Identification and Deletion of The Genes Responsible for Hydrogen Production in Thermoanaerobacter Ethanolicus JW200

Background Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. Due to the coupling of hydrogenase activity with fermentation product distribution, understanding hydrogen production of T. ethanolicus, particularly the genes responsible, is valuable for metabolic engineering of the species. Results Utilizing the hydrogenases reported in Thermoanaerobacterium saccharolyticum and Pyrococcus furiosus as templates, BLAST search identified five hydrogenase gene clusters, including two membrane-bound [NiFe] hydrogenases ech and mbh, two cytoplasmic [FeFe] hydrogenases hyd and hydII, and one cytoplasmic [NiFe] hydrogenase shi. The combined deletion of ech, mbh, shi and hydG resulted in a strain that did not produce hydrogen and showed no methyl viologen hydrogenase activity in cell extracts. Strains with deletions of all the hydrogenases except one showed normal hydrogen production. Methyl viologen hydrogenase activity was greatly reduced in all combined deletion strains except the strain with an intact hydG gene. Conclusion High hydrogen production and hydrogenase activities have been observed for T. ethanolicus. Five hydrogenases have been identified. Hydrogen production was eliminated by deleting genes required for all five hydrogenases. Each individual hydrogenase was verified to be capable of producing hydrogen during fermentation, indicating a high degree of redundancy and flexibility in the hydrogenase systems of T. ethanolicus. A large portion of hydrogenase activity is encoded by the [Fe-Fe] hydrogenases.

Genome Editing of the Anaerobic Thermophile Thermoanaerobacter ethanolicus Using Thermostable Cas9

ABSTRACT Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen, and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to the utilization of both pentose and hexose. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9-based system for genome editing of T. ethanolicus. As a proof of principle, three genes, including the thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE), and redox sensing protein gene (rsp), were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knockout and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome-editing efficiency was observed. Furthermore, our in vivo results revealed that redox sensing protein (RSP) plays a more important role in regulation of energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us with an effective strategy to identify genes involved in biosynthesis and energy metabolism. IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. On the basis of the biochemical experiments of previous investigation, the formation of ethanol was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus. Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here, we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus. The thermostable Cas9-based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system represents an important expansion of the genetic tool box of anaerobic thermophile T. ethanolicus strains.

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200

Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp.

Dual enzymatic dynamic kinetic resolution by Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase and Candida antarctica lipase B

The immobilization of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) using sol–gel method enables its use to racemize enantiopure alcohols in organic media, thus allows for a dual enzymatic dynamic kinetic resolution.