Screening and evolution of a novel protist xylose isomerase from the termite Reticulitermes speratus for efficient xylose fermentation in Saccharomyces cerevisiae

Risa Nagura,Nobuki Tada,Nobuhiko Muramoto,Kenro Tokuhiro,Satoshi Katahira,Toru Onishi,Moriya Ohkuma,Shigeharu Moriya,Noriko Yasutani

doi:10.1186/s13068-017-0890-1

Abstract

BackgroundThe yeast Saccharomyces cerevisiae, a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae, a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae, the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae. Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus.ResultsEight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes. The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans, similarly improved xylose utilization in S. cerevisiae.ConclusionsA novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the protists in the termite hindgut. Isolation of this XI gene and identification of the point mutation described in this study might contribute to improving the productivity of industrial bioethanol.

Highlights

Introduction of mutation in RsXIC1 and growth‐based screening of mutants To introduce random mutations in the xylose isomer‐ ase (XI) genes, an error-prone PCR was carried out with GeneMorph II kit (Agilent technologies, CA, USA) using pRS436GAP-RsXIC1O as the template and primer pair pRSSacII-AAA-ATG-F4 and pRSXhoI-TAA-R3; sequences of these primers are listed in Additional file 10: Table S4
Isolation of novel XI genes and their sequence analysis Novel XI genes were isolated from a cDNA library, which was constructed previously from the protists residing in the Reticulitermes speratus hindgut [31], using a PCR-based cloning method
The 5′- and 3′-regions of these XI gene-like fragments were amplified by PCR from the same cDNA library using primers that were designed based on the internal sequences of these XI gene-like fragments and the vector sequences flanking the 5′- and 3′-regions of the cDNA inserts

Summary

Introduction

C1 and growth‐based screening of mutants To introduce random mutations in the XI genes, an error-prone PCR was carried out with GeneMorph II kit (Agilent technologies, CA, USA) using pRS436GAP-RsXIC1O as the template and primer pair pRSSacII-AAA-ATG-F4 and pRSXhoI-TAA-R3; sequences of these primers are listed in Additional file 10: Table S4. The amplified DNA fragment was inserted into the PvuII-digested pRS316 (NBRP Accession Number: BYP562) to generate pRS316GAP (Additional file 11: Figure S7e) In this plasmid, the cloned genes are under the control of the TDH3 promoter and CYC1 terminator. For each XI gene, four different single amino acid substitution mutants were created; the respective asparagine residue in each XI was substituted with cysteine, threonine, valine, and alanine Corresponding plasmids harboring these substitution mutants are listed in Additional file 7: Table S3. Because of the differences in the cofactor preferences of NADPH-dependent XR and NAD+-dependent XDH, substantial amount of xylitol is accumulated, and only suboptimal yield of ethanol is obtained when a recombinant yeast harboring the XRXDH pathway is used for this purpose

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Conclusion