Acetylation of homogalacturonan and rhamnogalacturonan-I is catalyzed by a suite of trichome birefringence-like proteins.

Plant cell wall polysaccharides, including xylan, mannan, xyloglucan, and pectins, are often acetylated and members of the domain of unknown function 231 (DUF231)/trichome birefringence-like (TBL) family have been shown to be O-acetyltransferases mediating the acetylation of xylan, mannan, and xyloglucan. However, little is known about the O-acetyltransferases responsible for pectin acetylation. In this report, we biochemically characterized a suite of Arabidopsis DUF231/TBL proteins for their roles in pectin acetylation. We generated 24 TBL recombinant proteins in mammalian cells and demonstrated that 10 of them were able to transfer acetyl groups from acetyl-CoA onto the pectins homogalacturonan (HG) or rhamnogalacturonan-I (RG-I), and thus were named pectin O-acetyltransferase 1 to 10 (POAT1 to 10). It was found that POAT2,4,9,10 specifically acetylated HG and POAT5,6 acetylated RG-I, whereas POAT1,3,7,8 could act on both HG and RG-I. The acetylation of HG and RG-I by POATs was further corroborated by hydrolysis with pectin acetylesterases and by nuclear magnetic resonance spectroscopy. In addition, mutations of the conserved GDS and DXXH motifs in POAT3 and POAT8 were shown to lead to a loss of their ability to acetylate HG and RG-I. Furthermore, simultaneous RNA interference downregulation of POAT1,3,6,7,8 resulted in reduced cell expansion, impaired plant growth, and decreased pectin acetylation. Together, our findings indicate that these POATs are pectin O-acetyltransferases involved in acetylation of the pectin polysaccharides HG and RG-I.

Carnosol, a diterpene present in rosemary, increases ELP1 levels in familial dysautonomia patient-derived cells and healthy adults: a possible therapy for FD.

Recent research on familial dysautonomia (FD) has focused on the development of therapeutics that facilitate the production of the correctly spliced, exon 20-containing, transcript in cells and individuals bearing the splice-altering, FD-causing mutation in the elongator acetyltransferase complex subunit I (ELP1) gene. We report here the ability of carnosol, a diterpene present in plant species of the Lamiaceae family, including rosemary, to enhance the cellular presence of the correctly spliced ELP1 transcript in FD patient-derived fibroblasts by upregulating transcription of the ELP1 gene and correcting the aberrant splicing of the ELP1 transcript. Carnosol treatment also elevates the level of the RNA binding motif protein 24 (RBM24) and RNA binding motif protein 38 (RBM38) proteins, two multifunctional RNA-binding proteins. Transfection-mediated expression of either of these RNA binding motif (RBMs) facilitates the inclusion of exon 20 sequence into the transcript generated from a minigene-bearing ELP1 genomic sequence containing the FD-causing mutation. Suppression of the carnosol-mediated induction of either of these RBMs, using targeting siRNAs, limited the carnosol-mediated inclusion of the ELP1 exon 20 sequence. Carnosol treatment of FD patient peripheral blood mononuclear cells facilitates the inclusion of exon 20 into the ELP1 transcript. The increased levels of the ELP1 and RBM38 transcripts and the alternative splicing of the sirtuin 2 (SIRT2) transcript, a sentinel for exon 20 inclusion in the FD-derived ELP1 transcript, are observed in RNA isolated from whole blood of healthy adults following the ingestion of carnosol-containing rosemary extract. These findings and the excellent safety profile of rosemary together justify an expedited clinical study of the impact of carnosol on the FD patient population.

ELOVL5-mediated fatty acid elongation promotes cellular proliferation and invasion in renal cell carcinoma.

