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

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 Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis.

Respiratory chain plays a pivotal role in the energy and redox balance of aerobic bacteria. By engineering respiration, it is possible to alter the efficiency of energy generation and intracellular redox state, and thus affect the key bioprocess parameters: cell yield, productivity and stress resistance. Here we summarize the current metabolic engineering and synthetic biology approaches to bacterial respiratory metabolism, with a special focus on the respiratory chain of the ethanologenic bacterium Zymomonas mobilis. Electron transport in Z. mobilis can serve as a model system of bacterial respiration with low oxidative phosphorylation efficiency. Its application for redox balancing and relevance for improvement of stress tolerance are analyzed.

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.

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

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.

The Low Energy-Coupling Respiration in Zymomonas mobilis Accelerates Flux in the Entner-Doudoroff Pathway.

Performing oxidative phosphorylation is the primary role of respiratory chain both in bacteria and eukaryotes. Yet, the branched respiratory chains of prokaryotes contain alternative, low energy-coupling electron pathways, which serve for functions other than oxidative ATP generation (like those of respiratory protection, adaptation to low-oxygen media, redox balancing, etc.), some of which are still poorly understood. We here demonstrate that withdrawal of reducing equivalents by the energetically uncoupled respiratory chain of the bacterium Zymomonas mobilis accelerates its fermentative catabolism, increasing the glucose consumption rate. This is in contrast to what has been observed in other respiring bacteria and yeast. This effect takes place after air is introduced to glucose-consuming anaerobic cell suspension, and can be simulated using a kinetic model of the Entner-Doudoroff pathway in combination with a simple net reaction of NADH oxidation that does not involve oxidative phosphorylation. Although aeration hampers batch growth of respiring Z. mobilis culture due to accumulation of toxic byproducts, nevertheless under non-growing conditions respiration is shown to confer an adaptive advantage for the wild type over the non-respiring Ndh knock-out mutant. If cells get occasional access to limited amount of glucose for short periods of time, the elevated glucose uptake rate selectively improves survival of the respiring Z. mobilis phenotype.

Structure of the Zymomonas mobilis respiratory chain: oxygen affinity of electron transport and the role of cytochrome c peroxidase.

The genome of the ethanol-producing bacterium Zymomonas mobilis encodes a bd-type terminal oxidase, cytochrome bc1 complex and several c-type cytochromes, yet lacks sequences homologous to any of the known bacterial cytochrome c oxidase genes. Recently, it was suggested that a putative respiratory cytochrome c peroxidase, receiving electrons from the cytochrome bc1 complex via cytochrome c552, might function as a peroxidase and/or an alternative oxidase. The present study was designed to test this hypothesis, by construction of a cytochrome c peroxidase mutant (Zm6-perC), and comparison of its properties with those of a mutant defective in the cytochrome b subunit of the bc1 complex (Zm6-cytB). Disruption of the cytochrome c peroxidase gene (ZZ60192) caused a decrease of the membrane NADH peroxidase activity, impaired the resistance of growing culture to exogenous hydrogen peroxide and hampered aerobic growth. However, this mutation did not affect the activity or oxygen affinity of the respiratory chain, or the kinetics of cytochrome d reduction. Furthermore, the peroxide resistance and membrane NADH peroxidase activity of strain Zm6-cytB had not decreased, but both the oxygen affinity of electron transport and the kinetics of cytochrome d reduction were affected. It is therefore concluded that the cytochrome c peroxidase does not terminate the cytochrome bc1 branch of Z. mobilis, and that it is functioning as a quinol peroxidase.