Fatty Acid Biosynthetic Pathway Research Articles

Identification and Functional Characterization of a Novel Δ12 Fatty Acid Desaturase Gene from Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis accumulates large amounts of fatty acids in response to adverse conditions. However, the key fatty acid desaturase genes in H. pluvialis remain unknown. In this study, we cloned and functionally characterized a A12 fatty acid desaturase gene, and designated it as HpFAD2. The open reading frame of HpFAD2 consisted of 1137 base pairs and encoded 378 amino acids. The deduced polypeptide showed 70% identity to other endoplasmic reticulum Δ12 fatty acid desaturases, whereas it had only 44% identity to plastid Δ12 fatty acid desaturases. The PSORT algorithm and phylogenetic analysis further confirmed its affiliation to the endoplasmic reticulum Δ12 fatty acid desaturases. Heterologous expression was performed in Saccharomyces cerevisiae cells transformed with the recombinant plasmid pYES2-HpFAD2. Two additional fatty acids (C16:2 and C18:2) were detected in the yeast transformants. The results indicated Δ12 desaturation activity and substrate preference for C18:1 over C16:1. The transcriptional levels of H. pluvialis HpFAD2 at different growth stages were measured by quantitative polymerase chain reaction (PCR), indicating that the HpFAD2 transcriptional levels were significantly higher in red cells than those in green cells. Our study brings more insight into the fatty acid biosynthetic pathway of H. pluvialis.

Nonactive-Site Mutations in S. aureus FabI That Induce Triclosan Resistance.

The wide use of the antimicrobial agent/biocide, triclosan, promotes triclosan-resistant bacterial strains, including Staphylococcus aureus, as well as leads to accumulation in the aquatic and terrestrial environments. Knowledge of the molecular actions of triclosan on S. aureus is needed to understand the consequence of triclosan resistance and environmental accumulation of triclosan on S. aureus resistant strains, as well as to develop biphenyl ether analogs as antibiotic candidates. Triclosan inhibits an essential enzyme in the fatty acid biosynthetic pathway, the reduced nicotinamide adenine dinucleotide (NADH)/reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent enoyl-acyl carrier protein (enoyl-ACP) reductase, or FabI. In this study, we used error-prone polymerase chain reaction (epPCR) to generate mutations in the S. aureus FabI enzyme. Instead of using an elaborate FabI enzyme activity assay that involves ACP-linked substrates to determine whether triclosan inhibits the enzyme activities of individual FabI mutants, we used an efficient and economical assay that we developed, based on thermal shift principles, to screen for triclosan binding to FabI mutants in cells. We identified four active-site mutations. More interestingly, we also identified nine triclosan-resistant mutations distant from the active site (G113V, Y123H, S166N, N220I, G227C, A230T, V241I, F252I, and H253P) but located in disparate positions in the monomer–monomer and dimer–dimer interface regions in S. aureus FabI. We suggest that these sites may serve as potential allosteric sites for designing potential therapeutic inhibitors that offer advantages in selectivity since allosteric sites are less evolutionarily conserved.

OsPLS4 Is Involved in Cuticular Wax Biosynthesis and Affects Leaf Senescence in Rice

Leaf senescence is one of the most common factors that affects the growth and yield of rice. Although numerous genes affecting leaf senescence have been identified, few involved in cuticular wax synthesis have been described for rice premature leaf senescence. Here, we cloned and characterized Premature Leaf Senescence 4 (PLS4) in rice (Oryza sativa), which encodes a putative 3-oxoacyl-reductase in the fatty acid biosynthetic pathway. Subcellular localization of OsPLS4 was observed in the chloroplast. A single nucleotide substitution in OsPLS4 reduced leaf cuticular wax, and the expression levels of most wax biosynthesis-associated genes were downregulated. TEM showed chloroplast development were defective in the pls4 mutant. Further investigation revealed that the chlorophyll (Chl) content was reduced in the pls4 mutant compared with the WT and that the photosynthesis rate was lower, which caused ROS dramatic accumulation at the heading stage. These results confirmed premature leaf senescence in pls4 plants. Cold treatment indicated that the mutant was more sensitive than the WT was to cold stress. Together, all the above results indicate that the OsPLS4 mutation affects cuticular wax biosynthesis and chloroplast development in rice, causing reduced cuticular wax and premature leaf senescence.