Renal cell carcinoma (RCC) features altered lipid metabolism and accumulated polyunsaturated fatty acids (PUFAs). Elongation of very long-chain fatty acid (ELOVL) family enzymes catalyze fatty acid elongation, and ELOVL5 is indispensable for PUFAs elongation, but its role in RCC progression remains unclear. Here, we show that higher levels of ELOVL5 correlate with poor RCC clinical prognosis. Liquid chromatography/electrospray ionization-tandem mass spectrometry analysis showed decreases in ELOVL5 end products (arachidonic acid and eicosapentaenoic acid) under CRISPR/Cas9-mediated knockout of ELOVL5 while supplementation with these fatty acids partially reversed the cellular proliferation and invasion effects of ELOVL5 knockout. Regarding cellular proliferation and invasion, CRISPR/Cas9-mediated knockout of ELOVL5 suppressed the formation of lipid droplets and induced apoptosis via endoplasmic reticulum stress while suppressing renal cancer cell proliferation and in vivo tumor growth. Furthermore, CRISPR/Cas9-mediated knockout of ELOVL5 inhibited AKT Ser473 phosphorylation and suppressed renal cancer cell invasion through chemokine (C-C motif) ligand-2 downregulation by AKT-mTOR-STAT3 signaling. Collectively, these results suggest that ELOVL5-mediated fatty acid elongation promotes not only cellular proliferation but also invasion in RCC.

Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L.

Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 µM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.

BAHD acetyltransferase contributes to wound-induced biosynthesis of oleo-gum resin triterpenes in Boswellia.

Triterpenes (30-carbon isoprene compounds) represent a large and highly diverse class of natural products that play various physiological functions in plants. The triterpene biosynthetic enzymes, particularly those catalyzing the late-stage regio-selective modifications are not well characterized. The bark of select Boswellia trees, e.g., B. serrata exudes specialized oleo-gum resin in response to wounding, which is enriched with boswellic acids (BAs), a unique class of C3α-epimeric pentacyclic triterpenes with medicinal properties. The bark possesses a network of resin secretory structures comprised of vertical and horizontal resin canals, and amount of BAs in bark increases considerably in response to wounding. To investigate BA biosynthetic enzymes, we conducted tissue-specific transcriptome profiling and identified a wound-responsive BAHD acetyltransferase (BsAT1) of B. serrata catalyzing the late-stage C3α-O-acetylation reactions in the BA biosynthetic pathway. BsAT1 catalyzed C3α-O-acetylation of αBA, ßBA, and 11-keto-ßBA in vitro and in planta assays to produce all the major C3α-O-acetyl-BAs (3-acetyl-αBA, 3-acetyl-ßBA, and 3-acetyl-11-keto-ßBA) found in B. serrata bark and oleo-gum resin. BsAT1 showed strict specificity for BA scaffold, whereas it did not acetylate the more common C3ß-epimeric pentacyclic triterpenes. The analysis of steady-state kinetics using various BAs revealed distinct substrate affinity and catalytic efficiency. BsAT1 transcript expression coincides with increased levels of C3α-O-acetyl-BAs in bark in response to wounding, suggesting a role of BsAT1 in wound-induced biosynthesis of C3α-O-acetyl-BAs. Overall, the results provide new insights into the biosynthesis of principal chemical constituents of Boswellia oleo-gum resin.

Expression of a high-activity diacylglycerol acetyltransferase results in enhanced synthesis of acetyl-TAG in camelina seed oil.

Acetyl-triacylglycerols (acetyl-TAG) contain an acetate group in the sn-3 position instead of the long-chain fatty acid present in regular triacylglycerol (TAG). The acetate group confers unique physical properties such as reduced viscosity and a lower freezing point to acetyl-TAG, providing advantages for use as emulsifiers, lubricants, and 'drop-in' biofuels. Previously, the synthesis of acetyl-TAG in the seeds of the oilseed crop camelina (Camelina sativa) was achieved through the heterologous expression of the diacylglycerol acetyltransferase gene EaDAcT, isolated from Euonymus alatus seeds that naturally accumulate high levels of acetyl-TAG. Subsequent work identified a similar acetyltransferase, EfDAcT, in the seeds of Euonymus fortunei, that possesses higher in vitro activity compared to EaDAcT. In this study, the seed-specific expression of EfDAcT in camelina led to a 20 mol% increase in acetyl-TAG levels over that of EaDAcT. Coupling EfDAcT expression with suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation, up to 90 mol% in the best transgenic lines. Accumulation of high levels of acetyl-TAG was stable over multiple generations, with minimal effect on seed size, weight, and fatty acid content. Slight delays in germination were noted in transgenic seeds compared to the wild type. EfDAcT transcript and protein levels were correlated during seed development with a limited window of EfDAcT protein accumulation. In high acetyl-TAG producing lines, EfDAcT protein expression in developing seeds did not reflect the eventual acetyl-TAG levels in mature seeds, suggesting that other factors limit acetyl-TAG accumulation.

