A Modular Vector Toolkit with a Tailored Set of Thermosensors To Regulate Gene Expression in <i>Thermus thermophilus</i>

Verdú, Carlos; Sánchez, Esther; Ortega, Carmen; Hidalgo, Aurélio; Berenguer, José; Mencı́a, Mario

doi:10.1021/acsomega.9b02107

Cited by 12 publications

(9 citation statements)

References 36 publications

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“…T. thermophilus is an excellent source of thermophilic enzymes (including thermostable polymerases, which are widely used in PCR applications), and has found applications in the thermic degradation of biomass. The synthetic biology toolset of T. thermophilus is also limited; however, research groups around the world have enriched the toolset with elements for insertions/deletions [46], a modular vector toolkit for gene expression-which includes promoters, thermosensors and origins of replication [47]-and thermostable CRISPR Cas9 systems that work at 65 • C [48,49].…”

Section: Description Organism Referencesmentioning

confidence: 99%

Synthetic Biology of Thermophiles: Taking Bioengineering to the Extremes?

Vavitsas¹,

Glekas²,

Hatzinikolaou³

2022

Applied Microbiology

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Synthetic biology applications rely on a well-characterized set of microbial strains, with an established toolbox of molecular biology methods for their genetic manipulation. Since there are no thermophiles with such attributes, most biotechnology and synthetic biology studies use organisms that grow in the mesophilic temperature range. As a result, thermophiles, a heterogenous group of microbes that thrive at high (>50 °C) temperatures, are largely overlooked, with respect to their biotechnological potential, even though they share several favorable traits. Thermophilic bacteria tend to grow at higher rates compared to their mesophilic counterparts, while their growth has lower cooling requirements and is less prone to contamination. Over the last few years, there has been renewed interest in developing tools and methods for thermophile bioengineering. In this perspective, we explain why it is a good idea to invest time and effort into developing a thermophilic synthetic biology direction, which is the state of the art, and why we think that the implementation of a thermophilic synthetic biology platform—a thermochassis—will take synthetic biology to the extremes.

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Section: Description Organism Referencesmentioning

confidence: 99%

Synthetic Biology of Thermophiles: Taking Bioengineering to the Extremes?

Vavitsas¹,

Glekas²,

Hatzinikolaou³

2022

Applied Microbiology

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“…Efficient pathways for translocation via the outer membrane are not known, although certain proteins that are exported to the periplasm diffuse or leak into the extracellular medium. Passive transport across the outer membrane can be stimulated by external or internal destabilization of the structural components of E. coli [ 111 , 112 , 113 ]. Destabilization is achieved either by the lysis of proteins functioning at the interior of the cell using strains lacking structural membrane components or permeabilization directed from the cell exterior either mechanically, enzymatic, or chemically.…”

Section: Location Of Expressionmentioning

confidence: 99%

“…Co-expression of molecular chaperones may be advantageous for increasing the solubility and folding of proteins. While chaperone co-expression increases the development of many monomeric and multimeric proteins, the effectiveness of this strategy seems to be protein specific [ 111 , 128 , 129 ]. Various factors lead to the failure of overexpressed proteins to fold into their original form, including the presence of molecular chaperones, lack of disulfide links, and/or post-translational modifications.…”

Section: Co-expression Of Chaperonesmentioning

confidence: 99%

“…In E. coli , the following two mechanisms lead to the breakdown of disulfide bonds: Thioredoxin mechanism involving trxB and the glutaredoxin network containing glutathione reductase, glutathione, and three glutaredoxins [ 131 , 132 ]. The use of E. coli strains that are deficient involves techniques to generate a less reductive cytoplasmic environment that promotes the formation of disulfide bonds with trxB, which contributes to sulfhydryl reduction [ 111 , 133 , 134 ]. Finally, purifying target proteins from the cytoplasmic protein pool is a relatively challenging task, as this compartment comprises the vast majority of the total cell protein.…”

Section: Co-expression Of Chaperonesmentioning

confidence: 99%

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Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Lee

Park

2020

Antibiotics

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Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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“…In addition of being used as frequent sources of enzymes of biotechnological interest, few isolates of the genus Thermus have been employed as study model according to their easy adaptation to fast growth under laboratory conditions, both in complex and mineral media, and their highly efficient natural competence system [21], which has allowed the development of quite a complete set of genetic tools [20,[22][23][24][25].…”

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confidence: 99%

Nitrate Respiration in Thermus Thermophilus NAR1: From Horizontal Gene Transfer to Internal Evolution

Sánchez-Costa

Blesa

Berenguer

2020

Preprint

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Genes coding for enzymes of the denitrification pathway appear randomly distributed among isolates of the ancestral genus Thermus, but only in few strains of the species T. thermophilus the pathway has been studied to a certain detail. Here, we review the enzymes involved in this pathway present in T. thermophilus NAR1, a strain extensively employed as a model for nitrate respiration, on the light of its full sequence recently assembled through a combination of PacBio and Illumina technologies in order to counteract the systematic errors introduced by the former technique. The genome of this strain is divided in four replicons, a chromosome of 2,021,843 pb, two megaplasmids of 370,865 and 77,135 bp and a small plasmid of 9,799 pb. Nitrate respiration is encoded in the largest megaplasmid, pTTHNP4, within a region that includes operons for O2 and nitrate sensory systems, a nitrate reductase, nitrate and nitrite transporters and a nitrate specific NADH dehydrogenase, in addition to multiple insertion sequences (IS), suggesting its mobility-prone nature. Despite nitrite is the final product of nitrate respiration in this strain, the megaplasmid encodes two putative nitrite reductases of the cd1 and Cu-containing types, apparently inactivated by IS. No nitric oxide reductase genes have been found within this region, although the NorR sensory gene, needed for its expression, is found near the inactive nitrite respiration system. These data clearly support that partial denitrification in this strain is the consequence of recent deletions and IS insertions in genes involved in nitrite respiration. Based on these data, the capability of this strain to transfer or acquire denitrification clusters by horizontal gene transfer is discussed.

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A Modular Vector Toolkit with a Tailored Set of Thermosensors To Regulate Gene Expression in Thermus thermophilus

Cited by 12 publications

References 36 publications

Synthetic Biology of Thermophiles: Taking Bioengineering to the Extremes?

Synthetic Biology of Thermophiles: Taking Bioengineering to the Extremes?

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

Nitrate Respiration in Thermus Thermophilus NAR1: From Horizontal Gene Transfer to Internal Evolution

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