Hydrocarbon Biosynthesis Research Articles

Comparative genomics uncovers the evolutionary dynamics of detoxification and insecticide target genes across 11 phlebotomine sand flies.

Sand flies infect more than one million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: Cytochrome P450s (CYPs), Glutathione-S-Transferases (GSTs), UDP-Glycosyltransferases (UGTs), Carboxyl/Cholinesterases (CCEs) and ATP-Binding Cassette (ABC) transporters. Using this comparative approach we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of a) CYP4G genes involved in cuticular hydrocarbon biosynthesis, b) ABCB genes involved in xenobiotic toxicity and c) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and Voltage Gated Sodium Channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Strong resistance to β-cyfluthrin in a strain of the beetle Alphitobius diaperinus: a de novo transcriptome analysis.

The lesser mealworm, Alphitobius diaperinus, is an invasive tenebrionid beetle and a vector of pathogens. Due to the emergence of insecticide resistance and consequent outbreaks that generate significant phytosanitary and energy costs for poultry farmers, it has become a major insect pest worldwide. To better understand the molecular mechanisms behind this resistance, we studied a strain of A. diaperinus from a poultry house in Brittany that was found to be highly resistant to the β-cyfluthrin. The strain survived β-cyfluthrin exposures corresponding to more than 100 times the recommended dose. We used a comparative de novo RNA-Seq approach to explore genes expression in resistant versus sensitive strains. Our de novo transcriptomic analyses showed that responses to β-cyfluthrin likely involved a whole set of resistance mechanisms. Genes related to detoxification, metabolic resistance, cuticular hydrocarbon biosynthesis and proteolysis were found to be constitutively overexpressed in the resistant compared to the sensitive strain. Follow-up enzymatic assays confirmed that the resistant strain exhibited high basal activities for detoxification enzymes such as cytochrome P450 monooxygenase and glutathione-S-transferase. The in-depth analysis of differentially expressed genes suggests the involvement of complex regulation of signaling pathways. Detailed knowledge of these resistance mechanisms is essential for the establishment of effective pest control.

Cryoprotectant-Mediated Cold Stress Mitigation in Litchi Flower Development: Transcriptomic and Metabolomic Perspectives.

Temperature is vital in plant growth and agricultural fruit production. Litchi chinensis Sonn, commonly known as litchi, is appreciated for its delicious fruit and fragrant blossoms and is susceptible to stress when exposed to low temperatures. This study investigates the effect of two cryoprotectants that counteract cold stress during litchi flowering, identifies the genes that generate the cold resistance induced by the treatments, and hypothesizes the roles of these genes in cold resistance. Whole plants were treated with Bihu and Liangli cryoprotectant solutions to protect inflorescences below 10 °C. The soluble protein, sugar, fructose, sucrose, glucose, and proline contents were measured during inflorescence. Sucrose synthetase, sucrose phosphate synthetase, antioxidant enzymes (SOD, POD, CAT), and MDA were also monitored throughout the flowering stage. Differentially expressed genes (DEGs), gene ontology, and associated KEGG pathways in the transcriptomics study were investigated. There were 1243 DEGs expressed after Bihu treatment and 1340 in the control samples. Signal transduction pathways were associated with 39 genes in the control group and 43 genes in the Bihu treatment group. The discovery of these genes may contribute to further research on cold resistance mechanisms in litchi. The Bihu treatment was related to 422 low-temperature-sensitive differentially accumulated metabolites (DAMs), as opposed to 408 DAMs in the control, mostly associated with lipid metabolism, organic oxidants, and alcohols. Among them, the most significant differentially accumulated metabolites were involved in pathways such as β-alanine metabolism, polycyclic aromatic hydrocarbon biosynthesis, linoleic acid metabolism, and histidine metabolism. These results showed that Bihu treatment could potentially promote these favorable traits and increase fruit productivity compared to the Liangli and control treatments. More genomic research into cold stress is needed to support the findings of this study.

Dynamic relationships among pathways producing hydrocarbons and fatty acids of maize silk cuticular waxes.

