Design of non-ribosomal peptide production in microbial cells
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Journal Title
Journal ISSN
Volume Title
Kemian tekniikan korkeakoulu |
Master's thesis
Author
Date
2023-08-21
Department
Major/Subject
Biosystems and Biomaterials Engineering
Mcode
CHEM3028
Degree programme
Master’s Programme in Life Science Technologies
Language
en
Pages
47+7
Series
Abstract
Fungi produce a vast array of secondary metabolites including non-ribosomal peptides (NRP), synthesized at megasynthase complexes, so-called non-ribosomal peptide synthetases (NRPS). NRP are attractive targets for the pharmaceutical, agricultural and food industries due to their wide spectrum of bioactivities. The theoretical chemical potential of fungi is vast, but underexploited, partially due to the high rediscovery rate in traditional compound screenings, and challenges in pathway induction in the native host. The transfer of single core enzymes into a heterologous host has proven beneficial, but enzyme engineering and the identification of suitable enzyme variants with improved characteristics for industrial applications remain a challenge. The work presented here made use of recent technological advances in computational compound discovery, protein structure modelling and enzyme engineering with the aim to rapidly identify NRP with interesting bioactivities, and prioritize the most promising NRPS variants for future cell factory design. The antiSMASH pipeline, the current gold standard in in silico biosynthetic gene cluster (BGC) prediction and annotation, enabled the detection of nearly one thousand BGC in 23 whole fungal genomes, a subset of which encoded NRP biosynthetic machinery. Two NRP were chosen as targets for future heterologous expression: The antibiotic aspergillic acid with the NRPS asaC, and the yellow pigment chrysogine with chyA. Instead of experimentally generating and in vivo testing thousands of enzyme variants, a novel computational pipeline guided the identification of the most promising naturally occurring enzyme variants based on sequence and structural homology, and shared substrate binding site architecture with the seed protein. A priority set of enzyme variants, 24 for asaC and 4 for chyA, was determined, significantly speeding up the future design of the first known heterologous expression systems for the peptides aspergillic acid and chrysogine.Description
Supervisor
Jouhten, PaulaThesis advisor
Castillo, SandraKeywords
genome mining, non-ribosomal peptides, enzyme variants, AlphaFold2
