Development of a GIN11/FRT-based multiple-gene integration technique affording inhibitor-tolerant, hemicellulolytic, xylose-utilizing abilities to industrial Saccharomyces cerevisiae strains for ethanol production from undetoxified lignocellulosic hemicelluloses

Abstract

Background: Bioethanol produced by the yeast Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels. Lignocellulosic hemicelluloses obtained after hydrothermal pretreatment are important feedstock for bioethanol production. However, hemicellulosic materials cannot be directly fermented by yeast: xylan backbone of hemicelluloses must first be hydrolyzed by heterologous hemicellulases to release xylose, and the yeast must then ferment xylose in the presence of fermentation inhibitors generated during the pretreatment. Results: A GIN11/FRT-based multiple-gene integration system was developed for introducing multiple functions into the recombinant S. cerevisiae strains engineered with the xylose metabolic pathway. Antibiotic markers were efficiently recycled by a novel counter selection strategy using galactose-induced expression of both FLP recombinase gene and GIN11 flanked by FLP recombinase recognition target (FRT) sequences. Nine genes were functionally expressed in an industrial diploid strain of S. cerevisiae: endoxylanase gene from Trichoderma reesei, xylosidase gene from Aspergillus oryzae, β-glucosidase gene from Aspergillus aculeatus, xylose reductase and xylitol dehydrogenase genes from Scheffersomyces stipitis, and XKS1, TAL1, FDH1 and ADH1 variant from S. cerevisiae. The genes were introduced using the homozygous integration system and afforded hemicellulolytic, xylose-assimilating and inhibitor-tolerant abilities to the strain. The engineered yeast strain demonstrated 2.7-fold higher ethanol titer from hemicellulosic material than a xylose-assimilating yeast strain. Furthermore, hemicellulolytic enzymes displayed on the yeast cell surface hydrolyzed hemicelluloses that were not hydrolyzed by a commercial enzyme, leading to increased sugar utilization for improved ethanol production.

Yeast genetic interaction screens in the age of CRISPR/Cas

“…Efforts to do so are in their infancy (Haber et al 2013; Kuzmin et al 2018), largely because performing such systematic screens is technically demanding with current methods. So far, the multiplexing capabilities of CRISPR/Cas have been exploited mostly by metabolic engineers and synthetic biologists constructing complex genetic circuits and biosynthetic pathways in a handful of strains (Shao et al 2009; Mikkelsen et al 2012; Hasunuma et al 2014; Ryan et al 2014; Stovicek et al 2015; Tsai et al 2015; Jakočiunas et al 2015b; Ronda et al 2015; Walter et al 2016; Jessop-Fabre et al 2016; Shi et al 2016; Garst et al 2017; Kuivanen et al 2018). However, geneticists could also take advantage of the multiplexing capabilities of CRISPR/Cas to perform systematic GI screens to generate large numbers of strains carrying three or more alleles.…”

Accumulation of sugar from pulp and xylitol from xylose by pyruvate decarboxylase-negative white-rot fungus Phlebia sp. MG-60

“…Fermentation of lignocellulose is an effective method to convert non-edible materials to low-molecular-weight compounds that can be utilized as biofuels as well as raw materials. A number of researchers have investigated metabolically-engineered bacteria or yeasts that can ferment lignocellulose or xylose, a typical hemicellulose in lignocellulosic biomass (Hasunuma et al, 2014;Kim et al, 2014;Smith et al, 2014;Wang et al, 2013). However, almost of all these genetically-engineered microorganisms could barely exploit lignocellulosic biomass without additional enzymes or pretreatments for lignin degradation.…”

Rational design and evolutional fine tuning of Saccharomyces cerevisiae for biomass breakdown

“…To overcome these limitations, many researchers have attempted to develop recombinant microbial strains capable of hydrolyzing lignocellulose to glucose [8,14,15]. Heterologous expression of genes encoding cellulases and hemicellulases in recombinant strains can promote biomass utilization, which simplifies the biorefinery process by integrating biological processes (enzyme production, saccharification, and fermentation).…”

“…Lignocellulose is primarily composed of cellulose, hemicelluloses, and lignin. Cellulose forms highly crystalline microfibrils consisting of homopolysaccharides of b-1,4-linked glucose units embedded in hemicelluloses (heterologous polysaccharides including heteroxylans, xyloglucan, heteromannanns, and mixed-linkage glucans) and lignin (amorphous polymers formed by polymerization of aromatic alcohols, p-coumaryl, coniferyl, and synapyl alcohols) [6][7][8]. To utilize lignocellulose as a biorefinery feedstock, the structure must be swollen by chemical and physico-chemical pretreatment to increase its accessibility to cellulolytic enzymes for generating fermentable sugars [5].…”