Identification of novel antiplasmodial compound by hierarquical virtual screening and in vitro assays

Malaria is an infectious disease caused by protozoa of the genus Plasmodium spp. with approximately 219 million cases in 2017. P. falciparum is main responsible for the most severe form of the disease, cerebral malaria. Despite of public health impacts, chemotherapy against malaria is still limited due to the emergence of drug resistance cases used in monotherapy and combination therapies. Thus, the development of new antimalarial drugs becomes emergency. One way of achieve this goal is to explore essential and/or unique therapeutic targets of the parasite, or at least sufficiently different to ensure selective inhibition. Enoil-ACP reductase (ENR) is a NADH-dependent enzyme responsible for the limiting step of the type II fatty acid biosynthetic pathway (FAS II). Thus, pharmacophore and docking based virtual screening were applied to prioritize molecules for in vitro assays against P. falciparum W2 strain. The application of successive filters at OOCC database (n = 618) resulted in the identification of one molecule (13) (EC50 = 0.098 ± 0.021 μM) with similar biological activity to artemether. The molecule 13 is a typical drug repurposing case due to previous other approved therapeutic uses on Chinese medicine as a non-specific cholinergic antagonist, thus it could be accelerated the drug development process. Additionally, molecular dynamics studies were used to confirm stability of the molecular interactions identified by molecular docking. Thus, representative structures of P. falciparum ENR can be used in a study to propose new derivatives for evaluation of biological activity in vitro and in vivo. Communicated by Ramaswamy H. Sarma

AtMYB92 enhances fatty acid synthesis and suberin deposition in leaves of Nicotiana benthamiana.

Acyl lipids are important constituents of the plant cell. Depending on the cell type, requirements in acyl lipids vary greatly, implying a tight regulation of fatty acid and lipid metabolism. The discovery of the WRINKLED1 (WRI1) transcription factors, members of the AP2-EREBP (APETALA2-ethylene-responsive element binding protein) family, has emphasized the importance of transcriptional regulation for adapting the rate of acyl chain production to cell requirements. Here, we describe the identification of another activator of the fatty acid biosynthetic pathway, the Arabidopsis MYB92 transcription factor. This MYB andall themembers of the subgroups S10 and S24 of MYB transcription factors can directly activate the promoter of BCCP2 that encodes a component of the fatty acid biosynthetic pathway. Two adjacent MYB cis-regulatory elements are essential for the binding and activation of the BCCP2 promoter by MYB92. Overexpression of MYB92 or WRI1 in Nicotiana benthamiana induces the expression of fatty acid biosynthetic genes but results in the accumulation of different types of acyl lipids. In the presence of WRI1, triacylglycerol biosynthetic enzymes coded by constitutively expressed genes efficiently channel the excess fatty acids toward reserve lipid accumulation. By contrast, MYB92 activates both fatty acid and suberin biosynthetic genes; hence, the remarkable increase in suberin monomers measured in leaves expressing MYB92. These results provide additional insight into the molecular mechanisms that control the biosynthesis of an important cell wall-associated acylglycerol polymer playing critical roles in plants.

Linoleic acid induces metabolic stress in the intestinal microorganism Bifidobacterium breve DSM 20213

Despite clinical and research interest in the health implications of the conjugation of linoleic acid (LA) by bifidobacteria, the detailed metabolic pathway and physiological reasons underlying the process remain unclear. This research aimed to investigate, at the molecular level, how LA affects the metabolism of Bifidobacterium breve DSM 20213 as a model for the well-known LA conjugation phenotype of this species. The mechanisms involved and the meaning of the metabolic changes caused by LA to B. breve DSM 20213 are unclear due to the lack of comprehensive information regarding the responses of B. breve DSM 20213 under different environmental conditions. Therefore, for the first time, an untargeted metabolomics-based approach was used to depict the main changes in the metabolic profiles of B. breve DSM 20213. Both supervised and unsupervised statistical methods applied to the untargeted metabolomic data allowed confirming the metabolic changes of B. breve DSM 20213 when exposed to LA. In particular, alterations to the amino-acid, carbohydrate and fatty-acid biosynthetic pathways were observed at the stationary phase of growth curve. Among others, significant up-regulation trends were detected for aromatic (such as tyrosine and tryptophan) and sulfur amino acids (i.e., methionine and cysteine). Besides confirming the conjugation of LA, metabolomics suggested a metabolic reprogramming during the whole growth curve and an imbalance in redox status following LA exposure. Such redox stress resulted in the down-accumulation of peroxide scavengers such as low-molecular-weight thiols (glutathione- and mycothiol-related compounds) and ascorbate precursors, together with the up-accumulation of oxidized (hydroxy- and epoxy-derivatives) forms of fatty acids. Consistently, growth was reduced and the levels of the oxidative stress marker malondialdehyde were higher in LA-exposed B. breve DSM 20213 than in the control.

