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Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production.

ABSTRACT: Hydrogen (H?) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H? and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H? production for reductant disposal. To explore the reductant flux and possible competition for H? production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H? production and consumption. The results obtained establish that H? is generated predominantly by the membrane-bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H? consumption results in a substantial net increase in H? production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (?TK1086) to grow in the absence of S° and stimulated H? production, revealing a second mechanism to increase H? production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild-type growth and H? production. With S° present, they grew slower but synthesized more H? per unit biomass than T. kodakarensis TS517.

SUBMITTER: Santangelo TJ

PROVIDER: S-EPMC3179246 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production.

Santangelo Thomas J TJ Cuboňová L'ubomíra L Reeve John N JN

Molecular microbiology 20110713 4

Hydrogen (H₂) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H₂ and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H₂ production for reductant disposal. To explore the reductant flux and possible competition for H₂ production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the ...[more]

PMID: 21749486

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Project description: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-?-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 KC04?t. In both KC04 and KC04?t strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04?t was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04?tM1) 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 KC04?tM1 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 ?-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.

Dataset Information

Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production.

Publications

Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production.

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