Investigation and Optimisation of Fusarubins Production in Fusarium solani via Targeted Deletion
Author
Kabemba Kaniki, Samba Eveline
Term
4. term
Education
Publication year
2020
Submitted on
2020-06-04
Pages
79
Abstract
Fusarubins are red naphthoquinone pigments produced by Fusarium species and have been investigated for antimicrobial and anti-tuberculotic activities. Their biosynthesis is governed by the PKS3 gene cluster (fsr1–fsr6). In Fusarium solani, the cluster differs from other Fusarium species by containing 17 additional genes between fsr3 and fsr4. This study aimed to clarify the roles of PKS3 genes in F. solani and to elucidate the functions of the 17 extra genes as a basis for optimizing fusarubin production. The work combined (i) comparative bioinformatics, including synteny alignment within the Fusarium solani species complex (FSSC) to assess gene conservation, and protein function predictions; and (ii) targeted gene deletions via Agrobacterium tumefaciens-mediated transformation of F. solani. Individual knockouts of fsr genes and selected extra genes were generated, and resulting strains were evaluated for phenotypic changes in pigmentation and growth. Synteny analysis indicated that most genes, including the 17 additional ones, are conserved across the FSSC, and protein predictions suggested likely roles in secondary metabolism. Experimentally, eight gene deletions were achieved; these mutants displayed clear changes in pigmentation and growth, while chemical analysis of their metabolite profiles is pending. The thesis provides a detailed investigation of the PKS3 cluster in F. solani, a comparison with related Fusarium species, an initial phenotypic characterization of deletion mutants, and a concise protocol for F. solani transformation. Together, these results lay the groundwork for understanding and eventually tuning fusarubin production in F. solani.
Fusarubiner er røde naphthoquinon-pigmenter produceret af Fusarium-arter og undersøgt for antimikrobielle og anti-tuberkulotiske egenskaber. Deres biosyntese styres af PKS3-genklyngen (fsr1–fsr6). I Fusarium solani adskiller klyngen sig fra andre Fusarium-arter ved at rumme 17 ekstra gener mellem fsr3 og fsr4. Dette projekt havde til formål at undersøge rollerne af generne i PKS3-klyngen i F. solani og at belyse funktionerne af de 17 ekstra gener som grundlag for at kunne optimere fusarubinproduktionen. Arbejdet omfattede (i) komparative bioinformatiske analyser, herunder syntenialignment inden for Fusarium solani species-komplekset (FSSC), for at vurdere genkonservering, samt proteinfunktionsprediktioner; og (ii) målrettede gen-deletioner via Agrobacterium tumefaciens-medieret transformation af F. solani. Individuelle knockouts af fsr-generne og udvalgte ekstra gener blev konstrueret, og de resulterende stammer blev vurderet for fænotypiske ændringer i pigmentering og vækst. Syntenianalysen viste, at de fleste gener, herunder de 17 ekstra, er konserverede på tværs af FSSC, og proteinprediktioner indikerede sandsynlige roller i sekundærmetabolismen. Eksperimentelt blev deletion af otte gener opnået; disse mutanter udviste tydelige ændringer i pigmentering og vækst, mens kemisk analyse af deres metabolitter er igang/på vej. Rapporten præsenterer dermed en detaljeret undersøgelse af PKS3-klyngen i F. solani, en sammenligning med beslægtede Fusarium-arter, en første fænotypisk karakterisering af deletionsmutanter og en kort protokol for transformation af F. solani. Arbejdet lægger et vigtigt grundlag for at forstå og på sigt målrette produktionen af fusarubiner i F. solani.
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