BackgroundWood-feeding termite, Coptotermes formosanus Shiraki, represents a highly efficient system for biomass deconstruction and utilization. However, the detailed mechanisms of lignin modification and carbohydrate degradation in this system are still largely elusive.ResultsIn order to reveal the inherent mechanisms for efficient biomass degradation, four different organs (salivary glands, foregut, midgut, and hindgut) within a complete digestive system of a lower termite, C. formosanus, were dissected and collected. Comparative transcriptomics was carried out to analyze these organs using high-throughput RNA sequencing. A total of 71,117 unigenes were successfully assembled, and the comparative transcriptome analyses revealed significant differential distributions of GH (glycosyl hydrolase) genes and auxiliary redox enzyme genes in different digestive organs. Among the GH genes in the salivary glands, the most abundant were GH9, GH22, and GH1 genes. The corresponding enzymes may have secreted into the foregut and midgut to initiate the hydrolysis of biomass and to achieve a lignin-carbohydrate co-deconstruction system. As the most diverse GH families, GH7 and GH5 were primarily identified from the symbiotic protists in the hindgut. These enzymes could play a synergistic role with the endogenous enzymes from the host termite for biomass degradation. Moreover, twelve out of fourteen genes coding auxiliary redox enzymes from the host termite origin were induced by the feeding of lignin-rich diets. This indicated that these genes may be involved in lignin component deconstruction with its redox network during biomass pretreatment.ConclusionThese findings demonstrate that the termite digestive system synergized the hydrolysis and redox reactions in a programmatic process, through different parts of its gut system, to achieve a maximized utilization of carbohydrates. The detailed unique mechanisms identified from the termite digestive system may provide new insights for advanced design of future biorefinery.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1015-1) contains supplementary material, which is available to authorized users.
According to the amino acid sequence, a codonoptimized xylanase gene (xynA1) from Thermomyces lanuginosus DSM 5826 was synthesized to construct the expression vector pHsh-xynA1. After optimization of the mRNA secondary structure in the translational initiation region of pHsh-xynA1, free energy of the 70 nt was changed from -6.56 to -4.96 cal/mol, and the spacing between AUG and the Shine-Dalgarno sequence was decreased from 15 to 8 nt. The expression level was increased from 1.3 to 13% of total cell protein. A maximum xylanase activity of 47.1 U/ mL was obtained from cellular extract. The recombinant enzyme was purified 21.5-fold from the cellular extract of Escherichia coli by heat treatment, DEAE-Sepharose FF column and t-Butyl-HIC column. The optimal temperature and pH were 65°C and pH 6.0, respectively. The purified enzyme was stable for 30 min over the pH range of 5.0-8.0 at 60°C, and had a half-life of 3 h at 65°C.
The development of new procedures and protocols that allow researchers to obtain
recombinant proteins is of fundamental importance in the biotechnology field. A
strategy was explored to overcome inclusion-body formation observed when
expressing an aggregation-prone fungal xylanase in Escherichia
coli. pHsh is an expression plasmid that uses a synthetic
heat-shock (Hsh) promoter, in which gene expression is regulated by an
alternative sigma factor (σ32). A derivative of pHsh was
constructed by fusing a signal peptide to xynA2 gene to
facilitate export of the recombinant protein to the periplasm. The xylanase was
produced in a soluble form. Three factors were essential to achieving such
soluble expression of the xylanase: 1) the target gene was under the control of
the Hsh promoter, 2) the gene product was exported into the periplasm, and 3)
gene expression was induced by a temperature upshift. For the first time we
report the expression of periplasmic proteins under the control of an Hsh
promoter regulated by σ32. One unique feature of this approach
was that over 200 copies of the Hsh promoter in an E. coli cell
significantly increased the concentration of σ32. The growth
inhibition of the recombinant cells corresponded to an increase in the levels of
soluble periplasmic protein. Therefore, an alternative protocol was designed to
induce gene expression from pHsh-ex to obtain high levels of active soluble
enzymes.
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