Structural insight into Pichia pastoris fatty acid synthase

Joseph S Snowden,Lee Sherry,Jehad Alzahrani,Nicola J Stonehouse,Martin Stacey,David J Rowlands,Neil A Ranson

doi:10.1038/s41598-021-89196-2

Joseph S Snowden, Lee Sherry + Show 5 more

Open Access

https://doi.org/10.1038/s41598-021-89196-2

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Abstract

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. Serendipitously, we identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production. From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. While the overall organisation of the complex was typical of type I FASs, we identified several differences in both structural and enzymatic domains through comparison with the prototypical yeast FAS from S. cerevisiae. Using focussed classification, we were also able to resolve and model the mobile acyl-carrier protein (ACP) domain, which is key for function. Ultimately, the structure reported here will be a useful resource for further efforts to engineer yeast FAS for synthesis of alternate products.

Highlights

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products
Further inspection of individual micrographs revealed that FAS particles were well-separated from virus-like particles (VLPs), indicating that FAS was not co-purified as a result of an association with the VLPs (Fig. 1a)
While some particles were discarded after the initial 2D classification, ~ 67% of these were VLPs or areas of carbon film that were inadvertently selected during automated particle picking, and the remaining discarded classes mainly comprised apparently intact FAS particles over thick carbon

Summary

Introduction

Type I fatty acid synthases (FASs) are critical metabolic enzymes which are common targets for bioengineering in the production of biofuels and other products. We identified FAS as a contaminant in a cryoEM dataset of virus-like particles (VLPs) purified from P. pastoris, an important model organism and common expression system used in protein production From these data, we determined the structure of P. pastoris FAS to 3.1 Å resolution. No structures are available for Pichia pastoris FAS from either cryoEM or X-ray crystallography This represents an important gap in our knowledge, because P. pastoris (Komagataella spp.) is a methylotrophic yeast which is used extensively for recombinant protein p roduction[23]. P. pastoris has a major advantage over other yeast strains like S. cerevisiae, as it produces relatively

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