Metabolic Engineering of Saccharomyces cerevisiae: From p-Coumaric Acid to Rosmarinic Acid

Metabolic engineering aims to develop robust cell factories which can produce a wide range of high-value chemicals. Designing an efficient host – and, therefore, the commercial exploitation of an efficient microbial factory – is vital for reducing dependency on oil-based processes and developing a more sustainable society. Aromatic amino acid derivatives are phenolic compounds with the potential for industrial commercialisation. There is great interest in developing cost effective bioprocesses as extraction or traditional chemical synthesis of these compounds is challenging. In particular, engineering efficient microbial cell factories may enable more sustainable production of aromatic amino acid derivatives. The brewer’s yeast, Saccharomyces cerevisiae, is a well-characterised and robust microbial factory suitable for producing bio-based phenolic compounds, and the aromatic amino acid derivative p-coumaric acid is a precursor for more complex phenolic compounds with potentially numerous applications at the industrial level.
In Chapter 1 of this thesis, an S. cerevisiae strain capable of producing high levels of p-coumaric acid from xylose (as a carbon source) was engineered for the first time. A system biology approach was then used to investigate the gene expression of the resulting phenotype. The results show how system biology and metabolic engineering can obtain p-coumaric acid in yeast, demonstrating proof-of-principle of a robust cell factory for producing p-coumaric acid from xylose and developing a viable bioprocess. In Chapter 2, rosmarinic acid, a complex phenolic compound derived from p-coumaric acid, was produced by a metabolic engineering approach. The rosmarinic acid biosynthetic pathway was optimised by installing different gene variants and increasing the copy number of critical enzymes in the chimeric pathway. This study highlights the amenability to engineering complex aromatic amino acid derivatives using S. cerevisiae. However, the ability to systematically install, genome edit, and adjust the expression levels of a
specific pathway is vital for metabolic engineering projects. Therefore, in Chapter 3, a set of integrative vectors was developed to further expand and optimise the genome editing tools in S. cerevisiae. Tool development in metabolic engineering plays a crucial role in heterologous gene insertions, pathway optimisation, gene circuits, and the instalment of complex biochemical routes. In line with this, progress in the metabolic engineering field depends on designing available tools, building, and learning, which this thesis summarises as the “Design-Build-Test-Learn” cycle.