Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production

Mehwish Aslam; Ayumi Horiuchi; Jan-Robert Simons; Savyasachee Jha; Masahiro Yamada; Toru Odani; Rikako Fujimoto; Yasuyuki Yamamoto; Ryoma Gunji; Tadayuki Imanaka; Tamotsu Kanai; Haruyuki Atomi

doi:10.1128/AEM.00280-17

Engineering of the Hyperthermophilic Archaeon Thermococcus kodakarensis for Chitin-Dependent Hydrogen Production

Appl Environ Microbiol. 2017 Jul 17;83(15):e00280-17. doi: 10.1128/AEM.00280-17. Print 2017 Aug 1.

Authors

Mehwish Aslam¹, Ayumi Horiuchi¹, Jan-Robert Simons^{1

2}, Savyasachee Jha^{1

2}, Masahiro Yamada¹, Toru Odani¹, Rikako Fujimoto¹, Yasuyuki Yamamoto¹, Ryoma Gunji¹, Tadayuki Imanaka^{2

3}, Tamotsu Kanai^{1

2}, Haruyuki Atomi^{4

2}

Affiliations

¹ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan.
² JST, CREST, Tokyo, Japan.
³ Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Japan.
⁴ Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan atomi@sbchem.kyoto-u.ac.jp.

Abstract

Thermococcus kodakarensis is a hyperthermophilic archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N-,N'-diacetylchitobiose in the medium. To enhance N-,N'-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-β-d-glucosaminidase, and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (wt/vol) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and, moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has acquired the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation.IMPORTANCE Chitin is a linear homopolymer of β-1,4-linked N-acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the hyperthermophilic archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit of sugar consumed was slightly higher with swollen chitin than with starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.

Keywords: Archaea; Thermococcus; Thermococcus kodakarensis; archaea; chitin; chitinase; chitinases; hyperthermophile; hyperthermophiles.