The hydrophobic cuticle is the first line of defense between aerial portions of plants and the external environment. On maize (Zea mays L.) silks, the cuticular cutin matrix is infused with cuticular waxes, consisting of a homologous series of very long-chain fatty acids (VLCFAs), aldehydes, and hydrocarbons. Together with VLC fatty-acyl-CoAs (VLCFA-CoAs), these metabolites serve as precursors, intermediates and end-products of the cuticular wax biosynthetic pathway. To deconvolute the potentially confounding impacts of the change in silk microenvironment and silk development on this pathway, we profiled cuticular waxes on the silks of the inbreds B73 and Mo17, and their reciprocal hybrids. Multivariate interrogation of these metabolite abundance data demonstrates that VLCFA-CoAs and total free VLCFAs are positively correlated with the cuticular wax metabolome, and this metabolome is primarily affected by changes in the silk microenvironment and plant genotype. Moreover, the genotype effect on the pathway explains the increased accumulation of cuticular hydrocarbons with a concomitant reduction in cuticular VLCFA accumulation on B73 silks, suggesting that the conversion of VLCFA-CoAs to hydrocarbons is more effective in B73 than Mo17. Statistical modeling of the ratios between cuticular hydrocarbons and cuticular VLCFAs reveals a significant role of precursor chain length in determining this ratio. This study establishes the complexity of the product-precursor relationships within the silk cuticular wax-producing network by dissecting both the impact of genotype and the allocation of VLCFA-CoA precursors to different biological processes, and demonstrates that longer chain VLCFA-CoAs are preferentially utilized for hydrocarbon biosynthesis.

The polyketide to fatty acid transition in the evolution of animal lipid metabolism

Animals synthesize simple lipids using a distinct fatty acid synthase (FAS) related to the type I polyketide synthase (PKS) enzymes that produce complex specialized metabolites. The evolutionary origin of the animal FAS and its relationship to the diversity of PKSs remain unclear despite the critical role of lipid synthesis in cellular metabolism. Recently, an animal FAS-like PKS (AFPK) was identified in sacoglossan molluscs. Here, we explore the phylogenetic distribution of AFPKs and other PKS and FAS enzymes across the tree of life. We found AFPKs widely distributed in arthropods and molluscs (>6300 newly described AFPK sequences). The AFPKs form a clade with the animal FAS, providing an evolutionary link bridging the type I PKSs and the animal FAS. We found molluscan AFPK diversification correlated with shell loss, suggesting AFPKs provide a chemical defense. Arthropods have few or no PKSs, but our results indicate AFPKs contributed to their ecological and evolutionary success by facilitating branched hydrocarbon and pheromone biosynthesis. Although animal metabolism is well studied, surprising new metabolic enzyme classes such as AFPKs await discovery.

Fatty Acid Origin of Insect Pheromones.

Pheromones are utilized to a great extent in insects. Many of these pheromones are biosynthesized through a pathway involving fatty acids. This chapter will provide examples where the biosynthetic pathways of fatty acid-derived pheromones have been studied in detail. These include pheromones from Lepidoptera, Coleoptera, and Hymenoptera. Many species of Lepidoptera utilize fatty acids as precursors to pheromones with a functional group that include aldehydes, alcohols, and acetate esters. In addition, the biosynthesis of hydrocarbons will be briefly examined because many insects utilize hydrocarbons or modified hydrocarbons as pheromones.

Insights into unique features of Drosophila CYP4G enzymes

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most intriguing insect P450s in terms of structure and function. In Drosophila, CYP4G1 is highly expressed in the oenocytes and is the last enzyme in the biosynthesis of cuticular hydrocarbons, while CYP4G15 is expressed in the brain and is of unknown function. Both proteins have a CYP4G-specific and characteristic amino acid sequence insertion corresponding to a loop between the G and H helices whose function is unclear. Here we address these enigmatic structural and functional features of Drosophila CYP4Gs. First, we used reverse genetics to generate D. melanogaster strains in which all or part of the CYP4G-specific loop was removed from CYP4G1. We showed that the full loop was not needed for proper folding of the P450, but it is essential for function, and that just a short stretch of six amino acids is required for the enzyme’s ability to make hydrocarbons. Second, we confirmed by immunocytochemistry that CYP4G15 is expressed in the brain and showed that it is specifically associated with the cortex glia cell subtype. We then expressed CYP4G15 ectopically in oenocytes, revealing that it can produce of a blend of hydrocarbons, albeit to quantitatively lower levels resulting in only a partial rescue of CYP4G1 knockdown flies. The CYP4G1 structural variants studied here should facilitate the biochemical characterization of CYP4G enzymes. Our results also raise the question of the putative role of hydrocarbons and their synthesis by cortex glial cells.

