An overview of 25 years of research on Thermococcus kodakarensis, a genetically versatile model organism for archaeal research

Rashid, Naeem; Aslam, Mehwish

doi:10.1007/s12223-019-00730-2

Cited by 6 publications

(5 citation statements)

References 147 publications

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“…This has been supported by previous studies 39 . For example, Kropp et al reported that the structure of KOD pol has a long crack with an electro-positive potential extending from the DNA binding region at the thumb domain upwards along the β-hairpin and the palm domain to the N-terminal domain 11 . The increased positive charge introduced in the DNA binding region of the polymerase results in enhanced affinity towards the template DNA, leading to a more stable binding 53 .…”

Section: Resultsmentioning

confidence: 99%

“…KOD DNA polymerase (KOD pol) is an important representative of B family DNA polymerases 24,25 . Since the discovery and isolation of strain KOD1 by Morikawa et al in 1994 26 , an abundant number of related strains and their enzyme products were examined and reported from this archaeon 27,28 . Recombinantly produced KOD DNA polymerases are the key component of numerous biotechnological applications such as PCR 29 , RT-qPCR 30 , multiplex PCR 31 , incorporation of base/sugar modified nucleoside triphosphates 32 , and TNA synthesis 33,34,35 etc.…”

Section: Introductionmentioning

confidence: 99%

“…Challenges in using SBS with cleavable fluorescent nucleotide reversible terminators involve further improving the DNA polymerase that efficiently recognizes and incorporates the modified nucleotides 9 . Archaeal B-family DNA polymerases are widely engineered to be applied in catalyzing the addition of chemically modified nucleotide substrates in SBS sequencing, because of their potential for tunability of the active site and acceptance of broad substrate 11,12,13 . The engineering of B-family DNA polymerases relies on the development of various enzyme evolution techniques 14 .…”

Section: Introductionmentioning

confidence: 99%

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Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Zhai

Wang²,

Liu³

et al. 2023

Preprint

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Engineering B family DNA polymerases to utilize artificial nucleotides with unique large group labeling is limited by a general lack of understanding about the mechanism of the structural determinants that define substrate specificity. In this study, we report the engineering process of a natural DNA polymerase (KOD pol) from Thermococcus Kodakaraenis by using semi-rational design strategies. The essential residues around the active pocket and DNA binding site of KOD pol are characterized by using an established high-throughput microwell-based screening method. We obtained a five-mutant variant by performing site-directed saturation mutagenesis and combinatorial mutations of motif A and position 485, including two previously reported mutation sites (141/143) with inactive exonuclease activity. Then we selected more mutants, which were expected to increase the catalytic activity based on computational simulations, to perform experimental verification. By stepwise combinatorial mutation, we obtained an eleven-mutation variant E10 with over 25 times improvement of kinetic efficiency which has similar and satisfactory performances on both the BGI NGS platform (SE50) and MGI CoolMPSTM platform (PE100). The results indicated that E10 is a potential candidate for commercialization. The beneficial mutation sites identified in this study might be shared with other archaeal B-family DNA polymerases, which can provide a guide for the mutagenesis of other types of enzymes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Zhai

Wang²,

Liu³

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

Section: Introductionmentioning

confidence: 99%

“…Thermococcales are hyperthermophilic motile cocci that typically grow in media containing peptides, chitin, starch, or pyruvate, using sulfur or hydrogen as terminal electron acceptors ( Rashid and Aslam, 2020 ). Termococcus kodakarensis and Pyrococcus furiosus are most commonly used as models ( Leigh et al, 2011 ; Rashid and Aslam, 2020 ). These organisms are known to form contacts between cells of the same or even different species through their archaeallum filaments ( Nather et al, 2006 ; Schopf et al, 2008 ).…”

Section: Introductionmentioning

confidence: 99%

A Unique Gene Module in Thermococcales Archaea Centered on a Hypervariable Protein Containing Immunoglobulin Domains

et al. 2021

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Molecular mechanisms involved in biological conflicts and self vs nonself recognition in archaea remain poorly characterized. We apply phylogenomic analysis to identify a hypervariable gene module that is widespread among Thermococcales. These loci consist of an upstream gene coding for a large protein containing several immunoglobulin (Ig) domains and unique combinations of downstream genes, some of which also contain Ig domains. In the large Ig domain containing protein, the C-terminal Ig domain sequence is hypervariable, apparently, as a result of recombination between genes from different Thermococcales. To reflect the hypervariability, we denote this gene module VARTIG (VARiable Thermococcales IG). The overall organization of the VARTIG modules is similar to the organization of Polymorphic Toxin Systems (PTS). Archaeal genomes outside Thermococcales encode a variety of Ig domain proteins, but no counterparts to VARTIG and no Ig domains with comparable levels of variability. The specific functions of VARTIG remain unknown but the identified features of this system imply three testable hypotheses: (i) involvement in inter-microbial conflicts analogous to PTS, (ii) role in innate immunity analogous to the vertebrate complement system, and (iii) function in self vs nonself discrimination analogous to the vertebrate Major Histocompatibility Complex. The latter two hypotheses seem to be of particular interest given the apparent analogy to the vertebrate immunity.

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Biofuel Synthesis by Extremophilic Microorganisms

Mukhtar

Aslam

2020

Biofuel and Biorefinery Technologies

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An overview of 25 years of research on Thermococcus kodakarensis, a genetically versatile model organism for archaeal research

Cited by 6 publications

References 147 publications

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

A Unique Gene Module in Thermococcales Archaea Centered on a Hypervariable Protein Containing Immunoglobulin Domains

Biofuel Synthesis by Extremophilic Microorganisms

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