High copy and stable expression of the xylanase XynHB in Saccharomyces cerevisiae by rDNA-mediated integration

Cheng Fang,Yanhe Ma,Yuling Zhou,Lixin Ma,Qinhong Wang,Guimin Zhang,Jonathan Nimal Selvaraj

doi:10.1038/s41598-017-08647-x

Cheng Fang, Yanhe Ma + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-08647-x

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Abstract

Xylanase is a widely-used additive in baking industry for enhancing dough and bread quality. Several xylanases used in baking industry were expressed in different systems, but their expression in antibiotic free vector system is highly essential and safe. In the present study, an alternative rDNA-mediated technology was developed to increase the copy number of target gene by integrating it into Saccharomyces cerevisiae genome. A xylanase-encoding gene xynHB from Bacillus sp. was cloned into pHBM367H and integrated into S. cerevisiae genome through rDNA-mediated recombination. Exogenous XynHB expressed by recombinant S. cerevisiae strain A13 exhibited higher degradation activity towards xylan than other transformants. The real-time PCR analysis on A13 genome revealed the presence of 13.64 copies of xynHB gene. Though no antibiotics have been used, the genetic stability and the xylanase activity of xynHB remained stable up to 1,011 generations of cultivation. S. cerevisiae strain A13 expressing xylanase reduced the required kneading time and increased the height and diameter of the dough size, which would be safe and effective in baking industry as no antibiotics-resistance risk. The new effective rDNA-mediated technology without using antibiotics here provides a way to clone other food related industrial enzymes for applications.

Highlights

Chemical food additives were traditionally used to enlarge loaf volume, increase shelf life and improve the flavor of bread
Integration of exogenous genes into the S. cerevisiae genome through rDNA-mediated homologous recombination can lead to the generation of transformants containing different copy numbers of target gene
Higher copies of target gene in the genome will lead to higher enzyme expression, which was demonstrated by the size of the halos on the SC-Ura plates containing Remazol Brilliant Blue (RBB)-xylan

Summary

Introduction

Chemical food additives were traditionally used to enlarge loaf volume, increase shelf life and improve the flavor of bread. De Wijs et al.[20] reported that the stability of exogenous genes mainly depends on the size of integrated fragment, suggesting that if integrated plasmid is smaller than the rDNA fragment (9.1 kb), it will enhance the gene stability Based on this concept, a new rDNA-mediated vector (pHBM367H) was constructed and used to express a bacterial-origin xylanase gene xynHB in S. cerevisiae. This step shortened the integrated DNA fragment to be shorter than 9.1 kb and prevented any antibiotic resistance genes into the host Using this strategy, selecting the S. cerevisiae to express effective xylanase with high copy numbers was the primary objective of our study. The expressed exogenous xylanase was further tested for their effectiveness in bread production

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