Front Cover: Metalloenzymes for Fatty Acid‐Derived Hydrocarbon Biosynthesis: Nature's Cryptic Catalysts (Chem. Asian J. 10/2022)

Hydrocarbon biosynthesis has gained tremendous attention in recent years due to its implication in the development of next-generation advanced biofuels. In nature, various organisms such as plants, insects, bacteria, and cyanobacteria produce fatty acid-derived alka(e)nes. Most of the enzymes involved in fatty acid-derived hydrocarbon biosynthesis are metalloenzymes, which employ divergent and intriguing catalytic mechanisms. Herein, metalloenzymes are reviewed in detail with special emphasis on their structure and mechanism. More information can be found in the Review by Debasis Das et al.

Metalloenzymes for Fatty Acid-Derived Hydrocarbon Biosynthesis: Nature's Cryptic Catalysts.

Waning resources, massive energy consumption, ever-deepening global warming crisis, and climate change have raised grave concerns regarding continued dependence on fossil fuels as the predominant source of energy and generated tremendous interest for developing biofuels, which are renewable. Hydrocarbon-based 'drop-in' biofuels can be a proper substitute for fossil fuels such as gasoline or jet fuel. In Nature, hydrocarbons are produced by diverse organisms such as insects, plants, bacteria, and cyanobacteria. Metalloenzymes play a crucial role in hydrocarbons biosynthesis, and the past decade has witnessed discoveries of a number of metalloenzymes catalyzing hydrocarbon biosynthesis from fatty acids and their derivatives employing unprecedented mechanisms. These discoveries elucidated the enigma related to the divergent chemistries involved in the catalytic mechanisms of these metalloenzymes. There is substantial diversity in the structure, mode of action, cofactor requirement, and substrate scope among these metalloenzymes. Detailed structural analysis along with mutational studies of some of these enzymes have contributed significantly to identifying the key amino acid residues that dictate substrate specificity and catalytic intricacy. In this Review, we discuss the metalloenzymes that catalyze fatty acid-derived hydrocarbon biosynthesis in various organisms, emphasizing the active site architecture, catalytic mechanism, cofactor requirements, and substrate specificity of these enzymes. Understanding such details is essential for successfully implementing these enzymes in emergent biofuel research through protein engineering and synthetic biology approaches.

Synthetic metabolic pathways for conversion of CO2 into secreted short-to medium-chain hydrocarbons using cyanobacteria

The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of reaction ingredients, it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the reaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis even in the Synechocystis strains expressing GmLOX1. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8–C10 preferring thioesterases ('CaFatB3.5 and 'ChoFatB2.2). When UndB instead was combined with a C12-specific 'UcFatB1 thioesterase, this resulted in a ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Preliminary optimization of the RBS, promoter and gene order in some of the synthetic operons resulted in improved 1-alkene productivity, reaching a titer of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was implemented with several different metabolic systems. Optimal productivity was observed with UndB and a C12 chain-length specific thioesterase, although further optimization of the entire biosynthetic system is still possible.

Recombinant Expression and Purification of Large Bacterial Multienzyme Assemblies for Biosynthetic Processes.

Many biosynthetic transformations have strict spatial and temporal requirements that necessitate the physical association of multiple enzymes for proper function. Here, we describe protocols for obtaining large multienzyme assemblies (>500kDa) by recombinant expression in Escherichia coli. We focus on assemblies from stand-alone enzymes joined by intermolecular forces rather than multiple catalytic domains from a single polypeptide chain. Details are given for strategies to optimize protein expression and to design a multi-affinity tag purification scheme for large multienzyme assemblies. These insights are drawn from our study of bacterial hydrocarbon biosynthesis.

