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Construction of microcompartments in thermophilic Geobacillus thermoglucosidasius as nano-bioreactors for advanced biofuels production at high temperature

Periodic Reporting for period 1 - THERMCP (Construction of microcompartments in thermophilic Geobacillus thermoglucosidasius as nano-bioreactors for advanced biofuels production at high temperature)

Reporting period: 2016-05-09 to 2018-05-08

Due to the rapidly diminishing sources of conventional fossil fuels coupled with the large evidence-base for ecocide related to increased anthropogenic atmospheric carbon dioxide, there is an urgent need for rapid transition from non-renewable hydrocarbons to alternative novel pathways in order to secure, clean and efficient ‘green’ bio-energy production. This will have a major impact on individuals and the environment at a global scale. Thermophilic Gram-positive spore-forming bacterial Geobacillus species have an intrinsic ability to utilise a wide range of substrates including renewable resources, such as cell wall hemicellulose polymers, and are of biotechnologically and industrially important producers of ethanol and isobutanol. However, tolerance to biofuel-related alcohols by the host-producing organism is often lower than that of the exogenously supplied alcohol. This arises due to the toxicity of reactive intermediates, such as aldehydes. Co-localising the steps in alcohol production in an organelle designed to protect reactive intermediates from undesirable interactions provides a route to increased yields and decreased toxicity. Bacterial MicroComPartments (MCPs) are organelles that host specific biochemical reactions for both anabolic and catabolic functions. Engineered, morphologically-diverse MCPs bearing heterologous enzymatic pathways have shown enhanced productivity for commodity chemicals, which make MCPs an important focus for metabolic engineering. Gaining control of BMC assembly and incorporation of a heterologous enzymatic cargo has yet to be achieved in thermophiles. The Fellowship aimed to find new perspectives in the development of novel bacterial strains of thermophilic Geobacillus spp. to enhance the production of second-generation biofuels at high temperatures.

The THERMCP objectives were: 1). To search for shell protein homologs in the available sequenced genomes of Geobacillus species and perform bioinformatics analysis of the pdu operon from G. thermoglucosidasius; 2). To establish the protein engineering rules and the ultimate benefit of directing the Pdc and Adh enzymes required for ethanol production to the Pdu MCP shell of thermophilic G. thermoglucosidasius; 3). To engineer a strain of G. thermoglucosidasius capable of overproducing 2-ketoisovalerate (2-kiv) as described in the literature, in a strain where fermentation pathways have been deleted; 4). To use the strain developed in 3. as a platform to engineer a novel keto-acid dehydrogenase and acylating alcohol dehydrogenase route for isobutanol production, and introduce this into the protein MCP in thermophilic G. thermoglucosidasius. The project has achieved most of its objectives and milestones for the period, with relatively minor deviations.

Furthermore, a set of project training objectives were satisfied to reintegrate Dr. Yana Wade into the research environment after a career break. She also gained a thorough grounding in microbial engineering of thermophiles to complement her existing knowledge and skills in thermophilic environmental microbiology providing a foundation for a research career in industrial applied microbiology.
The genetic capacity of thermophilic bacterium G. thermoglucosidasius for MCPs formation was determined during the THERMCP project. The genes in G. thermoglucosidasius required to assemble empty recombinant MCPs and target recombinant proteins within the organelle were identified. The feasibility of constructing cell factories for products such as advanced biofuels and fine chemicals in industrially important Bacillus and Geobacillus spp. by heterologous cargo-carrying MCP production and assembly were established. The possibility of exploiting compartments in thermophiles were shown for the first time. The platform for repurposing the native organelles into nanobioreactors for enhance isobutanol production at elevated temperatures in Geobacillus spp. for future research was developed.

A manuscript describing heterologous microcompartment assembly in Bacillaceae and establishing the components necessary for scaffold formation was submitted to ACS Synthetic Biology [Under Review]. Research has been actively disseminated by the Fellow to academic beneficiaries at several high-profile conferences, maximising the impact of the project at an international level. The Fellow engaged with the wider community (e.g. general public, industry, stakeholders) to develop awareness on how her research has an impact in their lives and work, where efficient biofuel/high-value chemical production acts as a platform for a “green energy” future. Furthermore, the work of Dr. Wade was showcased as a successful female role model in science at events targeting the next generation of young female researchers.

Dr. Wade successfully reintegrated back into academia, broadened her research and technical skills in interdisciplinary fields such as bioinformatics, microbial engineering, synthetic biology and applied microbiology fields, and developed leadership and management skills relevant for future career.
The THERMCP project has facilitated the development of novel and state-of-the-art scientific and biotechnological knowledge on the bioengineering of microcompartments within bacteria for the implication to generate high-value products, such as biofuel. The study has identified genes encoding homologs of MCP proteins in thermophilic Geobacillus spp. including three new genes of unknown function, that are probably associated with the operation of MCPs in thermophiles and uniquely associated with members of the Bacillaceae family. The polyhedral MCPs closely resembled, in shape and size those reported previously, were produced in thermophilic G. thermoglucosidasius cells at 60ºC. As with recent studies on successful direction of pyruvate decarboxylases and an alcohol dehydrogenase into the lumen of BMCs in mesophilic Gram-positive and Gram-negative organisms, it is possible to incorporate thermostable proteins and generate an ethanol-producing bioreactor inside G. thermoglucosidasius cells at high temperatures especially with the encapsulation peptides that target proteins within MCPs in G. thermoglucosidasius. That was a major discovery in THERMCP. Large recombinant MCPs were discovered during the project that provide significant advantages for loading the organelle with multi-enzymatic pathways into their expanded volume for enhancing their metabolic efficiency. It will be possible to load the empty larger organelles developed in this work with multi-enzyme pathways and, subsequently, to utilise them as intracellular bioreactors within Bacillus subtilis. Coupled with the prospect of applying the compartmentalisation to deliver a commercial standard of products such as vaccines, advance biofuels and fine chemicals, the development of larger MCPs at the industrial-scale presents a significant and realistic opportunity for industry. Ultimately, the project and its outputs will lead to the ability to deliver more economic and productive processes for the generation of high-values chemicals and biofuels using the power of simple engineered microorganisms.
Graphical summary of work used in publication