Production of the Extremolyte Cyclic 2,3-Diphosphoglycerate Using Thermus thermophilus as a Whole-Cell Factory

Abstract

Osmolytes protect microbial cells against temperature, osmolarity and other stresses. The osmolyte cyclic 2,3-diphosphoglycerate, originally isolated from the thermophilic archaeon Methanothermus fervidus, naturally protects cellular proteins under extreme conditions. The biosynthetic pathway for cyclic 2,3-diphosphoglycerate has been introduced into the thermophilic bacterium Thermus thermophilus. The two enzymes in this synthetic pathway, 2-phosphoglycerate kinase and cyclic diphosphoglycerate synthetase, were incorporated into a newly designed modular BioBricks vector. The expression of this two-enzyme cascade resulted in the whole cell production of cyclic 2,3 diphosphoglycerate. In vivo production of cyclic 2,3-diphosphoglycerate was confirmed by mass spectrometry to a concentration up to 650 µM. This study demonstrates the feasibility of using this well studied thermophilic bacterium as a host in a whole-cell factory approach to produce cyclic 2,3 diphosphoglycerate. This raises the potential for commercialisation of cDPG for cosmetic and healthcare applications. Our work demonstrates the application of Thermus thermophilus as an alternative host for other high value small organic molecules of industrial interest.

Extremophiles in a changing world

“…(Lipscomb et al 2011 ; Sato et al 2005 ; Thiel et al 2014 ; Birien et al 2018 ) and Sulfolobus (Wagner et al 2012 ; Quehenberger et al 2017 ) as well as halophilic (Peck et al 2000 ; Bitan-Banin et al 2003 ; Allers et al 2004 ) and methanogenic (Susanti et al 2019 ) archaea. Within the extremophilic bacteria, it is now possible to over-produce oxygen-sensitive proteins; e.g ., in the anaerobe Thermoanaerobacter kivui (Basen et al 2018 ; Katsyv et al 2021a ), and a two-enzyme cascade from Methanothermus fervidus has been used successfully to produce the extremolyte cyclic di-phosphoglycerate, a small molecule that can stabilize proteins and DNA and has commercial application in the healthcare and cosmetic industries (de Rose et al 2021b ).…”

Teaching old dogs new tricks: genetic engineering methanogens

“…Although relatively few BioBrick-compatible parts have been developed for methanogens to date, some BioBrick-compatible shuttle vectors are available for M. maripaludis ( 84 – 86 ). Another BioBrick-compatible shuttle vector was developed in a study that evaluated the effect of high temperature and osmolarity conditions on Methanothermus fervidus ( 87 ). Further development of BioBrick and other standard genetic elements for methanogens will accelerate the pace of genetic discovery and innovation.…”

Structural characterization of a novel cyclic 2,3-diphosphoglycerate synthetase involved in extremolyte production in the archaeon Methanothermus fervidus

“…10.3389/fmicb.2023.1267570 Frontiers in Microbiology 08 frontiersin.org for proteins (Hensel and 1993;Borges et al, 2002) and DNA (Lentzen and Schwarz, 2006) has industrial applications for a variety of high value cosmetic and healthcare products. The biosynthesis of cDPG has already been demonstrated in a cascade reaction using the two enzymes 2PGK and cDPG both in vitro or in vivo (De Rose et al, 2021). The optimisation of these methodologies will allow the scale-up of the production of this extremolyte for new biotechnological applications.…”

“…In a recent study, we have established a process to produce cDPG using the thermophilic bacterium Thermus thermophilus as a whole-cell factory (De Rose et al, 2021). A protein BLAST (Altschul et al, 1990) search of the cDPGS and 2PGK sequences against the Protein Data Bank (PDB) revealed that there are no other known protein structures which share significant sequence similarity, making these enzymes of novelty and interest.…”