Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

AbstractThe thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content

Metabolic engineering of thermophilic bacteria for production of biotechnologically interesting compounds

Many thermophilic bacteria are efficient biomass degraders (producing polysaccharide degrading enzymes and utilizing a great variety of substrates, e.g. lignocellulosic polymers, pentoses, hexoses, as well sugar acids, and sugar alcohols). This makes them interesting organisms as potential cell factories in a circular bioeconomy. Lignocellulosic and marine macroalgal biomasses are regarded as sustainable biorefinery feedstocks for the production of energy carriers and platform and specialty chemicals, thereby meeting impending fossil fuel shortage and counteracting accumulation of greenhouse gasses. However, progress in using thermophilic bacteria that utilize these feedstocks as carbon sources has been hampered by the lack of suitable engineering tools to improve the production profiles of interesting target metabolites as specific synthetic production pathways need to be inserted/modified or existing pathways optimized by metabolic engineering. In this chapter, we review the progress on the use of thermophilic bacteria in metabolic engineering and the available engineering tools and give examples of species for which successful engineering has been accomplished. Today, the majority of thermophilic bacteria targeted for production of compounds of industrial interest by metabolic engineering belong to the phylum Firmicutes (e.g. Thermoanaerobacterium, Caldocellulosiruptor, Geobacillus, and Bacillus), taking advantage of anaerobic catabolic pathways producing organic acids and alcohols. However, there are additional and aerobic species gaining interest concerning biomass degradation and the ability of carbon dioxide fixation as well as production of molecules of interest, and some examples of this are also given

Engineering the carotenoid biosynthetic pathway in Rhodothermus marinus for lycopene production

Rhodothermus marinus has the potential to be well suited for biorefineries, as an aerobic thermophile that produces thermostable enzymes and is able to utilize polysaccharides from different 2nd and 3rd generation biomass. The bacterium produces valuable chemicals such as carotenoids. However, the native carotenoids are not established for industrial production and R. marinus needs to be genetically modified to produce higher value carotenoids. Here we genetically modified the carotenoid biosynthetic gene cluster resulting in three different mutants, most importantly the lycopene producing mutant TK-3 (ΔtrpBΔpurAΔcruFcrtB::trpBcrtBT.thermophilus). The genetic modifications and subsequent structural analysis of carotenoids helped clarify the carotenoid biosynthetic pathway in R. marinus. The nucleotide sequences encoding the enzymes phytoene synthase (CrtB) and the previously unidentified 1′,2′-hydratase (CruF) were found fused together and encoded by a single gene in R. marinus. Deleting only the cruF part of the gene did not result in an active CrtB enzyme. However, by deleting the entire gene and inserting the crtB gene from Thermus thermophilus, a mutant strain was obtained, producing lycopene as the sole carotenoid. The lycopene produced by TK-3 was quantified as 0.49 ​g/kg CDW (cell dry weight)