Insights into in vitro phenotypic plasticity, growth and secondary metabolites of the mycobiont isolated from the lichen Platygramme caesiopruinosa.

The choice of inoculum for successful isolation and establishment of axenic lichen mycobiont culture is a key step towards eliminating endolichenic and lichenicolous fungi and other microbial contamination. The nutritional requirements of each lichen species are unique. This work reports on the isolation, phenotypic plasticity, growth and secondary metabolites from mycobiont culture of the pantropical lichen Platygramme caesiopruinosa. Media composition [Malt yeast extract (MY), Modified Murashige and Skoog (MMS) and Lilly and Barnett (LB) media] was optimized to determine nutritional requirements for optimal growth of this species as assessed by dry biomass and the occurrence of secondary metabolite. Furthermore, the role of different carbon sources in affecting growth, growth stages, phenotypic plasticity, biomass and spectrum of secondary metabolites produced of this mycobiont was examined. The molecular identity of the mycobiont culture was confirmed by amplifying mitochondrial small subunit (mtSSU) sequences. Cultures showed optimum biomass production in MYmediumwith 10% sucrose. The secondary metabolite profiles for each culture treatment were characterized using High-performance Thin-Layer Chromatography (HPTLC) and Gas Chromatography with Mass Spectrometric (GC-MS) analysis. The HPTLC spectral comparison, phenolic and iodine confirmatory analysis revealed the absence of phenolic metabolites and the presence of non-phenolic metabolites in mycobiont extracts, while GC-MS analysis revealed the biosynthesis of side chain fatty acids, hydrocarbons and sugar alcohol in mycobiont cultures treated with 10% supplemented sucrose as a carbon source.

Exopolysaccharides frommicroalgae: production, characterization, optimization and techno-economic assessment.

Microalgae cultivation for exopolysaccharide production has getting more attention as a result of their high hydrocarbon biosynthesis skill. The aim of this study is to examine the exopolysaccharide production potential of different species of microalgae. In this context, exopolysaccharides were produced from Chlorella minutissima, Chlorella sorokiniana and Botryococcus braunii microalgae and the effects of carbon and nitrogen content in the growth medium and illumination time on exopolysaccharide production were analyzed statistically using Box-Behnken experimental design. In addition, techno-economic assessment of exopolysaccharide production were also performed by using the most productive microalgae and optimum conditions determined in this study. As a result of the experiments, it was seen that C. minutissima, C. sorokiniana and B. braunii produced 0.245 ± 0.0025g/L, 0.163 ± 0.0016g/L and 0.117 ± 0.0007g/L exopolysaccharide, respectively. Statistically, it was observed that there was an inverse relationship between the exopolysaccharide production and investigated parameters such as illumination period and carbon and nitrogen amounts of culture mediums. The techno-economic assessment comprising microalgal exopolysaccharide (EPS) bioprocess was carried out, and it showed that the system can be considered economically viable, yet can be improved with biorefinery approach.

A novel characteristic of a phytoplankton as a potential source of straight-chain alkanes

Biosynthesis of hydrocarbons is a promising approach for the production of alternative sources of energy because of the emerging need to reduce global consumption of fossil fuels. However, the suitability of biogenic hydrocarbons as fuels is limited because their range of the number of carbon atoms is small, and/or they contain unsaturated carbon bonds. Here, we report that a marine phytoplankton, Dicrateria rotunda, collected from the western Arctic Ocean, can synthesize a series of saturated hydrocarbons (n-alkanes) from C10H22 to C38H78, which are categorized as petrol (C10–C15), diesel oils (C16–C20), and fuel oils (C21–C38). The observation that these n-alkanes were also produced by ten other cultivated strains of Dicrateria collected from the Atlantic and Pacific oceans suggests that this capability is a common characteristic of Dicrateria. We also identified that the total contents of the n-alkanes in the Arctic D. rotunda strain increased under dark and nitrogen-deficient conditions. The unique characteristic of D. rotunda could contribute to the development of a new approach for the biosynthesis of n-alkanes.

