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Journal of Industrial Microbiology &... Apr 2022Alkanes are high-energy molecules that are compatible with enduring liquid fuel infrastructures, which make them highly suitable for being next-generation biofuels.... (Review)
Review
Alkanes are high-energy molecules that are compatible with enduring liquid fuel infrastructures, which make them highly suitable for being next-generation biofuels. Though biological production of alkanes has been reported in various microorganisms, the reports citing photosynthetic cyanobacteria as natural producers have been the most consistent for the long-chain alkanes and alkenes (C15-C19). However, the production of alkane in cyanobacteria is low, leading to its extraction being uneconomical for commercial purposes. In order to make alkane production economically feasible from cyanobacteria, the titre and yield need to be increased by several orders of magnitude. In the recent past, efforts have been made to enhance alkane production, although with a little gain in yield, leaving space for much improvement. Genetic manipulation in cyanobacteria is considered challenging, but recent advancements in genetic engineering tools may assist in manipulating the genome in order to enhance alkane production. Further, advancement in a basic understanding of metabolic pathways and gene functioning will guide future research for harvesting the potential of these tiny photosynthetically efficient factories. In this review, our focus would be to highlight the current knowledge available on cyanobacterial alkane production, and the potential aspects of developing cyanobacterium as an economical source of biofuel. Further insights into different metabolic pathways and hosts explored so far, and possible challenges in scaling up the production of alkanes will also be discussed.
Topics: Alkanes; Alkenes; Biofuels; Cyanobacteria; Metabolic Engineering
PubMed: 34718648
DOI: 10.1093/jimb/kuab075 -
Microbial Cell Factories Nov 2020The development of sustainable routes to the bio-manufacture of gaseous hydrocarbons will contribute widely to future energy needs. Their realisation would contribute... (Review)
Review
The development of sustainable routes to the bio-manufacture of gaseous hydrocarbons will contribute widely to future energy needs. Their realisation would contribute towards minimising over-reliance on fossil fuels, improving air quality, reducing carbon footprints and enhancing overall energy security. Alkane gases (propane, butane and isobutane) are efficient and clean-burning fuels. They are established globally within the transportation industry and are used for domestic heating and cooking, non-greenhouse gas refrigerants and as aerosol propellants. As no natural biosynthetic routes to short chain alkanes have been discovered, de novo pathways have been engineered. These pathways incorporate one of two enzymes, either aldehyde deformylating oxygenase or fatty acid photodecarboxylase, to catalyse the final step that leads to gas formation. These new pathways are derived from established routes of fatty acid biosynthesis, reverse β-oxidation for butanol production, valine biosynthesis and amino acid degradation. Single-step production of alkane gases in vivo is also possible, where one recombinant biocatalyst can catalyse gas formation from exogenously supplied short-chain fatty acid precursors. This review explores current progress in bio-alkane gas production, and highlights the potential for implementation of scalable and sustainable commercial bioproduction hubs.
Topics: Alkanes; Biofuels; Biosynthetic Pathways; Butanes; Carboxy-Lyases; Fatty Acids; Gases; Genetic Engineering; Industrial Microbiology; Metabolic Engineering; Oxidation-Reduction; Oxygenases; Propane; Synthetic Biology
PubMed: 33187524
DOI: 10.1186/s12934-020-01470-6 -
Nature Communications Apr 2023Alkanes are the most energy-rich form of carbon and are widely dispersed in the environment. Their transformation by microbes represents a key step in the global carbon...
Alkanes are the most energy-rich form of carbon and are widely dispersed in the environment. Their transformation by microbes represents a key step in the global carbon cycle. Alkane monooxygenase (AlkB), a membrane-spanning metalloenzyme, converts straight chain alkanes to alcohols in the first step of the microbially-mediated degradation of alkanes, thereby playing a critical role in the global cycling of carbon and the bioremediation of oil. AlkB biodiversity is attributed to its ability to oxidize alkanes of various chain lengths, while individual AlkBs target a relatively narrow range. Mechanisms of substrate selectivity and catalytic activity remain elusive. Here we report the cryo-EM structure of AlkB, which provides a distinct architecture for membrane enzymes. Our structure and functional studies reveal an unexpected diiron center configuration and identify molecular determinants for substrate selectivity. These findings provide insight into the catalytic mechanism of AlkB and shed light on its function in alkane-degrading microorganisms.
Topics: Alkanes; Biodegradation, Environmental; Carbon; Oxidation-Reduction; AlkB Enzymes
PubMed: 37069165
DOI: 10.1038/s41467-023-37869-z -
The ISME Journal Apr 2022The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain...
The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.
