Molecular Cloning and Overexpression of an Endo-β-1,4-xylanase Gene from Aspergillus niger in Industrial Saccharomyces cerevisiae YS2 Strain

Abstract

The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to overexpress the xylanase by constructing a homology-driven integration vector. The total mRNA from a xylanase-producing strain of Aspergillus niger IME-216 was extracted and used as the template for the production of endo-β-1,4-xylanase cDNA by reverse transcription. The fusion fragment containing the phosphoglycerate kinase promoter, α-factor signal peptide, xylanase gene encoding the mature peptide, and CYC1 terminator was first generated by overlap extension polymerase chain reaction. Then, the vector pUPX was constructed by inserting the fusion fragment into the S. cerevisiae plasmid pUG6. Then, A 2.2-kb rDNA sequence was further cloned and attached to the SalI-digested pUPX to obtain the integration plasmid pUPXR. The pUPXR was linearized by KpnI, transformed into the industrial strain S. cerevisiae YS2 using the lithium acetate method and integrated into the S. cerevisiae chromosome. The maximum yield of the recombinant xylanase produced by the engineered S. cerevisiae strain YS2_2 was 74.8 U per microliter, which was about 1.5-fold higher than the original 50 U per microliter by Aspergillus niger IME-216 strain under the flask culture at 28 °C for 72 h. The findings of our study can be used for further development of industrial S. cerevisiae strain for producing interested enzymes, or improving the achievement of metabolism, for example, simultaneous fermentation of glucose and xylose to producing bioethanol.

High-level expression of improved thermo-stable alkaline xylanase variant in Pichia Pastoris through codon optimization, multiple gene insertion and high-density fermentation

“…Several studies have reported the role of different host strains produce high level of exogenous xylanase, like Bacillus , Trichoderma reesei , Pichia pastoris , Saccharomyces cerevisiae and Escherichia coli 2324252627. But, P. pastoris is known for its properties of efficient enzyme secretion, cellulase free, and fast growth with high cell density in simple media28.…”

Application of the Cre/loxP Site-Specific Recombination System for Gene Transformation in Aurantiochytrium limacinum

“…Finally, the candidate recombinant was sub-cultured in medium without antibiotics for 10 generations during which plasmid pSH65 could be lost [17]. The cultures were spread on two solid media with and without zeocin, respectively.…”

Trends in recombinant protein use in animal production

“…S38 [ 131 ], among others [ 126 ]. Albeit at a lesser extent, S. cerevisiae has also been studied for the production of fungal xylanases [ 112 , 132 , 133 ]. Different enzymes in different yeast-based cell factories have been evaluated under diverse production conditions aiming to optimize enzyme production yields [ 133 ].…”