Research progress of the decarboxylation mechanisms of fatty acids to generate aliphatic hydrocarbons catalyzed by iron-containing enzymes

With the progress of human society, the consumption of energy materials is increasing in particularly the fossil fuels, which accounts for 84.7% of the total global consumption in 2018. Thus, excessive exploitation of fossil fuels not only brings about an increasingly serious energy crisis, but also leads to a series of environmental problems, such as the air quality deterioration and soil acidification, which are not conducive to the sustainable development of society. Since the 21st Annual Conference of the China Association for Science and Technology in 2019, “renewable synthetic fuels” has become one of the focuses of attention, and it also puts forward higher requirements for the research and development of renewable energy. About 60 years ago, many plants, insects, microorganisms and microalgae were found to metabolize hydrocarbons. In 2010, Schirmer’s groups recognized the alkane biosynthesis pathways of marine chlorobacteria, which aroused great interest in hydrocarbon biosynthesis. Recently, more proteases to catalyze the synthesis of hydrocarbons have been successively discovered, such as aldehyde deformylase (ADO, CYP4G and CER), fatty acid decarboxylase (UndA, UndB and OleT), polyketone synthase (Ols) and thiolytic enzyme complex system (OleABCD). In the past decades, many studies have been conducted to explore the biosynthesis of hydrocarbon, and useful information regarding the reaction mechanism has been obtained, including the kinetic rate constants and the crystal structures of the enzymes. In addition, on the basis of crystal structure, quantum chemistry method (QM) and molecular dynamics simulations (MD) were also used to explore the reaction mechanism of fatty acid decarboxylation. Since most enzymes involved in the hydrocarbon biosynthesis are iron-containing enzymes, which employ the Fe-coordinate dioxygen to trigger the reaction. In general, the whole reaction cycle contains the binding of substrates and dioxygen, and decarboxylation usually involves the hydrogen atom abstraction and breaking of C−C bond. During the reaction, complex electron transfer takes place among the Fe center, O2 and substrate, and many factors were found to affect the catalytic activity. According to the previous experimental and theoretical studies, different enzymes show different activity and substrate scope. In general, the catalytic efficiency is still very low for most enzymes, and thus, understanding the mechanism of these catalytic reactions is undoubtedly an important basis for engineering optimization and all applications, and can also provide ideas for the design and synthesis of biomimetic catalysts. In this paper, we review the research progress of five representative iron enzymes that catalyze the biosynthesis of aliphatic hydrocarbon, including OleT, UndA, UndB, ADO and CYP4G, and discuss the future development and application of enzyme catalysis in renewable synthetic fuels. This paper may offer some ideas and guidelines for sustainable energy supply in the future.

Connection between cuticular hydrocarbons and melanization in Harmonia axyridis revealed by RNAi-mediated silencing of the CYP4G79

The insect cuticle is a multifunctional shield providing shape stabilization and threat protection. It has a complex composition of cuticular components. Although cuticular melanization is essential and melanin spots are common for many insects, information about the connection between melanization and other cuticular components is limited. The ladybird Harmonia axyridis adults and pupae exhibit regular and predictable melanin spots on their cuticles. Here, RNAi-mediated silencing of CYP4G79 , which functions as an oxidative decarbonylase in hydrocarbon biosynthesis, decreased the cuticular hydrocarbon contents at different levels of H. axyridis , resulted in enlarging (mild) or breaking (severe) melanin spots on cuticle. In addition, transcriptional analysis showed key genes of cuticular melanization up-regulated under CYP4G79 RNAi; Scanning electron microscope (SEM) and Oil Red O (ORO)-staining results suggested a distribution correlation among Cuticular hydrocarbons (CHCs), lipids and melanin. For severe phenotypes with broken melanin spots of treated beetle’s pupal cuticle, histological examinations showed that abnormal pigmentation in cuticular layers were physiologically caused by cuticular microstructural change. In brief, these two cuticular components of H. axyridis , may function together; and cuticular hydrocarbons are essential to the formation of melanin spots. Furthermore, the orderly formations of cuticular components are important to cuticle construction; this study furthers our understanding of insect cuticle formation and functions.

Chemical Ecology, Biochemistry, and Molecular Biology of Insect Hydrocarbons.