Dietary L-carnitine affects the expression of genes involved in apoptosis and fatty acid metabolism in rooster testes.

Thirty-six 12-week-old breeder roosters (Ross 308) were randomly allocated into three groups to receive L-carnitine (LC): LC-0, LC-250 or LC-500 mg/kg of diet to evaluate the effects of dietary LC on the expression of apoptotic-related genes and desaturases and elongase mRNA transcript levels, in the cockerel testicles. Alteration of Bak (Bcl2 antagonist/killer), Bcl2, Cas3, Cas8, Cas9, Elovl2, Elovl4, Elovl5, Fads1, Fads2 and Scd expression at 24 and 34 weeks of age was compared by real-time quantitative PCR. The expression of Bcl2 and Elovl5 was significantly up-regulated (p < .05), while Cas8 expression (p < .05) and Bak/Bcl2 ratio were reduced (p < .02) in the cockerel testicles at 24 weeks of age. Although Bak mRNA abundance decreased by dietary LC, Bak/Bcl2 ratio was not affected by the treatments at 34 weeks of age. The expression of Cas3 was down-regulated, while Fads2 was up-regulated in the cockerel testicles by dietary LC at 34 weeks of age (p < .05). The results demonstrate the beneficial effects of LC supplementation in suppression of the Bak/Bcl2 ratio by altering Bak and Bcl2 mRNA abundance and, ultimately, prevention of apoptosis. Furthermore, LC increased the expression of Elovl5 and Fads2 genes which are involved in the metabolism of long chain fatty acids.

Juniperonic Acid Biosynthesis is Essential in Caenorhabditis Elegans Lacking Δ6 Desaturase (fat-3) and Generates New ω-3 Endocannabinoids.

In eukaryotes, the C20:4 polyunsaturated fatty acid arachidonic acid (AA) plays important roles as a phospholipid component, signaling molecule and precursor of the endocannabinoid-prostanoid axis. Accordingly, the absence of AA causes detrimental effects. Here, compensatory mechanisms involved in AA deficiency in Caenorhabditis elegans were investigated. We show that the ω-3 C20:4 polyunsaturated fatty acid juniperonic acid (JuA) is generated in the C. elegansfat-3(wa22) mutant, which lacks Δ6 desaturase activity and cannot generate AA and ω-3 AA. JuA partially rescued the loss of function of AA in growth and development. Additionally, we observed that supplementation of AA and ω-3 AA modulates lifespan of fat-3(wa22) mutants. We described a feasible biosynthetic pathway that leads to the generation of JuA from α-linoleic acid (ALA) via elongases ELO-1/2 and Δ5 desaturase which is rate-limiting. Employing liquid chromatography mass spectrometry (LC-MS/MS), we identified endocannabinoid-like ethanolamine and glycerol derivatives of JuA and ω-3 AA. Like classical endocannabinoids, these lipids exhibited binding interactions with NPR-32, a G protein coupled receptor (GPCR) shown to act as endocannabinoid receptor in C. elegans. Our study suggests that the eicosatetraenoic acids AA, ω-3 AA and JuA share similar biological functions. This biosynthetic plasticity of eicosatetraenoic acids observed in C. elegans uncovers a possible biological role of JuA and associated ω-3 endocannabinoids in Δ6 desaturase deficiencies, highlighting the importance of ALA.

Antidepressant Activity of Euparin: Involvement of Monoaminergic Neurotransmitters and SAT1/NMDAR2B/BDNF Signal Pathway.