Research advances of WRINKLED1 (WRI1) in plants.

WRINKLED 1 (WRI1), a member of the AP2/EREBP class of transcription factors, regulates carbon allocation between the glycolytic and fatty acid biosynthetic pathways and plays important roles in other biological events. Previous studies have suggested that post-translational modifications and interacting partners modulate the activity of WRI1. We systematically summarised the structure of WRI1 as well as its molecular interactions during transcription and translation in plants. This work elucidates the genetic evolution and regulatory functions of WRI1 at the molecular level and describes a new pathway involving WRI1 that can be used to produce triacylglycerols (TAGs) in plants.

Phenomics reveals a novel putative chloroplast fatty acid transporter in the marine diatom Skeletonema marinoi involved in temperature acclimation

Diatoms are the dominant phytoplankton in temperate oceans and coastal regions and yet little is known about the genetic basis underpinning their global success. Here, we address this challenge by developing the first phenomic approach for a diatom, screening a collection of randomly mutagenized but identifiably tagged transformants. Based upon their tolerance to temperature extremes, several compromised mutants were identified revealing genes either stress related or encoding hypothetical proteins of unknown function. We reveal one of these hypothetical proteins is a novel putative chloroplast fatty acid transporter whose loss affects several fatty acids including the two omega-3, long-chain polyunsaturated fatty acids - eicosapentaenoic and docosahexaenoic acid, both of which have medical importance as dietary supplements and industrial significance in aquaculture and biofuels. This mutant phenotype not only provides new insights into the fatty acid biosynthetic pathways in diatoms but also highlights the future value of phenomics for revealing specific gene functions in these ecologically important phytoplankton.

Novel fatty acid-thiadiazole derivatives as potential antimycobacterial agents.

The discovery of antibiotics around the middle twentieth century led to a decrease in the interest in antimycobacterial fatty acids. In order to re-establish the importance of naturally abundant fatty acid, a series of fatty acid-thiadiazole derivatives were designed and synthesized based on molecular hybridization approach. In vitro antimycobacterial potential was established by a screening of synthesized compounds against Mycobacterium tuberculosis H37Rv strain. Among them, compounds 5a, 5d, 5h, and 5j were the most active, with compound 5j exhibiting minimum inhibitory concentration of 2.34μg/ml against M.tb H37Rv. Additionally, the compounds were docked to determine the probable binding interactions and understand the mechanism of action of most active molecules on enoyl-acyl carrier protein reductases (InhA), which is involved in the mycobacterium fatty acid biosynthetic pathway.

Engineering Escherichia coli FAB system using synthetic plant genes for the production of long chain fatty acids

BackgroundSustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition.ResultsIn this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli.ConclusionThe efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Summary Chlorella vulgaris is a fast‐growing fresh‐water microalga cultivated on the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed us to assemble the nuclear genome in 14 pseudo‐molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA‐seq data obtained at two different irradiances of growth (high light, HL versus low light, LL) enabled us to identify 10 724 nuclear genes, coding for 11 082 transcripts. Moreover, 121 and 48 genes, respectively, were found in the chloroplast and mitochondrial genome. Functional annotation and expression analysis of nuclear, chloroplast and mitochondrial genome sequences revealed particular features of Chlorella vulgaris. Evidence of horizontal gene transfers from chloroplast to mitochondrial genome was observed. Furthermore, comparative transcriptomic analyses of LL versus HL provided insights into the molecular basis for metabolic rearrangement under HL versus LL conditions leading to enhanced de novo fatty acid biosynthesis and triacylglycerol accumulation. The occurrence of a cytosolic fatty acid biosynthetic pathway could be predicted and its upregulation upon HL exposure was observed, consistent with the increased lipid amount under HL conditions. These data provide a rich genetic resource for future genome editing studies, and potential targets for biotechnological manipulation of Chlorella vulgaris or other microalgae species to improve biomass and lipid productivity.

Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival.

Chrysin reduces the activity and protein level of mature forms of sterol regulatory element-binding proteins.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of genes involved in fatty acid and cholesterol biosynthetic pathways. The present study showed that the flavonoid chrysin impairs the fatty acid synthase promoter. Chrysin reduces the expression of SREBP target genes, such as fatty acid synthase, in human hepatoma Huh-7 cells and impairs de novo synthesis of fatty acids and cholesterol. Moreover, it reduces the endogenous mature, transcriptionally active forms of SREBPs, which are generated by the proteolytic processing of precursor forms. In addition, chrysin reduces the enforced expressing mature forms of SREBPs and their transcriptional activity. The ubiquitin-proteasome system is not involved in the chrysin-mediated reduction of SREBPs mature forms. These results suggest that chrysin suppresses SREBP activity, at least partially, via the degradation of SREBPs mature forms. Abbreviations: ACC1: acetyl-CoA carboxylase 1; DMEM: Dulbecco's modified Eagle's medium; FAS: fatty acid synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 25-HC: 25-hydroxycholesterol; HMGCS: HMG-CoA synthase; LDH: lactate dehydrogenase; LPDS: lipoprotein-deficient serum; PI3K: phosphatidylinositol 3-kinase; SCD1: stearoyl-CoA desaturase; SREBPs: sterol regulatory element-binding proteins.

Chemical synthesis, molecular modeling and pharmacophore mapping of new pyrrole derivatives as inhibitors of InhA enzyme and Mycobacterium tuberculosis growth

Substituted phenylthiazolyl benzamide and pyrrolyl benzamide derivatives were developed using molecular hybridization technique to create novel lead antimycobacterial molecules used to fight against Mycobacteriumtuberculosis. The newly synthesized molecules have inhibited InhA, the enoyl-ACP reductase enzyme from the mycobacterial type II fatty acid biosynthetic pathway. Of these, compound 3b showed H-bonding interactions with Tyr158 and co-factor NAD+ that binds the active site of InhA. All the molecules were screened for in vitro antitubercular activity against M. tuberculosis H37Rv, as well as some representative molecules as the inhibitors of InhA. Thirteen compounds exhibited good anti-TB activities (MIC = 1.6 μg/mL), but only few representative molecules showed the moderate InhA enzyme inhibition activity.

Maternal BMI Modulates Placental Expression of Lipid Transport, Metabolism and Innate Immune Response Genes at Term (OR35-01-19)

ObjectivesMaternal body composition during pregnancy is an important determinant of offspring obesity risk. One hypothesized mechanism linking maternal and offspring obesity is via alterations in placental gene expression and function. Here we examined the influence of early pregnancy maternal obesity on placental gene expression at term using transcriptomic approaches. MethodsWe examined placenta collected from a longitudinal observational study of mothers and infants where maternal anthropometrics, body composition, dietary intake (3 d food records) and other variables were collected every trimester in the mothers. Placentas were collected within 30 min of delivery and archived for molecular and histologic analysis. For the current analyses, transcriptomic analysis of villous placental tissues from normal weight (NW, N = 71), and overweight/obese women (OW, N = 70, based on BMI < 10 wk of pregnancy) were performed using strand-specific mRNA-seq. Data analyses were carried out using the STAR-Seqmonk-Limma pipeline. ResultsRNA-seq analysis provided quantitative information on expression of 13,831 transcripts (> 2 counts/million in 50% of samples). Assessment via principal component analysis identified significant effects of maternal BMI (in male infants), infant sex and mode of delivery on placental gene expression. Linear regression of mRNA expression with early pregnancy maternal BMI (adjusting for mode of delivery and sex) identified significant association with 230 transcripts (P < 0.05). Likewise, regression analysis of maternal body fat identified significant associations with 271 genes, with 99 genes shared in common between BMI and body fat. These transcripts were enriched for cell morphogenesis, fatty acid biosynthetic and lipid transport related pathways. Of note, expression of ABCG1 (cholesterol transporter), MMP1, CEBPα and PRKAG2 (AMPKγ2) were negatively associated with maternal BMI (P < 0.05). mRNA expression of MHC Class II antigens DRB1 and DRB5, critical in antigen presentation and immune response was increased (> 1.8-fold) in male placenta of OW women. ConclusionsIn a large cohort of well-characterized pregnant women maternal adiposity status in early pregnancy influences term placental expression of genes involved in lipid transport, metabolism and innate immune response in a sexually dimorphic manner. Funding SourcesSupported by USDA-ARS Project 6026-51000-010-05S.

