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3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD + regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD + , were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower K m values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD + , less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).

Anaerobic microorganisms are thought to play a critical role in regulating the flux of short-chain gaseous alkanes (SCGAs; including ethane, propane and butane) from terrestrial and aquatic ecosystems to the atmosphere. Sulfate has been confirmed to act as electron acceptor supporting microbial anaerobic oxidation of SCGAs, yet several other energetically more favourable acceptors co-exist with these gases in anaerobic environments. Here, we show that a bioreactor seeded with biomass from a wastewater treatment facility can perform anaerobic propane oxidation coupled to nitrate reduction to dinitrogen gas and ammonium. The bioreactor was operated for more than 1000 days, and we used 13 C- and 15 N-labelling experiments, metagenomic, metatranscriptomic, metaproteomic and metabolite analyses to characterize the microbial community and the metabolic processes. The data collectively suggest that a species representing a novel order within the bacterial class Symbiobacteriia is responsible for the observed nitrate-dependent propane oxidation. The closed genome of this organism, which we designate as ‘ Candidatus Alkanivorans nitratireducens’, encodes pathways for oxidation of propane to CO 2 via fumarate addition, and for nitrate reduction, with all the key genes expressed during nitrate-dependent propane oxidation. Our results suggest that nitrate is a relevant electron sink for SCGA oxidation in anaerobic environments, constituting a new microbially-mediated link between the carbon and nitrogen cycles. Anaerobic microorganisms can oxidize short-chain gaseous alkanes such as ethane, propane and butane using sulfate as electron acceptor. Here, the authors show that a bioreactor enrichment of a wastewater microbial community can perform anaerobic propane oxidation coupled to nitrate reduction.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane—despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing “Candidatus Alkanivorans nitratireducens” was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that “Ca. A. nitratireducens” is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

The Deepwater Horizon event resulted in suspension of oil in the Gulf of Mexico water column because the leakage occurred at great depth. The distribution and fate of other abundant hydrocarbon constituents, such as natural gases, are also important in determining the impact of the leakage but are not yet well understood. From 11 to 21 June 2010, we investigated dissolved hydrocarbon gases at depth using chemical and isotopic surveys and on-site biodegradation studies. Propane and ethane were the primary drivers of microbial respiration, accounting for up to 70% of the observed oxygen depletion in fresh plumes. Propane and ethane trapped in the deep water may therefore promote rapid hydrocarbon respiration by low-diversity bacterial blooms, priming bacterial populations for degradation of other hydrocarbons in the aging plume.

Glycerol/diol dehydratases catalyze the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA), the basis of a multi-component system called reuterin. Reuterin has antimicrobial properties and undergoes chemical conjugation with dietary heterocyclic amines (HCAs). In aqueous solution reuterin is in dynamic equilibrium with the toxicant acrolein. It was the aim of this study to investigate the extent of acrolein formation at various physiological conditions and to determine its role in biological and chemical activities. The application of a combined novel analytical approach including IC-PAD, LC-MS and NMR together with specific acrolein scavengers suggested for the first time that acrolein, and not 3-HPA, is the active compound responsible for HCA conjugation and antimicrobial activity attributed to reuterin. As formation of the HCA conjugate was observed in vivo, our results imply that acrolein is formed in the human gut with implications on detoxification of HCAs. We propose to re-define the term reuterin to include acrolein.

Vanadium nitrogenase not only reduces dinitrogen to ammonia but also reduces carbon monoxide to ethylene, ethane, and propane. The parallelism between the two reactions suggests a potential link in mechanism and evolution between the carbon and nitrogen cycles on Earth.