Depression is the most significant risk factor for suicide, yet the causes are complex and disease mechanism remains unclear. The incidence and disability rate of depression are very high and the efficacy of some traditional antidepressants is not completely satisfactory. Recently, some studies have found that benzofurans have anti-oxidation and anti-monoamine oxidase properties, which are related to depression. Euparin is a monomer compound of benzofuran, previous work by our team found that it improves the behavior of depressed mice. However, additional antidepressant effects and mechanisms of Euparin have not been reported. In this study, the Chronic Unpredictable Mild Stress (CUMS) model of mice was used to further investigate the effect and mechanism of Euparin on depression. Results showed that Euparin (8, 16 and 32 mg/kg) reduced depression-like behavior in mice compared with the model group. Meanwhile, all doses of Euparin were found to increase the contents of monoamine neurotransmitter and decrease monoamine oxidase and reactive oxygen species (ROS) levels in brain of depression mice. Additionally, Euparin restored CUMS-induced decrease of Spermidine/Spermine N1-Acetyltransferase 1 (SAT1), N-methyl-D-aspartate receptor subtype 2B (NMDAR2B) and brain derived neurotrophic factor (BDNF) expression. These findings demonstrate that Euparin has antidepressant properties, and its mechanism involves the SAT1/NMDAR2B/BDNF signaling pathway.

Identification of a novel gene argJ involved in arginine biosynthesis critical for persister formation in Staphylococcus aureus.

Staphylococcus aureus can cause both acute and recurrent persistent infections such as peritonitis, endocarditis, abscesses, osteomyelitis, and chronic wound infections. Effective therapies to treat persistent disease are paramount. However, the mechanisms of S. aureus persistence are poorly understood. In this study, we performed a comprehensive and unbiased high-throughput mutant screen against a transposon-insertion mutant library of S. aureus USA300 and focused on the role of argJ encoding an acetyltransferase in the arginine biosynthesis pathway, whose transposon insertion caused a significant defect in persister formation using multiple drugs and stresses. Genetic complementation and arginine supplementation restored persistence in the argJ transposon insertion mutant while generation of mutations on the active site of the ArgJ protein caused a defect in persistence. Quantitative RT-PCR analysis showed that the genes encoded in the arg operon were over-expressed under drug stressed conditions and in stationary phase cultures. In addition, the argJ mutant had attenuated virulence in both mouse and C. elegans. Our studies identify a new mechanism of persistence mediated by arginine metabolism in S. aureus. These findings provide not only novel insights about the mechanisms of S. aureus persistence but also offer novel therapeutic targets that may help to develop more effective treatment of persistent S. aureus infections.

Enhancement of Production of D-Glucosamine in Escherichia coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc.

D-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that D-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The D-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of D-glucosamine by a factor of 2.16. Moreover, for further increasing D-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of D-glucosamine in E. coli BL21 glmU-Δgpa', E. coli BL21ΔmurQ', and E. coli BL21ΔnanE' were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.

Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice.

Marked elevation in the brain concentration of N-acetyl-L-aspartate (NAA) is a characteristic feature of Canavan disease, a vacuolar leukodystrophy resulting from deficiency of the oligodendroglial NAA-cleaving enzyme aspartoacylase. We now demonstrate that inhibiting NAA synthesis by intracisternal administration of a locked nucleic acid antisense oligonucleotide to young-adult aspartoacylase-deficient mice reverses their pre-existing ataxia and diminishes cerebellar and thalamic vacuolation and Purkinje cell dendritic atrophy. Ann Neurol 2020;87:480-485.

YY1-mediated overexpression of long noncoding RNA MCM3AP-AS1 accelerates angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis.

