AbstractInitially Aspergillus oryzae was used for heterologous expression of the pleuromutilin biosynthesis pathway. In an attempt to increase flux through the pathway, the pdc promoter was chosen to replace the weak eno promoter. The pdc promoter could effectively drive the GFP gene in A. oryzae, however transformants containing biosynthetic genes under control of the pdc promoter did not show any improvement in accumulation of the desired metabolites and the reasons for this are still unclear. Plant-based production systems may provide an alternative to filamentous fungi, so Nicotiana tabacum was explored as a heterologous host. Using the Golden Gate assembly method, expression vectors were developed to express part or all of the seven gene pleuromutilin biosynthesis pathway. Through transient Agrobacterium infiltration of leaves, it was demonstrated that candidate pleuromutilin intermediates were likely being produced, however this was correlated with chlorosis in the leaf tissues suggesting a toxic effect in planta, limiting this direction of research. Given others have had success using yeast-based expression systems for terpene products, this was also employed as a platform for the heterologous expression of pleuromutilin biosynthesis genes. The pathway-specific GGS and Cyclase were expressed under regulation of the GAL promoters and a product consistent with the initial pleuromutilin precursor was detected, however GAL-based induction was not reliable and unexpectedly, yields were not enhanced in the yeast host AH109 which ought to give elevated titres for diterpene products.
Whilst all three host systems proved to be amenable to manipulation, in all cases, further optimisation of gene expression is required in order to obtain the desired levels of enzyme activity needed to improve pleuromutilin production.
|Date of Award||23 Jun 2020|
|Supervisor||Andy M Bailey (Supervisor) & Gary Foster (Supervisor)|