Metabolically engineered Caldicellulosiruptor bescii as a platform for producing acetone and hydrogen from lignocellulose.

The production of volatile industrial chemicals utilizing metabolically engineered extreme thermophiles offers the potential for processes with simultaneous fermentation and product separation. An excellent target chemical for such a process is acetone (Tb = 56°C), ideally produced from lignocellulosic biomass. Caldicellulosiruptor bescii (Topt 78°C), an extremely thermophilic fermentative bacterium naturally capable of deconstructing and fermenting lignocellulose, was metabolically engineered to produce acetone. When the acetone pathway construct was integrated into a parent strain containing the bifunctional alcohol dehydrogenase from Clostridium thermocellum, acetone was produced at 9.1 mM (0.53 g/L), in addition to minimal ethanol 3.3 mM (0.15 g/L), along with net acetate consumption. This demonstrates that C. bescii can be engineered with balanced pathways in which renewable carbohydrate sources are converted to useful metabolites, primarily acetone and H2 , without net production of its native fermentation products, acetate and lactate.

Determinants of sulphur chemolithoautotrophy in the extremely thermoacidophilic Sulfolobales.

Species in the archaeal order Sulfolobales thrive in hot acid and exhibit remarkable metabolic diversity. Some species are chemolithoautotrophic, obtaining energy through the oxidation of inorganic substrates, sulphur in particular, and acquiring carbon through the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) CO2 -fixation cycle. The current model for sulphur oxidation in the Sulfolobales is based on the biochemical analysis of specific proteins from Acidianus ambivalens, including sulphur oxygenase reductase (SOR) that disproportionates S° into H2 S and sulphite (SO3 2- ). Initial studies indicated SOR catalyses the essential first step in oxidation of elemental sulphur, but an ancillary role for SOR as a 'recycle' enzyme has also been proposed. Here, heterologous expression of both SOR and membrane-bound thiosulphate-quinone oxidoreductase (TQO) from Sulfolobus tokodaii 'restored' sulphur oxidation capacity in Sulfolobus acidocaldarius DSM639, but not autotrophy, although earlier reports indicate this strain was once capable of chemolithoautotrophy. Comparative transcriptomic analyses of Acidianus brierleyi, a chemolithoautotrophic sulphur oxidizer, and S. acidocaldarius DSM639 showed that while both share a strong transcriptional response to elemental sulphur, S. acidocaldarius DSM639 failed to upregulate key 3-HP/4-HB cycle genes used by A. brierleyi to drive chemolithoautotrophy. Thus, the inability for S. acidocaldarius DSM639 to grow chemolithoautotrophically may be rooted more in gene regulation than the biochemical capacity.

A synthetic enzymatic pathway for extremely thermophilic acetone production based on the unexpectedly thermostable acetoacetate decarboxylase from Clostridium acetobutylicum.

One potential advantage of an extremely thermophilic metabolic engineering host (T opt ≥ 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 ß-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.

Biotechnology of extremely thermophilic archaea.

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.

Extremely thermophilic energy metabolisms: biotechnological prospects.

New strategies for metabolic engineering of extremely thermophilic microorganisms to produce bio-based fuels and chemicals could leverage pathways and physiological features resident in extreme thermophiles for improved outcomes. Furthermore, very recent advances in genetic tools for these microorganisms make it possible for them to serve as metabolic engineering hosts. Beyond providing a higher temperature alternative to mesophilic platforms, exploitation of strategic metabolic characteristics of high temperature microorganisms grants new opportunities for biotechnological products. This review considers recent developments in extreme thermophile biology as they relate to new horizons for energy biotechnology.

Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms.

The current upper thermal limit for life as we know it is approximately 120°C. Microorganisms that grow optimally at temperatures of 75°C and above are usually referred to as 'extreme thermophiles' and include both bacteria and archaea. For over a century, there has been great scientific curiosity in the basic tenets that support life in thermal biotopes on earth and potentially on other solar bodies. Extreme thermophiles can be aerobes, anaerobes, autotrophs, heterotrophs, or chemolithotrophs, and are found in diverse environments including shallow marine fissures, deep sea hydrothermal vents, terrestrial hot springs-basically, anywhere there is hot water. Initial efforts to study extreme thermophiles faced challenges with their isolation from difficult to access locales, problems with their cultivation in laboratories, and lack of molecular tools. Fortunately, because of their relatively small genomes, many extreme thermophiles were among the first organisms to be sequenced, thereby opening up the application of systems biology-based methods to probe their unique physiological, metabolic and biotechnological features. The bacterial genera Caldicellulosiruptor, Thermotoga and Thermus, and the archaea belonging to the orders Thermococcales and Sulfolobales, are among the most studied extreme thermophiles to date. The recent emergence of genetic tools for many of these organisms provides the opportunity to move beyond basic discovery and manipulation to biotechnologically relevant applications of metabolic engineering. WIREs Syst Biol Med 2017, 9:e1377. doi: 10.1002/wsbm.1377 For further resources related to this article, please visit the WIREs website.

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus.

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO3- and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO2 . Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the ß-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO2 -sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO2 into 3HP production in metabolically engineered P. furiosus, and hint at the important role that CA and BPL likely play in the native 3HP/4HB pathway in M. sedula. Biotechnol. Bioeng. 2016;113: 2652-2660. © 2016 Wiley Periodicals, Inc.

Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals.

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.

Alcohol Selectivity in a Synthetic Thermophilic n-Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes.

n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (ß-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from Caldanaerobacter subterraneus subsp. tengcongensis; trans-2-enoyl-CoA reductase [Ter] from Spirochaeta thermophila; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from Clostridium thermocellum; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures.

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production.

Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T(opt) 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (T(opt) 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.