Lung cancer is famous as an aggressive malignant tumor and is the main cause of cancer-associated mortality globally. Tumor angiogenesis is a vital part in cancer, which influences cell proliferation and metastasis. Increasing studies have claimed that long noncoding RNAs (lncRNAs) were involved in the progression of several cancers. Based on previous studies, this study focused on the role and mechanism of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in lung cancer. At first, MCM3AP-AS1 expression was found to be elevated in lung cancer cells. Depletion of MCM3AP-AS1 repressed cell proliferation, migration, and angiogenesis in lung cancer cells. YY1 was confirmed to mediate MCM3AP-AS1 transcription in lung cancer cells. Moreover, the molecular mechanism investigation revealed that MCM3AP-AS1 could sponge miR-340-5p and elevate KPNA4 expression. On the basis of rescue assays, we identified that the overexpression of KPNA4 partly counteracted the suppressed effect of MCM3AP-AS1 knockdown on angiogenesis and progression in lung cancer cells. Conclusively, the YY1-mediated overexpression of MCM3AP-AS1 accelerated angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis, which highlighted the possibility of MCM3AP-AS1 as a promising therapeutic target for lung cancer.

Discovery of novel oxoindolin derivatives as atypical dual inhibitors for DNA Gyrase and FabH.

The antibacterial agents and therapies today are facing serious problems such as drug resistance. Introducing dual inhibiting effect is a valid approach to solve this trouble and bring advantages including wide adaptability, favorable safety and superiority of combination. We started from potential DNA Gyrase inhibitory backbone isatin to develop oxoindolin derivatives as atypical dual Gyrase (major) and FabH (assistant) inhibitors via a two-round screening. Aiming at blocking both duplication (Gyrase) and survival (FabH), most of synthesized compounds indicated potency against Gyrase and some of them inferred favorable inhibitory effect on FabH. The top hit I18 suggested comparable Gyrase inhibitory activity (IC50 = 0.025 µM) and antibacterial effect with the positive control Novobiocin (IC50 = 0.040 µM). FabH inhibitory activity (IC50 = 5.20 µM) was also successfully introduced. Docking simulation hinted possible important interacted residues and binding patterns for both target proteins. Adequate Structure-Activity Relation discussions provide the future orientations of modification. With high potency, low initial toxicity and dual inhibiting strategy, advanced compounds with therapeutic methods will be developed for clinical application.

N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

N-acetylaspartate (NAA) is synthesized by aspartate N-acetyltransferase (gene: Nat8l) from acetyl-coenzyme A and aspartate. In the brain, NAA is considered an important energy metabolite for lipid synthesis. However, the role of NAA in peripheral tissues remained elusive. Therefore, we characterized the metabolic phenotype of knockout (ko) and adipose tissue-specific (ako) Nat8l-ko mice as well as NAA-supplemented mice on various diets. We identified an important role of NAA availability in the brain during adolescence, as 75% of Nat8l-ko mice died on fat-free diet (FFD) after weaning but could be rescued by NAA supplementation. In adult life, NAA deficiency promotes a beneficial metabolic phenotype, as Nat8l-ko and Nat8l-ako mice showed reduced body weight, increased energy expenditure, and improved glucose tolerance on chow, high-fat, and FFDs. Furthermore, Nat8l-deficient adipocytes exhibited increased mitochondrial respiration, ATP synthesis, and an induction of browning. Conversely, NAA-treated wild-type mice showed reduced adipocyte respiration and lipolysis and increased de novo lipogenesis, culminating in reduced energy expenditure, glucose tolerance, and insulin sensitivity. Mechanistically, our data point to a possible role of NAA as modulator of pancreatic insulin secretion and suggest NAA as a critical energy metabolite for adipocyte and whole-body energy homeostasis.-Hofer, D. C., Zirkovits, G., Pelzmann, H. J., Huber, K., Pessentheiner, A. R., Xia, W., Uno, K., Miyazaki, T., Kon, K., Tsuneki, H., Pendl, T., Al Zoughbi, W., Madreiter-Sokolowski, C. T., Trausinger, G., Abdellatif, M., Schoiswohl, G., Schreiber, R., Eisenberg, T., Magnes, C., Sedej, S., Eckhardt, M., Sasahara, M., Sasaoka, T., Nitta, A., Hoefler, G., Graier, W. F., Kratky, D., Auwerx, J., Bogner-Strauss, J. G. N-acetylaspartate availability is essential for juvenile survival on fat-free diet and determines metabolic health.

