Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high temperature industrial biotechnology.

Although the extremely thermophilic archaea (Topt ≥ 70°C) may be the most primitive extant forms of life, they have been studied to a limited extent relative to mesophilic microorganisms. Many of these organisms have unique biochemical and physiological characteristics with important biotechnological implications. These include methanogens that generate methane, fermentative anaerobes that produce hydrogen gas with high efficiency, and acidophiles that can mobilize base, precious and strategic metals from mineral ores. Extremely thermophilic archaea have also been a valuable source of thermoactive, thermostable biocatalysts, but their use as cellular systems has been limited because of the general lack of facile genetics tools. This situation has changed recently, however, thereby providing an important avenue for understanding their metabolic and physiological details and also opening up opportunities for metabolic engineering efforts. Along these lines, extremely thermophilic archaea have recently been engineered to produce a variety of alcohols and industrial chemicals, in some cases incorporating CO2 into the final product. There are barriers and challenges to these organisms reaching their full potential as industrial microorganisms but, if these can be overcome, a new dimension for biotechnology will be forthcoming that strategically exploits biology at high temperatures.

Microbial fermentation of lignocellulosic biomass to produce industrial chemicals is exacerbated by the recalcitrant network of lignin, cellulose and hemicelluloses comprising the plant secondary cell wall. In this study, we show that transgenic poplar ( Populus trichocarpa ) lines can be solubilized without any pretreatment by the extreme thermophile Caldicellulosiruptor bescii that has been metabolically engineered to shift its fermentation products away from inhibitory organic acids to ethanol. Carbohydrate solubilization and conversion of unpretreated milled biomass is nearly 90% for two transgenic lines, compared to only 25% for wild-type poplar. Unexpectedly, unpretreated intact poplar stems achieved nearly 70% of the fermentation production observed with milled poplar as the substrate. The nearly quantitative microbial conversion of the carbohydrate content of unpretreated transgenic lignocellulosic biomass bodes well for full utilization of renewable biomass feedstocks.

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One potential advantage of an extremely thermophilic metabolic engineering host (T ≥ 70°C) is facilitated recovery of volatile chemicals from the vapor phase of an active fermenting culture. This process would reduce purification costs and concomitantly alleviate toxicity to the cells by continuously removing solvent fermentation products such as acetone or ethanol, a process we are calling "bio-reactive distillation." Although extremely thermophilic heterologous metabolic pathways can be inspired by existing mesophilic versions, they require thermostable homologs of the constituent enzymes if they are to be utilized in extremely thermophilic bacteria or archaea. Production of acetone from acetyl-CoA and acetate in the mesophilic bacterium Clostridium acetobutylicum utilizes three enzymes: thiolase, acetoacetyl-CoA: acetate CoA transferase (CtfAB), and acetoacetate decarboxylase (Adc). Previously reported biocatalytic pathways for acetone production were demonstrated only as high as 55°C. Here, we demonstrate a synthetic enzymatic pathway for acetone production that functions up to at least 70°C in vitro, made possible by the unusual thermostability of Adc from the mesophile C. acetobutylicum, and heteromultimeric acetoacetyl-CoA:acetate CoA-transferase (CtfAB) complexes from Thermosipho melanesiensis and Caldanaerobacter subterraneus, composed of a highly thermostable α-subunit and a thermally labile β-subunit. The three enzymes produce acetone in vitro at temperatures of at least 70°C, paving the way for bio-reactive distillation of acetone using a metabolically engineered extreme thermophile as a production host.