Topics: Alkanes; Ammonia; Archaea; Bacteria; In Situ Hybridization, Fluorescence; Mixed Function Oxygenases; Oxidation-Reduction; Phylogeny
PubMed: 34743174
DOI: 10.1038/s41396-021-01144-0 -
World Journal of Microbiology &... Feb 2023Yarrowia lipolytica, a dimorphic yeast belonging to the Ascomycota, has potent abilities to utilize hydrophobic compounds, such as n-alkanes and fatty acids, as carbon... (Review)
Review
Yarrowia lipolytica, a dimorphic yeast belonging to the Ascomycota, has potent abilities to utilize hydrophobic compounds, such as n-alkanes and fatty acids, as carbon and energy sources. Yarrowia lipolytica can synthesize and accumulate large amounts of lipids, making it a promising host to produce various lipids and convert n-alkanes to useful compounds. For advanced use of Y. lipolytica in these applications, it is necessary to understand the metabolism of these hydrophobic compounds in this yeast and the underlying molecular mechanisms. In this review, current knowledge on the n-alkane metabolism and how this is regulated in Y. lipolytica is summarized. Furthermore, recent studies revealed that lipid transfer proteins are involved in the utilization of n-alkanes and the regulation of cell morphology in response to n-alkanes. This review discusses the roles of membrane lipids in these processes in Y. lipolytica.
Topics: Yarrowia; Alkanes; Fatty Acids
PubMed: 36781616
DOI: 10.1007/s11274-023-03541-3 -
The Science of the Total Environment Mar 2022Monitoring environmental status through molecular investigation of microorganisms in the marine environment is suggested as a potentially very effective method for...
Monitoring environmental status through molecular investigation of microorganisms in the marine environment is suggested as a potentially very effective method for biomonitoring, with great potential for automation. There are several hurdles to that approach with regards to primer design, variability across geographical locations, seasons, and type of environmental pollution. Here, qPCR analysis of genes involved in the initial activation of aliphatic and aromatic hydrocarbons were used in a laboratory setup mimicking realistic oil leakage at sea. Seawater incubation experiments were carried out under two different seasons with two different oil types. Degenerate primers targeting initial oxygenases (alkane 1-monooxygenase; alkB and aromatic-ring hydroxylating dioxygenase; ARHD) were employed in qPCR assays to quantify the abundance of genes essential for oil degradation. Shotgun metagenomics was used to map the overall community dynamics and the diversity of alkB and ARHD genes represented in the microbial community. The amplicons generated through the qPCR assays were sequenced to reveal the diversity of oil-degradation related genes captured by the degenerate primers. We identified a major mismatch between the taxonomic diversity of alkB and ARHD genes amplified by the degenerate primers and those identified through shotgun metagenomics. More specifically, the designed primers did not amplify the alkB genes of the two most abundant alkane degraders that bloomed in the experiments, Oceanobacter and Oleispira. The relative abundance of alkB sequences from shotgun metagenomics and 16S rRNA-based Oleispira-specific qPCR assay were better signals for oil in water than the tested qPCR alkB assay. The ARHD assay showed a good agreement with PAHs degradation despite covering only 25% of the top 100 ARHD genes and missing several abundant Cycloclasticus sequences that were present in the metagenome. We conclude that further improvement of the degenerate primer approach is needed to rely on the use of oxygenase-related qPCR assays for oil leakage detection.
Topics: Alkanes; Bacteria; Biodegradation, Environmental; Petroleum; Petroleum Pollution; Phylogeny; RNA, Ribosomal, 16S
PubMed: 34896501
DOI: 10.1016/j.scitotenv.2021.152238 -
Biochemistry May 2022Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications.... (Review)
Review
Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications. Archaea are proposed to degrade short- and long-chain alkanes to CO by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural knowledge of alkyl-CoM reductases and offers fresh ideas about the expected mechanistic and chemical differences among members of this broad family. We conclude with the challenges of the investigation of these particular enzymes, which might one day generate biofuels for our modern society.