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens.

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [14 C]-acetyl-CoA to oligogalacturonides. Through site-directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.

Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.

A Novel Gene Contributing to the Initiation of Fatty Acid Biosynthesis in Escherichia coli.

Type II fatty acid biosynthesis in bacteria can be broadly classified into the initiation and elongation phases. The biochemical functions defining each step in the two phases have been studied in vitro Among the ß-ketoacyl-acyl carrier protein (ACP) synthases, FabH catalyzes the initiation reaction, while FabB and FabF, which primarily catalyze the elongation reaction, can also drive initiation as side reactions. A role for FabB and FabF in the initiation of fatty acid biosynthesis would be supported by the viability of the ΔfabH mutant. In this study, we show that the ΔfabH and ΔyiiD mutations were synthetically lethal and that ΔfabH ΔrelA ΔspoT and ΔfabH ΔdksA synthetic lethality was rescued by the heterologous expression of yiiD In the ΔfabH mutant, the expression of yiiD was positively regulated by (p)ppGpp. The growth defect, reduced cell size, and altered fatty acid profile of the ΔfabH mutant and the growth defect of the ΔfabH ΔfabF fabB15(Ts) mutant in oleate- and palmitate-supplemented medium at 42°C were rescued by the expression of yiiD from a multicopy plasmid. Together, these results indicate that the yiiD-encoded function supported initiation of fatty acid biosynthesis in the absence of FabH. We have renamed yiiD as fabYIMPORTANCE Fatty acid biosynthesis is an essential process conserved across life forms. ß-Ketoacyl-ACP synthases are essential for fatty acid biosynthesis. FabH is a ß-ketoacyl-ACP synthase that contributes to the initiation of fatty acid biosynthesis in Escherichia coli In this study, we present genetic and biochemical evidence that the yiiD (renamed fabY)-encoded function contributes to the biosynthesis of fatty acid in the absence of FabH activity and that under these conditions, the expression of FabY was regulated by the stringent response factors (p)ppGpp and DksA. Combined inactivation of FabH and FabY resulted in growth arrest, possibly due to the loss of fatty acid biosynthesis. A molecule(s) that inhibits the two activities can be an effective microbicide.

Molecular Cloning, Characterization, and Nutritional Regulation of Elovl6 in Large Yellow Croaker (Larimichthys crocea).

Elongation of very long chain fatty acids protein 6 (Elovl6) is a key enzyme in fatty acid synthesis, which participates in converting palmitate (C16:0) to stearate (C18:0). Although studies of Elovl6 have been carried out in mammals, the nutritional regulation of elovl6 in fish remains poorly understood. In the present study, the cloning and nutritional regulation of elovl6 were determined in large yellow croaker. Sequence and phylogenetic analysis revealed that the full-length cDNA of elovl6 was 1360 bp, including an open reading frame of 810 bp encoding a putative protein of 269 amino acid that possesses the characteristic features of Elovl proteins. The transcript level of elovl6 was significantly increased in the liver of croaker fed the diets with soybean oil (enriched with 18: 2n-6, LA) or linseed oil (enriched with 18: 3n-3, ALA) than that in croaker fed the diet with fish oil (enriched with 20: 5n-3 and 22: 6n-3). Correspondingly, the elovl6 expression in croaker's hepatocytes treated with ALA or LA was remarkably increased compared to the controls. Furthermore, the transcription factors including hepatocyte nuclear factor 1α (HNF1α), CCAAT-enhancer-binding protein ß (CEBPß), retinoid X receptor α (RXRα), and cAMP response element-binding protein 1 (CREB1) greatly enhanced promoter activity of elovl6 in large yellow croaker, and the expression of transcription factors is consistent with the changes of elovl6 expression in response to fatty acids in vivo and in vitro. In conclusion, this study revealed that elovl6 expression in large yellow croaker could be upregulated by dietary ALA or LA via the increased transcriptional expression of transcription factors including hnf1α, cebpß, rxrα, and creb1.