Development of high-throughput genome editing tools towards ethylene production in Cupriavidus species

Depletion of natural hydrocarbon resources has catalysed research interest into sustainable routes for the production of bulk chemicals. Cupriavidus necator H16 has been extensively studied for the production of polyhydroxybutyrate (PHB), a biopolymer utilised as an alternative to petroleum-based plastics. However, the strain lacks efficient, fast, and user-friendly strain engineering tools. A single mutant is typically generated via a conjugation/counterselection method, which requires multiple steps and results in a maximum efficiency of 50%, necessitating extensive screening via colony PCR. Here is presented the development of HTP (high-throughput) editing tools in C. necator. These tools were then employed for the metabolic engineering of Cupriavidus metallidurans (C. metallidurans), another chassis utilised within the Synthetic Biology Research Center (SBRC) of Nottingham along with C. necator. In particular, engineering efforts focused on implementing for the first time the ethylene-forming enzyme (EFE) pathway in C. metallidurans and improving production of ethylene, a platform chemical of the SBRC, in that strain. The assessment of Lambda-Red (λ-Red) and RecET recombineering systems were inconclusive and highlighted the difficulty to adapt λ-Red outside of Escherichia coli (E. coli). The implementation of CRISPR/Cas9 required many optimisation steps before the emergence of a mutant, with an overall efficiency of 40%. Additional HTP tools were further designed for introduction and optimisation of the Ethylene-Forming Enzyme (EFE) pathway in Cupriavidus metallidurans CH34. These HTP tools were first applied in E. coli as proof of concept and enabled a 6.3-fold increase in ethylene productivity, compared to the highest ethylene productivity reported to date in E. coli (Lynch et al., 2016). The global Transcriptional Machinery Engineering (gTME) technique involved the semi-automated creation of an rpoD mutant library and ultimately participating in the emergence of ethylene overproducing strains. To maximise the selection of mutants with desirable traits, ethylene synthesis was coupled to proline formation via a growth couple and cells were maintained in a proline-free growth medium during Adapted Laboratory Evolution (ALE) fermentation. The gTME and ALE engineering methods are readily available for transfer into C. metallidurans and by extension, to other Cupriavidus strains. Altogether, the development of genomic, transcriptomic and metabolomic engineering tools described in this work will boost the strain engineering potential of these non-model chassis for both current and novel chemical production