Engineering microbes for consolidated bioprocessing: new approaches in the light of synthetic biology

Mitigating climate change calls for a reduction in emissions of greenhouse gases, mainly CO2. Twenty-seven per cent of all CO2 emissions come from sources hard to eliminate, including aviation, shipping, and long-distance land transportation. To scale-up production, a transition from first-generation biofuels (edible plant biomass feedstock) to second-generation biofuels (non-edible lignocellulosic plant biomass) is required. However, lignocellulosic bioprocesses struggle to reach economic viability due to the high cost of added enzymes required to digest cellulose. A microbe that was able to generate the cellulase to digest cellulose and able to generate desired products (in what is called a consolidated bioprocess) is not known to exist in nature. Decades of research in synthetic biology have not been successful in creating it due to multiple challenges, such as generating candidate microbial strains and testing them. In this work, novel methodological approaches were developed to overcome these challenges, thus increasing the likelihood of future research finding the ideal microbe. Firstly, the generation of candidate strains was improved by a re-design of the paradigm of modular cloning, based on a vector design called JUMP (Joint Universal Modular Plasmids). Complex multi-gene plasmids can be built from standard DNA parts in a reliable and automation-friendly way using modular cloning systems, based on Golden Gate cloning (which uses type IIS restriction enzymes). However, current standards lack the flexibility to change the microbial host and to perform assemblies to optimise genes of a group. JUMP vector backbones are based on the Standard European Vector Architecture (SEVA), and also have additional modular cloning sites to modify vectors for specific purposes. The experimental results presented here showed that these features allowed use of the vector system in different organisms and reduced the number of assemblies required to optimise and test multi-gene constructs. Secondly, among different explored approaches to test strains, using fluorescent growth reporters was found to have the properties required to screen strains in a faster and more relevant way. Cellulose is insoluble in water, and consequently, previous analytical methodologies to assess the cellulolytic capacity of microorganisms used soluble analogues of cellulase substrates or depended on separation steps which are difficult to do in a fast and high-throughput way. The data presented here showed that expression of fluorescence genes, providing a direct measure of growth, could be measured without separation of cellulose. An Escherichia coli strain expressing cellulases CenA and Chu2268, previously shown to bestow cellulolytic ability on E. coli, was confirmed to be able to grow using cellulose as sole carbon source, which demonstrated the use of fluorescent growth reporters to detect cellulolytic activity. Finally, a strain benchmark was built and characterised to allow screening of expression of cellulolytic genes. A collection of modular parts encoding cellulolytic proteins and secretion signal peptides was built to allow future screening, and the effect of genetic and environmental factors in the measurement of fluorescence as a reporter of growth was investigated. It was found that volumetric conditions and medium additives critically affected the capacity of E. coli to grow at the expense of cellulose. A methodology was developed to allow growth measurement in 96-well plates. In conclusion, this study lays the foundations to establish a faster research cycle to generate and screen microbes that can utilise cellulosic biomass to produce valuable bioproducts and biofuels.