Principal Supervisor: Dr. Maria Makarova

Co-supervisor: Prof. Dylan Owen

PhD project title: Addressing the global appetite for organic molecules through the rational engineering of microbial biochemistry

University of Registration: University of Birmingham

Project outline:

Sustainable future industries require sourcing biomolecules as raw materials without consuming finite petrochemicals or employing ecologically damaging land use. In this light, industrial microbial production of bulk chemicals via renewable sources of biomass is a sustainable and commercially attractive solution. One family of compounds where microbial production is used are medium-chain fatty acids (MCFAs)¹. These are widely used as herbicides, antimicrobials, lubricants, surfactants, food additives and biofuels and the market is projected to grow to over $1bn by 2026². However microbial production has never been able to match the yield or economy of plant or petroleum-based sourcing because the engineered species are not evolutionarily adapted to the high yield of MCFA production. A microorganism that naturally produces MCFAs would have the potential to be a game changer. We found such organism and propose a project to develop cell factories based on our fundamental findings³.

We will elucidate the mechanisms of synthesis, degradation and trafficking of MCFAs and to customise metabolic pathways inside the fission yeast Schizosaccharomyces japonicus in order to build a new biotechnology platform. I have shown that this organism has evolved a natural tolerance to high MCFAs concentrations, in order to adapt to anaerobic environments⁴. First, we will determine how this organism is able to generate and tolerate MCFAs. We will then employ multistep metabolic engineering to generate a rationally-designed strain capable of competing against current unsustainable production strategies. Success will be measured by comparing obtained yields vs current state-of-the-art microbial and plant-based production. Ultimately, this will generate the knowledge and materials to produce commercially viable MCFAs using sustainable and adaptable microbial “cell factories”.

The general strategy will employ two stages; first, rationally designed and directed mutagenesis and the second a period of laboratory-based evolutionary optimisation. We will employ this strategy in 3 consecutive stages:

• Enhanced synthesis: We will discover metabolic machinery responsible for MCFA synthesis and optimise these for enhanced MCFA production.
• Reduced degradation: We will elucidate the routes of fatty acid degradation to engineer enhanced retention.
• Optimised traffic and extraction: We will investigate fatty acid traffic and optimise the route for efficient purification of MCFAs from japonicus.

The main methodology will include molecular biology (cloning and genetic transformation), biochemistry (analysis of fatty acids via gas chromatography and mass spectrometry), cell biology (microscopy to visualise fatty acids) and genomics (analysis of laboratory-assisted evolution experiments).

References:

1. Sarria, S.et al. Microbial synthesis of MC chemicals from renewables. Biotechnol. (2017). 2. Global Medium-Chain Triglycerides Market. https://www.marketsandmarkets.com/Market-Reports/medium-chain-triglycerides-market-248063458.html. 3. Makarova, M. et al. Delineating the Rules for Structural Adaptation of Membrane-Associated Proteins to Evolutionary Changes in Membrane Lipidome. Curr. Biol. (2020). 4. Panconi, L., et al. Phospholipid tail asymmetry allows cellular adaptation to anoxic environments. bioRxiv (2022) doi:10.1101/2022.08.04.502790.

BBSRC Strategic Research Priority: Renewable Resources and Clean Growth – Industrial Biotechnology

Techniques that will be undertaken during the project:
Cloning and cell transformation

Fluorescence microscopy

GC-MS mass spectrometry

Laboratory-assisted evolution

Genomics

Contact: Dr. Maria Makarova