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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.

This work presents an evaluation of batch, fed-batch, and sequential batch cultivation techniques for production of R. marinus DSM 16675 and its exopolysaccharides (EPSs) and carotenoids in a bioreactor, using lysogeny broth (LB) and marine broth (MB), respectively, in both cases supplemented with 10 g/L maltose. Batch cultivation using LB supplemented with maltose (LBmalt) resulted in higher cell density (OD620 = 6.6) than use of MBmalt (OD620 = 1.7). Sequential batch cultivation increased the cell density threefold (OD620 = 20) in LBmalt and eightfold (OD620 = 14) in MBmalt. In both single and sequential batches, the production of carotenoids and EPSs using LBmalt was detected in the exponential phase and stationary phase, respectively, while in MBmalt formation of both products was detectable in both the exponential and stationary phases of the culture. Heteropolymeric EPSs were produced with an overall volumetric productivity (QE) of 0.67 (mg/L h) in MBmalt and the polymer contained xylose. In LB, QE was lower (0.1 mg/L h) and xylose could not be detected in the composition of the produced EPSs. In conclusion, this study showed the importance of a process design and medium source for production of R. marinus DSM 16675 and its metabolites.

Rhodothermus marinus, a marine aerobic thermophile, was first isolated from an intertidal hot spring in Iceland. In recent years, the R. marinus strain PRI 493 has been genetically modified, which opens up possibilities for targeted metabolic engineering of the species, such as of the carotenoid biosynthetic pathway. In this study, the carotenoids of the R. marinus type‐strain DSM 4252T, strain DSM 4253, and strain PRI 493 were characterized. Bioreactor cultivations were used for pressurized liquid extraction and analyzed by ultra‐high performance supercritical fluid chromatography with diode array and quadropole time‐of‐flight mass spectrometry detection (UHPSFC‐DAD‐QTOF/MS). Salinixanthin, a carotenoid originally found in Salinibacter ruber and previously detected in strain DSM 4253, was identified in all three R. marinus strains, both in the hydroxylated and nonhydroxylated form. Furthermore, an additional and structurally distinct carotenoid was detected in the three strains. MS/MS fragmentation implied that the mass difference between salinixanthin and the novel carotenoid structure corresponded to the absence of a 4‐keto group on the ß‐ionone ring. The study confirmed the lack of carotenoids for the strain SB‐71 (ΔtrpBΔpurAcrtBI’::trpB) in which genes encoding two enzymes of the proposed pathway are partially deleted. Moreover, antioxidant capacity was detected in extracts of all the examined R. marinus strains and found to be 2–4 times lower for the knock‐out strain SB‐71. A gene cluster with 11 genes in two operons in the R. marinusDSM 4252T genome was identified and analyzed, in which several genes were matched with carotenoid biosynthetic pathway genes in other organisms., Vetenskapsrådet, Grant/Award Number: 622-2010-333; Svenska Forskningsrådet Formas, Grant/Award Number: 239-2013-971 and 942-2015-1952; Seventh Framework Programme, Grant/Award Number: 311932

The 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.