Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

Abstract

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.

Introduction

Fuel ethanol production from biomass at the industrial level using Saccharomyces cerevisiae shows great promise for satisfying future energy demands, but the limited range of materials that can be fermented remains an obstacle to cost-effective bioethanol production (Farrell et al., 2006, Saha, 2003). Although several genetically engineered strains of S. cerevisiae have been developed that will ferment xylose to ethanol (Karhumaa et al., 2007, Sedlak and Ho, 2004, Wisselink et al., 2007), further optimization is needed. It will require the simultaneous expression, at sufficiently high level, of all the enzymes and proteins needed to allow industrial yeast strains to grow efficiently on pentose as well as hexose sugars anaerobically. In addition, for cost-effective industrial ethanol production from biomass it will be necessary to express the enzymes required to saccharify the lignocellulosic feedstocks that are the source of hexose and pentose sugars. Genes considered necessary for complete fermentation of xylose and arabinose, the two major pentose sugar constituents of lignocellulosic biomass, include those encoding xylose isomerase (XI), xylulokinase (XKS), arabinose A, arabinose B, and arabinose D (Karhumaa et al., 2007, Wisselink et al., 2007), which may be obtained from a microorganism naturally capable of fermenting these sugars. Saccharification of lignocellulosic feedstocks also requires utilization of hydrolytic enzymes including cellulases and hemicellulases after initial chemical pre-treatment (Rudolf et al., 2007, Saha et al., 2005). The cost-effectiveness of the fuel ethanol fermentation process could be further enhanced by obtaining high-value co-products and by-products from the process, such as monomers for polymer production and commercially important proteins and peptides. Genes for these proteins and peptides can be mutagenized, placed in an expression system capable of producing high levels of functional proteins or peptides, and screened in high throughput to optimize desired characteristics. An integrated automated platform is available to carry out all the processes for gene assembly, plasmid library production, expression of proteins, enzymes, and value-added proteins or peptides, and growth testing of the transformed yeast strains (Hughes et al., 2005, Hughes et al., 2006, Hughes et al., 2007). In addition, a system to express numerous genes simultaneously is essential. A yeast plasmid system was designed to accomplish this using three small ubiquitin-like modifier (SUMO) yeast expression vectors (Butt et al., 2005, Malakhov et al., 2004). We integrated this three-plasmid vector system into the automated protocols on the plasmid-based robotic workcell to engineer and screen improved ethanologenic yeast strains expressing (1) xylose isomerase, (2) a library of mutagenized peptides that are putative bioinsecticides, and (3) an enzyme of the pentose phosphate pathway to enhance xylose metabolism. This set of plasmids used on the automated platform offers the possibility of expressing pentose-utilization enzymes and commercially important peptides in yeast, and screening the resulting yeast strains in high throughput for those that grow rapidly anaerobically and produce ethanol at sufficiently high levels for industrial application.