The genetic basis of 3-hydroxypropanoate metabolism in Cupriavidus necator H16

Abstract

Background: 3-Hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this study, the native 3-HP metabolism of Cupriavidus necator was investigated and manipulated as it represents a promising chassis for the production of 3-HP and other fatty acid derivatives from CO 2 and H 2. Results: When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy, a highly undesirable trait in the context of biological 3-HP production which required elimination. Inactivation of the methylcitrate pathway needed for propionate utilisation did not affect the organism's ability to grow on 3-HP. Putative genes involved in 3-HP degradation were identified by bioinformatics means and confirmed by transcriptomic analyses, the latter revealing considerably increased expression in the presence of 3-HP. Genes identified in this manner encoded three putative (methyl)malonate semialdehyde dehydrogenases (mmsA1, mmsA2 and mmsA3) and two putative dehydrogenases (hpdH and hbdH). These genes, which are part of three separate mmsA operons, were inactivated through deletion of the entire coding region, either singly or in various combinations, to engineer strains unable to grow on 3-HP. Whilst inactivation of single genes or double deletions could only delay but not abolish growth, a triple ∆mmsA1∆mmsA2∆mmsA3 knockout strain was unable utilise 3-HP as the sole source of carbon and energy. Under the used conditions this strain was also unable to co-metabolise 3-HP alongside other carbon and energy sources such as fructose and CO 2 /H 2. Further analysis suggested primary roles for the different mmsA operons in the utilisation of β-alanine generating substrates (mmsA1), degradation of 3-HP (mmsA2), and breakdown of valine (mmsA3). Conclusions: Three different (methyl)malonate semialdehyde dehydrogenases contribute to 3-HP breakdown in C. necator H16. The created triple ∆mmsA1∆mmsA2∆mmsA3 knockout strain represents an ideal chassis for autotrophic 3-HP production.

Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus

“…During shake-flask cultivation the methylmalonate-semialdehyde dehydrogenase (g153; Ap ald6 ) is strongly up-regulated at all times in the engineered strains. This enzyme, normally involved in branched-chain amino acid degradation, has been implicated in 3HP metabolism in bacteria ( Zhou et al, 2014 ; Arenas-Lopez et al, 2019 ) and the yeast C. albicans ( Otzen et al, 2014 ) by also acting as a malonate-semialdehyde dehydrogenase and diverting flux toward acetyl-CoA rather than 3HP. Here we deleted a putative malonate-semialdehyde dehydrogenase Ap ald6 and found that 3HP yield was drastically improved ( Figure 4 ).…”

A genome-scale metabolic model of Cupriavidus necator H16 integrated with TraDIS and transcriptomic data reveals metabolic insights for biotechnological applications

“…necator H16 competent cells and for the TraDIS experiment. Chemically defined minimal medium (MM) [ 68 ] was supplemented with 4 g/l of either D-fructose (FMM), when used to grow the C . necator H16 transposon mutant library for TraDIS, or sodium D-gluconate (SGMM), when used to select C .…”

Phenotypic and Genetic Characterization of Temperature-Induced Mutagenesis and Mortality in Cupriavidus metallidurans

“…Interestingly, this degradation only occurs at room temperature and not at 37 • C, probably due to the inadequate ability to remove toxic malonic semialdehyde at higher temperatures (Loh et al, 2006;Kim et al, 2010). Currently, we do not know if Rmet_1009, similar to RutE, reduces malonic semialdehyde to 3-hydroxypropionic acid and if C. metallidurans CH34 carries other genes that encode for proteins that enable the removal of malonic semialdehyde, similar to Cupriavidus necator H16 (Arenas-López et al, 2019). Nevertheless, our results point toward metabolic poisoning of C. metallidurans grown in high-concentration nitrogencontaining media at 37 • C.…”