Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices

Abstract

Anaerobic fungi are emerging biotechnology platforms with genomes rich in biosynthetic potential. Yet, the heterologous expression of their biosynthetic pathways has had limited success in model hosts like E. coli. We find one reason for this is that the genome composition of anaerobic fungi like P. indianae are extremely AT-biased with a particular preference for rare and semi-rare AT-rich tRNAs in E coli, which are not explicitly predicted by standard codon adaptation indices (CAI). Native P. indianae genes with these extreme biases create drastic growth defects in E. coli (up to 69% reduction in growth), which is not seen in genes from other organisms with similar CAIs. However, codon optimization rescues growth, allowing for gene evaluation. In this manner, we demonstrate that anaerobic fungal homologs such as PI.atoB are more active than S. cerevisiae homologs in a hybrid pathway, increasing the production of mevalonate up to 2.5 g/L (more than two-fold) and reducing waste carbon to acetate by ~90% under the conditions tested. This work demonstrates the bioproduction potential of anaerobic fungal enzyme homologs and how the analysis of codon utilization enables the study of otherwise difficult to express genes that have applications in biocatalysis and natural product discovery.

A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis

“…10−12 These industrially relevant properties include thermostability, resistance to protease degradation, and preferential cofactor requirements, among others. 10,13 Addi-tional efforts reveal signaling and/or transport proteins from gut fungi that may be leveraged as orthogonal systems in model organisms. 14−17 However, heterologous expression of many anaerobic fungal proteins remains challenging due to the following: (1) differences in host reducing conditions, which inhibit proper folding, 18 (2) a compositional bias toward amino acids encoded by AT-rich codons, 13 which can present a metabolic burden that is not fully addressed by codon optimization, 13,19 and (3) glycosylation and other posttranslational modifications required for function that are not easily replicated in model hosts.…”

“…Efforts to characterize and tap into this biotechnological potential have revealed many proteins from anaerobic fungi that offer superior properties that are desirable for industrial processes. − These industrially relevant properties include thermostability, resistance to protease degradation, and preferential cofactor requirements, among others. , Additional efforts reveal signaling and/or transport proteins from gut fungi that may be leveraged as orthogonal systems in model organisms. − However, heterologous expression of many anaerobic fungal proteins remains challenging due to the following: (1) differences in host reducing conditions, which inhibit proper folding, (2) a compositional bias toward amino acids encoded by AT-rich codons, which can present a metabolic burden that is not fully addressed by codon optimization, , and (3) glycosylation and other post-translational modifications required for function that are not easily replicated in model hosts . Consequently, developing genetic tools to manipulate AGF proteins in their native context will enable the discovery of novel anaerobic fungal genes and metabolic products of biotechnological importance.…”

Editorial for Special Issue “Unleashing the Hidden Potential of Anaerobic Fungi”

“…Finally, with respect to the lack of genetic tools, Hillman and his co-authors [ 11 ] identified the AT-richness of AF genomes being a major obstacle for the heterologous expression of AF pathways in model hosts. Their findings open new doors to the exploitation of the biosynthetic potential of AF via codon usage and optimization.…”