SignificanceMicroorganisms can oxidize hydrocarbons anaerobically, but the detection and quantification of this process in natural settings remains difficult, impeding reliable estimation of these processes at the global scale. We have used the technique of position-specific isotope analysis of propane and show that anaerobic biological degradation of propane leads to a specific signature that differs from that of propane produced from thermal decomposition of higher hydrocarbons. When applied to natural gas reservoirs, we show that anaerobic bacterial oxidation of propane can be detected and quantified, which is not the case with the use of conventional methods. Our findings are thus of importance for the detection of subsurface biology, for the understanding of the carbon cycle, and more broadly for environmental sciences. Microbial anaerobic oxidation of hydrocarbons is a key process potentially involved in a myriad of geological and biochemical environments yet has remained notoriously difficult to identify and quantify in natural environments. We performed position-specific carbon isotope analysis of propane from cracking and incubation experiments. Anaerobic bacterial oxidation of propane leads to a pronounced and previously unidentified 13C enrichment in the central position of propane, which contrasts with the isotope signature associated with the thermogenic process. This distinctive signature allows the detection and quantification of anaerobic oxidation of hydrocarbons in diverse natural gas reservoirs and suggests that this process may be more widespread than previously thought. Position-specific isotope analysis can elucidate the fate of natural gas hydrocarbons and provide insight into a major but previously cryptic process controlling the biogeochemical cycling of globally significant greenhouse gases.

The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O 2 supply and removal of native aldehyde reductases. Propane is the main component of liquid petroleum gas and has a wide variety of commercial applications. Here, the authors engineer a synthetic metabolic pathway in E. coli , and demonstrate for the first time the renewable production of propane.

Commensal-derived probiotic bacteria inhibit enteric pathogens and regulate host immune responses in the gastrointestinal tract, but studies examining specific functions of beneficial microbes in the context of biofilms have been limited in scope. Lactobacillus reuteri formed biofilms that retained functions potentially advantageous to the host including modulation of cytokine output and the production of the antimicrobial agent, reuterin. Immunomodulatory activities of biofilms were demonstrated by the abilities of specific L. reuteri strains to suppress human TNF production by LPS-activated monocytoid cells. Quantification of the antimicrobial glycerol derivative, reuterin, was assessed in order to document the antipathogenic potential of probiotic biofilms. L. reuteri biofilms differed in the quantities of reuterin secreted in this physiological state. L. reuteri biofilms secreted factors that confer specific health benefits such as immunomodulation and pathogen inhibition. Future probiotic selection strategies should consider a strain's ability to perform beneficial functions as a biofilm.

Distinct groups of microorganisms have been thought to grow on methane and on short-chain alkanes; now, the methanotroph Methylocella silvestris is shown to express two distinct soluble di-iron centre monooxygenases that allow it to use either methane or propane as a carbon and energy source. Methanotroph adds protein to menu Methane is a potent greenhouse gas, and increasing anthropogenic emissions of methane to the atmosphere could have a major impact on climate. Microorganisms can use methane and short-chain alkanes such as propane and butane as an energy source, and it had been assumed that these gases were oxidized by completely different groups of organisms. Now Andrew Crombie and John Murrell show that the facultative methanotroph Methylocella silvestris BL2 encodes two distinct soluble di-iron centre monooxygenase gene clusters, equipping it to use either methane or propane as a carbon and energy source. The ability of an environmentally widespread strain to degrade both methane and propane underlines the importance of microbial processes in climate modelling as well as in oil spills, unconventional methods of gas extraction and other human activities. The climate-active gas methane is generated both by biological processes and by thermogenic decomposition of fossil organic material, which forms methane and short-chain alkanes, principally ethane, propane and butane 1 , 2 . In addition to natural sources, environments are exposed to anthropogenic inputs of all these gases from oil and gas extraction and distribution. The gases provide carbon and/or energy for a diverse range of microorganisms that can metabolize them in both anoxic 3 and oxic zones. Aerobic methanotrophs, which can assimilate methane, have been considered to be entirely distinct from utilizers of short-chain alkanes, and studies of environments exposed to mixtures of methane and multi-carbon alkanes have assumed that disparate groups of microorganisms are responsible for the metabolism of these gases. Here we describe the mechanism by which a single bacterial strain, Methylocella silvestris , can use methane or propane as a carbon and energy source, documenting a methanotroph that can utilize a short-chain alkane as an alternative to methane. Furthermore, during growth on a mixture of these gases, efficient consumption of both gases occurred at the same time. Two soluble di-iron centre monooxygenase (SDIMO) gene clusters were identified and were found to be differentially expressed during bacterial growth on these gases, although both were required for efficient propane utilization. This report of a methanotroph expressing an additional SDIMO that seems to be uniquely involved in short-chain alkane metabolism suggests that such metabolic flexibility may be important in many environments where methane and short-chain alkanes co-occur.