Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Abstract

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R -form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The ( R )-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as ( R )-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO 2 ) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of ( R )-1,3-BDO. Implementation of ( R )-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of ( R )-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol −1 h −1 autotrophically. This is first report of ( R )-1,3-BDO production from CO 2 .

Biosensor-informed engineering of Cupriavidus necator H16 for autotrophic D-mannitol production

“…Autotrophic batch fermentation of C. necator was performed as reported previously by Gascoyne and co-workers (Gascoyne et al, 2021) with slight modifications. Briefly, to prepare the fermenter culture, individual colonies of freshly streaked C. necator were used to inoculate 5 mL of LB medium containing gentamycin in 50-mL conical centrifuge tubes.…”

“…Due to its ability to store large amounts of reduced carbon in the form of polyhydroxybutyrate (PHB) (Schlegel et al, 1961a;Steinbüchel and Schlegel, 1991), C. necator is considered a promising host organism for the sustainable production of value-added compounds from CO 2 . In the last decade, with an extensive genetic toolkit available, allowing genome editing and the controlled expression of heterologous genes, C. necator has been engineered for the autotrophic production of methyl ketones (Müller et al, 2013), alka(e)nes (Crépin et al, 2016), isopropanol (Marc et al, 2017), α-humulene (Krieg et al, 2018), acetoin (Windhorst and Gescher, 2019), trehalose (Löwe et al, 2021), lipochitooligosaccharides (Nangle et al, 2020), 2,3-butanediol (Bommareddy et al, 2020) and 1, 3-butanediol (Gascoyne et al, 2021). A majority of these compounds are biosynthesised utilising precursors such as pyruvate and acetyl-CoA derived from the Entner-Doudoroff (ED) pathway.…”

Rebooting life: engineering non-natural nucleic acids, proteins and metabolites in microorganisms

“…Metabolically engineered S. cerevisiae has shown to improve 1,2,4-butanetriol production by expressing 2-ketoacid decarboxylase from Lactococcus lactis [ 16 , 17 ]. Several other host microorganisms have been employed for the production of non-natural metabolites such as, the examples of which are Corynebacterium glutamicum and C. crenatum for 2-methyl-1-butanol [ 18 , 19 ], S. cerevisiae for 1,2,4-butanetriol [ 20 ], and Cupriavidus necator for 1,3-butanediol [ 21 ], etc.…”

Charting a narrow course for direct electron uptake-facilitated electromicrobial production

“…Recently, uncertainty has grown about the promise of dEMP. 16,35 Mediated systems continue to make gains in scale-up, [36][37][38] product spectrum diversity, [39][40][41] and process modelling; 5,9 they rely on wellcharacterized metabolic pathways (i.e., those of Knallgas, formatotrophic, or aceto-/methanogenic microbes); and, in the case of H2and CO-mediated systems, they avoid challenges associated with incompatible electrolyte/medium requirements by transferring reducing power through the gas phase. 42 Moreover, questions about the pH and salt tolerance, in addition to the productive capacity of cathodic biofilms, have also raised serious questions about the viability of dEMP.…”