Metabolic engineering of isoprenoids

McElroy, Christopher; Blank, Lars M. (Thesis advisor); Fischer, Rainer (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

The cornerstones of modern medicine are arguably antibiotics, chemotherapeutics, antibodies and vaccines. Commonplace infections which laid our ancestors low are taken for granted as being easily treated with antibiotics in the modern era. In recent times efforts have been made in the pursuit of new active compounds for human exploitation within a medicinal setting. No greater source of such compounds is available than that found within nature. It is from nature that many approved medicines have been isolated, and it is here that we should return for novel activities using a combination of high throughout and knowledge based approaches. Within this setting, the investigation of a fascinating natural product biosynthesis pathway was undertaken. The obscure armillyl orsellinate, melleolide and armillane biosynthesis pathways possess a multitude of novel structures. Members of this isoprenoid family can have cytotoxic, antimicrobial and phytotoxic properties. Hindering exploitation of these compounds for society is their largely uncharacterized biosynthesis pathway. Thus at the start of this work the transcriptomic and genomic sequencing reads generated previously within our lab were assembled to elucidate the whole genome sequence of A. gallicaFU02472, along with its transcriptomic profile. Comparison of this assembly with a previously identified melleolide gene cluster, revealed additional putative biosynthesis genes. After investigation of these novel genes found within this extended gene cluster, a heterologous cDNA expression library screen was performed to identify genes encoding additional melleolide pathway enzymes. Despite the successful construction of the expression library, no novel enzymes responsible for the isomerization reaction could be identified. However, the successful isomerization of armillyl orsellinate precursors was demonstrated non‐enzymatically within a heterologous yeast system, thereby circumventing the requirement of an enzyme within this host. Subsequently a metabolically engineered armillyl orsellinate/melleolide platform yeast strain was constructed using an artificial biosynthesis pathway. Its capabilities were investigated, leading to the detection of multiple novel protoilludene products and trace levels of orsellinic acid. Furthermore, metabolic engineering of this strain was implemented for the construction of a second generation strain. Additionally, the identification of several novel hydroxylated protoilludenes was achieved and for one of the novel products (13‐hydroxy‐6‐protoilludene), the respective enzyme (CYPArm2) was determined. Finally a novel medium and production method was developed for increasing product titers and for simplifying the generation of reaction substrates.