Assessing Cupriavidus necator H16 as a chassis for the production of PQQ dependent enzymes using membrane bound glucose dehydrogenase.

Birtalan Andrei Mihai

Assessing Cupriavidus necator H16 as a chassis for the production of PQQ dependent enzymes using membrane bound glucose dehydrogenase.

Publication Date

July 27, 2026

Creator

Birtalan Andrei Mihai

University of Nottingham

Abstract

Rising global temperature caused by greenhouse gas emissions has reached an environmental tipping point signifying the uphill struggle presented by undoing centuries of explosive economic and societal growth fuelled by the fossil fuel industry and the titanic amounts of greenhouse gas emissions released. A major contributor to this is the chemical industry which has adopted a linear model, utilising non-renewable fossil fuel stocks as the basis for the majority of chemical production worldwide including plastics, pharmaceuticals, and solvents. An emerging and promising solution for this problem is the employment of microbial cell factories capable of producing chemicals from feedstocks such as biomass, industrial off gasses, municipal waste, and atmospheric CO2. Cupriavidus necator H16 provides an attractive model platform due to its metabolic robustness and redundancy making it conducive of modifications and subsequent optimisation of metabolic pathways; as well as a rapidly expanding synthetic biology toolkit. The diverse C. necator H16 genome possesses a pyrroloquinoline quinone biosynthesis operon under an unknown promoter, as well as putative quinoproteins, making this a promising chassis for quinoprotein pathways in order to expand available feedstocks. This study aimed to evaluate C. necator H16 as a chassis for quinoprotein production, and PQQ dependent metabolic pathways, through the coupling of the native PQQ biosynthetic operon with a medium strength synthetic promoter, and using membrane bound-PQQ dependent glucose dehydrogenase (mGDH) from Gluconacetobacter diazotrophicus as a non-toxic, proof of concept, which is also easily selectable due to the inability of C. necator to utilise glucose except when specific mutations arise spontaneously in the N-acetylglucosamine phosphotransferase system to confer glucose utilisation. If possible, the resultant strain would be subjected to adaptive laboratory evolution to elucidate potential bottlenecks and strategies for further optimisation. Firstly, the prevalence of glucose utilisation arising spontaneously was determined in a medium relevant to this study, mGDH was successfully introduced into C. necator on a broad-host range vector for simplicity, and by chromosomal insertion to replace the locus of possible mutations providing glucose utilisation. The PQQ biosynthetic operon was coupled with the P21 synthetic promoter; alongside this as a back-up for supplying intracellular PQQ, a putative PQQ import system was identified but shown to not be constitutively active. Active mGDH was not observed in any strain presumably due to species specific hurdles such as DNA sequence or chaperone incompatibilities.

Item Type

ethesis

Thesis Type

MRes

Supervisors

Winzer, Klaus