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

Biotechnology for Biofuels

Jessica Locker¹  Diego Orol¹  Frederik Walter¹  Christian Arenas-López¹  Katalin Kovács¹  Klaus Winzer¹  Nigel P. Minton¹  Tobias Busche²  Jörn Kalinowski² 

[1] BBSRC/EPSCR Synthetic Biology Research Centre (SBRC), School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham;Center for Biotechnology (CeBiTec), Bielefeld University;

关键词: 3-Hydroxypropionic acid;    Metabolic engineering;    Cupriavidus necator;    Ralstonia eutropha;    Co-metabolism;    Carbon fixation;   

DOI  :  10.1186/s13068-019-1489-5

来源: DOAJ

【 摘 要 】

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 CO2 and H2. 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 knock-out 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 CO2/H2. 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 knock-out strain represents an ideal chassis for autotrophic 3-HP production.

【 授权许可】

Unknown