Metabolic Engineering Strategies for the Enhanced Microalgal Production of Long-Chain Polyunsaturated Fatty Acids (LC-PUFAs).

Long-chain polyunsaturated fatty acids (LC-PUFAs), largely obtained from fish oil, serve as valuable dietary supplements with many health benefits, especially arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. In recent years, interest in the sustainable production of LC-PUFAs using heterologous production hosts has drastically increased because overfishing and polluted oceans have led to a gradual decline in the supply of LC-PUFAs from fish oil in the last few decades. Some species of microalgae have been considered to be an ideal producer of LC-PUFAs as they inherently accumulate large amounts of LC-PUFAs and utilize CO2 using light energy. Also, a growing number of genetic toolboxes have become available, and the microalgal cultivation process is amenable to a scale-up. In fact,tremendous progress has been achieved in the development of high-performance strains of microalgae through metabolic engineering that has led to the efficient production of fatty acids and various derivatives in the last decade. This review discusses metabolic engineering strategies that have contributed to the enhanced production of LC-PUFAs using microalgae, including optimizing fatty acid biosynthetic pathway and intracellular supply of precursors and cofactors, as well as engineering transcription factors.

The Discrepancy of Fatty Acid Composition of Astaxanthin Esters and Total Fatty Acids in Photoautotrophic and Heterotrophic Chlorella zofingiensis

Abstract Chlorella zofingiensis has great potential for astaxanthin and fatty acid production. This study investigated the accumulation of astaxanthin and total fatty acids (TFA), the fatty acid compositions of astaxanthin esters and TFA in photoautotrophic and heterotrophic C. zofingiensis, respectively. The results displayed that in photoautotrophic cells, 18:3 was the most predominant fatty acid, followed by 16:0, 18:2, and 18:1, while astaxanthin mono‐esters (mono‐AE) contained only 16:0 and 18:0, and astaxanthin di‐esters (di‐AE) contained 16:0 mostly, followed by 18:1, 18:0, 18:2, and 18:3. C. zofingiensis accumulated the highest level of astaxanthin and TFA under heterotrophic conditions fed with 30 g L−1 glucose. The percentage of 18:1 in TFA, mono‐AE, and di‐AE increased from 10.05%, 0%, and 17.76% to 35.80%, 14.84%, and 50.12%, respectively; in contrast, the percentages of 18:3 in TFA and di‐AE were observed to decrease from 40.42% and 7.59% to 9.56% and 1.30%, respectively, in comparison with those in photoautotrophic ones. The RNA sequencing (RNA‐seq) results showed that the expressions of the genes in astaxanthin and fatty acid biosynthetic pathways were increased under this heterotrophic condition. These results indicated differential accumulation of AE and TFA, especially fatty acid compositions of TFA and AE in photoautotrophic and heterotrophic cells, suggesting a positive correlation between astaxanthin and fatty acid biosynthesis.

Birth-and-Death Evolution of the Fatty Acyl-CoA Reductase (FAR) Gene Family and Diversification of Cuticular Hydrocarbon Synthesis in Drosophila.

