Deciphering and Pushing Whole-Cell Catalysis

The global market of chemicals accounts for more than €3.6 trillion yearly. However, 95% of the chemical production is covered by synthetic processes, which often employ rare-metal catalysts, in combination with energy-intensive procedures and polluting solvents. As a results, it is estimated that the chemical industry alone is responsible for around 15% of the global greenhouse gas direct emissions. Microorganisms have enormous potential in the production of chemicals, and alone, they could cover the entire manufacture of most sectors of the market. Biocatalysts, employed in organic chemical reactions, are an attractive alternative, as they are safe, biodegradable and perform efficiently in water at mild temperatures and pressures. These properties render biocatalysts more sustainable and cost-efficient and represent a pragmatic contribution to the Green Transition. Additionally, the biggest advantage of biocatalysts is their high regio-, chemo- and stereo-selectivity, that easily yields high-quality products, and which makes biocatalysis particularly suitable for the synthesis of drugs and fine chemicals. The establishment of biocatalysis in the chemical market is limited by the small number of microorganisms explored and by the lack of fast and non-invasive analytical methods to track mechanistic responses in the intracellular environment, to replace laborious blind approaches. The work carried out during this PhD project addressed many of the aforementioned limitations, by proposing suitable solutions, with particular contributions in whole cell bioproduction and development of real-time Nuclear Magnetic Resonance (NMR) and Hyperpolarized NMR analytical tools. An unconventional approach to reroute or fine-tune cellular metabolism toward industrially relevant compounds to support or replace genetic engineering when tools are not available, was proposed by means of substrate mixtures. The provision of different carbon sources to Baker´s yeast led to unexpected formation of valuable chemicals. Acyloins were produced, by rerouting fermentation towards C-C bond formation on furfural, a by-product of the pre-treatment of lignocellulosic biomass. A new metabolic regime towards C4 industrial precursors was discovered, yielding 2,3-butanediol, a widely employed precursor in the chemical industry. Upon addition of a mix of (fluorinated) benzaldehydes, fluorinated phenylacetyl carbinols were formed, renowned building blocks of drugs. The attempt of genetically engineer C. glutamicum with polyketide synthases was also pursued, with the aim of producing valerolactams, precursors of the commercial polymer nylon 5. Furthermore, novel analytical methods, i.e. in-cell NMR and DNP NMR were developed and optimized to track altered metabolism in cancer cells and distinctive pathways usage in Baker´s yeast and less conventional whole-cell catalysts.