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

Abstract

Background:The 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. Results:Eight 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. Conclusions:A 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.

A Bibliometric Study of Scientific Publications regarding Hemicellulose Valorization during the 2000–2016 Period: Identification of Alternatives and Hot Topics

“…The most practical approach proposes the introduction of the pentose-utilizing capability into efficient ethanol producers such as S. cerevisiae and Z. mobilis [135,188,189]. The introduction of xylose isomerases that function well in other xylose-fermenting yeasts is the preferred solution to overcome the limitations caused by xylose to these host organisms [190,191].…”

Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

“…The corresponding positions in PirXI are distant from the active site and the mutations were not examined here. We constructed the two earlier PirXI mutants [9,14], E15D-T142S and N338C, and confirmed that the variants are beneficial for PirXI-mediated growth of strain DS75543 on d-xylose as well (Fig. 10).…”

“…However, their properties exhibited disconnection between in vivo performance and in vitro catalytic properties. We further explored the ambiguous relation between in vivo and in vitro enzyme properties by studying PirXI mutants discovered independently in previous studies, using different S. cerevisiae host strains [9,14]. [11].…”

“…formation of side products, but combining the pathways may also have certain benefits [5]. Several efforts to find suitable xylose isomerases have been reported [6][7][8][9][10][11]. A xylose isomerase discovered by Kuyper et al [12,13] in the fungal strain Piromyces E2 through genome mining (PirXI) is an attractive candidate for xylose isomerization in engineered S. cerevisiae strains, and is used in several studies [14].…”

“…This approach does not require structural information. Here, one can take advantage of the fact that xylose isomerase activities limit the growth of S. cerevisiae on d-xylose and employ a random mutagenesis method with in vivo selection for improved growth [9,14]. This allowed the discovery of unexpected xylose isomerase mutations, some of which were far away from the active site.…”