Topics: Alkanes; Anaerobiosis; Archaea; Catalysis; Mesna; Methane; Oxidation-Reduction; Oxidoreductases; Phylogeny
PubMed: 35500274
DOI: 10.1021/acs.biochem.2c00135 -
Environmental Science and Pollution... Sep 2022Chemical signature of airborne particulates and deposition dusts is subject of study since decades. Usually, three complementary composition markers are investigated,... (Review)
Review
Chemical signature of airborne particulates and deposition dusts is subject of study since decades. Usually, three complementary composition markers are investigated, namely, (i) specific organic compounds; (ii) concentration ratios between congeners, and (iii) percent distributions of homologs. Due to its intrinsic limits (e.g., variability depending on decomposition and gas/particle equilibrium), the identification of pollution sources based on molecular signatures results overall restricted to qualitative purposes. Nevertheless, chemical fingerprints allow drawing preliminary information, suitable for successfully approaching multivariate analysis and valuing the relative importance of sources. Here, the state-of-the-art is presented about the molecular fingerprints of non-polar aliphatic, polyaromatic (PAHs, nitro-PAHs), and polar (fatty acids, organic halides, polysaccharides) compounds in emissions. Special concern was addressed to alkenes and alkanes with carbon numbers ranging from 12 to 23 and ≥ 24, which displayed distinct relative abundances in petrol-derived spills and exhausts, emissions from microorganisms, high vegetation, and sediments. Long-chain alkanes associated with tobacco smoke were characterized by a peculiar iso/anteiso/normal homolog fingerprint and by n-hentriacontane percentages higher than elsewhere. Several concentration ratios of PAHs were identified as diagnostic of the type of emission, and the sources of uncertainty were elucidated. Despite extensive investigations conducted so far, the origin of uncommon molecular fingerprints, e.g., alkane/alkene relationships in deposition dusts and airborne particles, remains quite unclear. Polar organics resulted scarcely investigated for pollution apportioning purposes, though they looked as indicative of the nature of sources. Finally, the role of humans and living organisms as actual emitters of chemicals seems to need concern in the future.
Topics: Air Pollutants; Alkanes; Alkenes; Carbon; Dust; Environmental Monitoring; Fatty Acids; Humans; Particulate Matter; Polycyclic Aromatic Hydrocarbons; Tobacco Smoke Pollution
PubMed: 35876994
DOI: 10.1007/s11356-022-21531-0 -
International Journal of Environmental... Jun 2021Strain sw-1, isolated from 7619-m seawater of the Mariana Trench, was identified as by 16S rRNA gene and whole-genome sequencing. sw-1 was able to efficiently utilize...
Strain sw-1, isolated from 7619-m seawater of the Mariana Trench, was identified as by 16S rRNA gene and whole-genome sequencing. sw-1 was able to efficiently utilize long-chain -alkanes (C-C), but not short- and medium-chain -alkanes (C-C). The degradation rate of C was 91.25%, followed by C, C, C, C, and C with the degradation rates of 89.30%, 84.03%, 80.29%, 30.29%, and 13.37%, respectively. To investigate the degradation mechanisms of -alkanes for this strain, the genome and the transcriptome analyses were performed. Four key alkane hydroxylase genes (, , , and ) were identified in the genome. Transcriptomes of strain sw-1 grown in C or CHCOONa (NaAc) as the sole carbon source were compared. The transcriptional levels of and , respectively, increased 78.28- and 3.51-fold in C compared with NaAc, while and did not show obvious change. The expression levels of other genes involved in the synthesis of unsaturated fatty acids, permeases, membrane proteins, and sulfur metabolism were also upregulated, and they might be involved in -alkane uptake. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed that expression was significantly induced by C, C, and C, and induction extent by C and C was higher than that with C Furthermore, expression was only induced by C, and expression was not induced by any of -alkanes. In addition, sw-1 could grow with 0%-3% NaCl or 8 out of 10 kinds of the tested heavy metals and degrade -alkanes at 15 °C. Taken together, these results provide comprehensive insights into the degradation of long-chain -alkanes by isolated from the deep ocean environment.
Topics: Acinetobacter; Alkanes; Biodegradation, Environmental; Gene Expression Profiling; RNA, Ribosomal, 16S
PubMed: 34208299
DOI: 10.3390/ijerph18126365 -
Microbial Cell Factories Dec 2022The use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in... (Review)
Review
The use of biologically produced alkanes has attracted considerable attention as an alternative energy source to petroleum. In 2010, the alkane synthesis pathway in cyanobacteria was found to include two small globular proteins, acyl-(acyl carrier protein [ACP]) reductase (AAR) and aldehyde deformylating oxygenase (ADO). AAR produces fatty aldehydes from acyl-ACPs/CoAs, which are then converted by ADO to alkanes/alkenes equivalent to diesel oil. This discovery has paved the way for alkane production by genetically modified organisms. Since then, many studies have investigated the reactions catalyzed by AAR and ADO. In this review, we first summarize recent findings on structures and catalytic mechanisms of AAR and ADO. We then outline the mechanism by which AAR and ADO form a complex and efficiently transfer the insoluble aldehyde produced by AAR to ADO. Furthermore, we describe recent advances in protein engineering studies on AAR and ADO to improve the efficiency of alkane production in genetically engineered microorganisms such as Escherichia coli and cyanobacteria. Finally, the role of alkanes in cyanobacteria and future perspectives for bioalkane production using AAR and ADO are discussed. This review provides strategies for improving the production of bioalkanes using AAR and ADO in cyanobacteria for enabling the production of carbon-neutral fuels.
Topics: Cyanobacteria; Oxygenases; Alkanes; Oxidoreductases; Escherichia coli; Aldehydes
PubMed: 36503511
DOI: 10.1186/s12934-022-01981-4