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

Author summaryGenome-scale metabolic models (GSMs) provide a tool for unravelling the complex metabolic behaviour of bacteria and how they adapt to changing environments and genetic perturbations, and thus offer invaluable insights for biotechnology applications. For a GSM to be used efficiently for strain development purposes, however, the model must be easily readable and reusable by other researchers, whilst being able to predict metabolic behaviour with a high level of accuracy. In this work, we developed a GSM for Cupriavidus necator H16 that is linked to the BioCyc database, which provides an efficient way of application, model update, integration of experimental data and network visualisation for other researchers. Using our model, we demonstrate how integrating experimental observations, including Transposon-directed Insertion site Sequencing (TraDIS) and omics data, can be used to compensate for the lack of regulatory, kinetic and thermodynamic information in GSMs, and thus improve model accuracy. Importantly, we found that TraDIS in vivo screening and GSM analysis are complementary approaches, which can be used in combination to provide reliable gene essentiality predictions. Overall, our results offer an informed strategy for the deliberate manipulation of C. necator H16 metabolic capabilities, towards its industrial application to convert greenhouse gases into biochemicals and biofuels. Exploiting biological processes to recycle renewable carbon into high value platform chemicals provides a sustainable and greener alternative to current reliance on petrochemicals. In this regard Cupriavidus necator H16 represents a particularly promising microbial chassis due to its ability to grow on a wide range of low-cost feedstocks, including the waste gas carbon dioxide, whilst also naturally producing large quantities of polyhydroxybutyrate (PHB) during nutrient-limited conditions. Understanding the complex metabolic behaviour of this bacterium is a prerequisite for the design of successful engineering strategies for optimising product yields. We present a genome-scale metabolic model (GSM) of C. necator H16 (denoted iCN1361), which is directly constructed from the BioCyc database to improve the readability and reusability of the model. After the initial automated construction, we have performed extensive curation and both theoretical and experimental validation. By carrying out a genome-wide essentiality screening using a Transposon-directed Insertion site Sequencing (TraDIS) approach, we showed that the model could predict gene knockout phenotypes with a high level of accuracy. Importantly, we indicate how experimental and computational predictions can be used to improve model structure and, thus, model accuracy as well as to evaluate potential false positives identified in the experiments. Finally, by integrating transcriptomics data with iCN1361 we create a condition-specific model, which, importantly, better reflects PHB production in C. necator H16. Observed changes in the omics data and in-silico-estimated alterations in fluxes were then used to predict the regulatory control of key cellular processes. The results presented demonstrate that iCN1361 is a valuable tool for unravelling the system-level metabolic behaviour of C. necator H16 and can provide useful insights for designing metabolic engineering strategies.