Insect cuticular hydrocarbons (CHCs) consist of complex mixtures of straight-chain alkanes and alkenes, and methyl-branched hydrocarbons. In addition to restricting water loss through the cuticle and preventing desiccation, they have secondarily evolved to serve a variety of functions in chemical communication and play critical roles as signals mediating the life histories of insects. In this review, we describe the physical properties of CHCs that allow for both waterproofing and signaling functions, summarize their roles as inter- and intraspecific chemical signals, and discuss the influences of diet and environment on CHC profiles. We also present advances in our understanding of hydrocarbon biosynthesis. Hydrocarbons are biosynthesized in oenocytes and transported to the cuticle by lipophorin proteins. Recent work on the synthesis of fatty acids and their ultimate reductive decarbonylation to hydrocarbons has taken advantage of powerful new tools of molecular biology, including genomics and RNA interference knockdown of specific genes, to provide new insights into the biosynthesis of hydrocarbons.

P-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA

The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C8) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and β-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p-nitrophenyl alkanoate esters as surrogate substrates. In the present study, p-nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p-Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p-nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p-nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p-nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p-nitrophenyl 1-13C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and β-lactone natural products for biotechnological applications using cheap and easily synthesized p-nitrophenyl esters.

Engineering an Alcohol-Forming Fatty Acyl-CoA Reductase for Aldehyde and Hydrocarbon Biosynthesis in Saccharomyces cerevisiae.

Aldehydes are a class of highly versatile chemicals that can undergo a wide range of chemical reactions and are in high demand as starting materials for chemical manufacturing. Biologically, fatty aldehydes can be produced from fatty acyl-CoA by the action of fatty acyl-CoA reductases. The aldehydes produced can be further converted enzymatically to other valuable derivatives. Thus, metabolic engineering of microorganisms for biosynthesizing aldehydes and their derivatives could provide an economical and sustainable platform for key aldehyde precursor production and subsequent conversion to various value-added chemicals. Saccharomyces cerevisiae is an excellent host for this purpose because it is a robust organism that has been used extensively for industrial biochemical production. However, fatty acyl-CoA-dependent aldehyde-forming enzymes expressed in S. cerevisiae thus far have extremely low activities, hence limiting direct utilization of fatty acyl-CoA as substrate for aldehyde biosynthesis. Toward overcoming this challenge, we successfully engineered an alcohol-forming fatty acyl-CoA reductase for aldehyde production through rational design. We further improved aldehyde production through strain engineering by deleting competing pathways and increasing substrate availability. Subsequently, we demonstrated alkane and alkene production as one of the many possible applications of the aldehyde-producing strain. Overall, by protein engineering of a fatty acyl-CoA reductase to alter its activity and metabolic engineering of S. cerevisiae, we generated strains with the highest reported cytosolic aliphatic aldehyde and alkane/alkene production to date in S. cerevisiae from fatty acyl-CoA.

Deciphering the role of Rhodnius prolixus CYP4G genes in straight and methyl-branched hydrocarbon formation and in desiccation tolerance.

Insect cuticle hydrocarbons are involved primarily in waterproofing the cuticle, but also participate in chemical communication and regulate the penetration of insecticides and microorganisms. The last step in insect hydrocarbon biosynthesis is carried out by an insect-specific cytochrome P450 of the 4G subfamily (CYP4G). Two genes (CYP4G106 and CYP4G107) have been reported in the triatomines Rhodnius prolixus and Triatoma infestans. In this work, their molecular and functional characterization is carried out in R. prolixus, and their relevance to insect survival is assessed. Both genes are expressed almost exclusively in the integument and have an expression pattern dependent on the developmental stage and feeding status. CYP4G106 silencing diminished significantly the straight-chain hydrocarbon production while a significant reduction - mostly of methyl-branched chain hydrocarbons - was observed after CYP4G107 silencing. Molecular docking analyses using different aldehydes as hydrocarbon precursors predicted a better fit of straight-chain aldehydes with CYP4G106 and methyl-branched aldehydes with CYP4G107. Survival bioassays exposing the silenced insects to desiccation stress showed that CYP4G107 is determinant for the waterproofing properties of the R. prolixus cuticle. This is the first report on the in vivo specificity of two CYP4Gs to make mostly straight or methyl-branched hydrocarbons, and also on their differential contribution to insect desiccation.