The birth-and-death evolutionary model proposes that some members of a multigene family are phylogenetically stable and persist as a single copy over time, whereas other members are phylogenetically unstable and undergo frequent duplication and loss. Functional studies suggest that stable genes are likely to encode essential functions, whereas rapidly evolving genes reflect phenotypic differences in traits that diverge rapidly among species. One such class of rapidly diverging traits are insect cuticular hydrocarbons (CHCs), which play dual roles in chemical communications as short-range recognition pheromones as well as protecting the insect from desiccation. Insect CHCs diverge rapidly between related species leading to ecological adaptation and/or reproductive isolation. Because the CHC and essential fatty acid biosynthetic pathways share common genes, we hypothesized that genes involved in the synthesis of CHCs would be evolutionary unstable, whereas those involved in fatty acid-associated essential functions would be evolutionary stable. To test this hypothesis, we investigated the evolutionary history of the fatty acyl-CoA reductases (FARs) gene family that encodes enzymes in CHC synthesis. We compiled a unique data set of 200 FAR proteins across 12 Drosophila species. We uncovered a broad diversity in FAR content which is generated by gene duplications, subsequent gene losses, and alternative splicing. We also show that FARs expressed in oenocytes and presumably involved in CHC synthesis are more unstable than FARs from other tissues. Taken together, our study provides empirical evidence that a comparative approach investigating the birth-and-death evolution of gene families can identify candidate genes involved in rapidly diverging traits between species.

Yeast engineering to express sex pheromone gland genes of the oriental fruit moth, Grapholita molesta

Abstract Mating disruption by using sex pheromone is an ecofriendly alternative way to control insect pests. To be effective, large amounts of sex pheromone are needed, leading to a relatively high production cost. To reduce the cost for chemical synthesis of sex pheromone, yeast engineering technology has been devised. This study used a baker's yeast, Saccharomyces cerevisiae, to express genes associated with sex pheromone biosynthesis of the Oriental fruit moth, Grapholita molesta. Compared to other fatty acid biosynthetic pathways, two steps that are unique to pheromone gland of G. molesta are proposed: desaturation at even number catalyzed by desaturase (Gm-DES) and terminal reduction catalyzed by fatty acyl reductase (Gm-FAR). Gm-DES and Gm-FAR were cloned into a yeast expression vector, pYES2.1. They were used to transform S. cerevisiae by a double transfection method. The transformed yeast was induced with 2% galactose to over-express these two exogenous genes. Their expression was confirmed by RT-PCR and western blotting. To facilitate pheromone production, transformed yeasts were supplied with myristic acid during over-expression. Resulting fatty acid composition was analyzed by GC-MS after fatty acid methyl ester derivatization. Control yeast produced mostly saturated fatty acids. However, a single gene (Gm-DES)-transformed yeast produced unsaturated fatty acids at ∆ 9 such as Z9-tetradecenoic acid (Z9-14:1), palmitoleic acid (Z9-16:1), and oleic acid (Z9-18:1) in addition to saturated fatty acids. The double-transformed yeast produced an additional component, alcohol form of oleic acid (Z9-18:OH). These results suggest that Gm-DES can catalyze desaturation of fatty acids at ∆ 9 and Gm-FAR can reduce terminal carboxylic acid into alcohol.

Computational structural enzymology methodologies for the study and engineering of fatty acid synthases, polyketide synthases and nonribosomal peptide synthetases.

Various computational methodologies can be applied to enzymological studies on enzymes in the fatty acid, polyketide, and non-ribosomal peptide biosynthetic pathways. These multi-domain complexes are called fatty acid synthases, polyketide synthases, and non-ribosomal peptide synthetases. These mega-synthases biosynthesize chemically diverse and complex bioactive molecules, with the intermediates being chauffeured between catalytic partners via a carrier protein. Recent efforts have been made to engineer these systems to expand their product diversity. A major stumbling block is our poor understanding of the transient protein-protein and protein-substrate interactions between the carrier protein and its many catalytic partner domains and product intermediates. The innate reactivity of pathway intermediates in two major classes of polyketide synthases has frustrated our mechanistic understanding of these interactions during the biosynthesis of these natural products, ultimately impeding the engineering of these systems for the generation of engineered natural products. Computational techniques described in this chapter can aid data interpretation or used to generate testable models of these experimentally intractable transient interactions, thereby providing insight into key interactions that are difficult to capture otherwise, with the potential to expand the diversity in these systems.