acyl-CoA Synthetase Gene Research Articles

Identification of long-chain acyl-CoA synthetase gene family reveals that SlLACS1 is essential for cuticular wax biosynthesis in tomato

Long-chain acyl-CoA synthetases (LACSs), belonging to the acyl-activating enzyme superfamily, play crucial roles in lipid biosynthesis and fatty acid catabolism. Here, we identified 11 LACS genes in the tomato reference genome, and these genes were clustered into six subfamilies. Gene structure and conserved motif analyses indicated that LACSs from the same subfamily shared conserved gene and protein structures. Expression analysis revealed that SlLACS1 was highly expressed in the outer epidermis of tomato fruits and leaves. Subcellular localization assay results showed that SlLACS1 was located in the endoplasmic reticulum. Compared with wild-type plants, the wax content on leaves and fruits decreased by 22.5–34.2 % in SlLACS1 knockout lines, confirming that SlLACS1 was involved in wax biosynthesis in both leaves and fruits. Water loss, chlorophyll extraction, water-deficit, and toluidine blue assays suggested that cuticle permeability was elevated in SlLACS1 knockout lines, resulting in reduction in both drought stress resistance and fruit shelf-life. Overall, our analysis of the LACSs in tomato, coupled with investigations of SlLACS1 function, yielded a deeper understanding of the evolutionary patterns of LACS members and revealed the involvement of SlLACS1 in wax accumulation contribute to drought resistance and extended fruit shelf-life in tomato.

Evolutionary origin and gradual accumulation with plant evolution of the LACS family

BackgroundLACS (long-chain acyl-CoA synthetase) genes are widespread in organisms and have multiple functions in plants, especially in lipid metabolism. However, the origin and evolutionary dynamics of the LACS gene family remain largely unknown.ResultsHere, we identified 1785 LACS genes in the genomes of 166 diverse plant species and identified the clades (I, II, III, IV, V, VI) of six clades for the LACS gene family of green plants through phylogenetic analysis. Based on the evolutionary history of plant lineages, we found differences in the origins of different clades, with Clade IV originating from chlorophytes and representing the origin of LACS genes in green plants. The structural characteristics of different clades indicate that clade IV is relatively independent, while the relationships between clades (I, II, III) and clades (V, VI) are closer. Dispersed duplication (DSD) and transposed duplication (TRD) are the main forces driving the evolution of plant LACS genes. Network clustering analysis further grouped all LACS genes into six main clusters, with genes within each cluster showing significant co-linearity. Ka/Ks results suggest that LACS family genes underwent purifying selection during evolution. We analyzed the phylogenetic relationships and characteristics of six clades of the LACS gene family to explain the origin, evolutionary history, and phylogenetic relationships of different clades and proposed a hypothetical evolutionary model for the LACS family of genes in plants.ConclusionsOur research provides genome-wide insights into the evolutionary history of the LACS gene family in green plants. These insights lay an important foundation for comprehensive functional characterization in future research.

Acyl-CoA synthetase 1 plays an important role on pollen development and male fertility in tomato

The development of pollen is critical to male reproduction in flowering plants. Acyl-CoA synthetase (ACOS) genes play conserved functions in regulating pollen development in various plants. Our previous work found that knockout of the SlACOS1 gene in tomato might decrease fruit setting. The current study further revealed that SlACOS1 was important to pollen development and male fertility. The SlACOS1 gene was preferentially expressed in the stamen of the flower with the highest expression at the tetrad stage of anther development. Mutation of the SlACOS1 gene by the CRISPR/Cas9-editing system reduced pollen number and viability as well as fruit setting. The tapetum layer exhibited premature degradation and the pollen showed abnormal development appearing irregular, shriveled, or anucleate in Slacos1 mutants at the tetrad stage. The fatty acid metabolism in anthers was significantly impacted by mutation of the SlACOS1 gene. Furthermore, targeted fatty acids profiling using GC-MS found that contents of most fatty acids except C18:1 and C18:2 were reduced. Yeast complementation assay demonstrated that the substrate preferences of SlACOS1 were C16:0 and C18:0 fatty acids. Male fertility of Slacos1 mutant could be slightly restored by applying exogenous palmitic acid, a type of C16:0 fatty acid. Taken together, SlACOS1 played important roles on pollen development and male fertility by regulating the fatty acid metabolism and the development of tapetum and tetrad. Our findings will facilitate unraveling the mechanism of pollen development and male fertility in tomato.

Efficient synthesis of malonyl-CoA by an acyl-CoA synthetase from Streptomyces sp.

Malonyl-CoA is a precursor of fatty acids, polyketides, and bio-based chemicals with potential applications in medicine, antibiotics, and fuels. However, its low intracellular concentration and high cost have led to difficulties in research and production. To develop an efficient method for producing malonyl-CoA, we screened the acyl-CoA synthetase (ACS) gene from Streptomyces sp. using sequence-structure alignment. This protein contains conserved sequences and active sites for malonyl-CoA synthetases. The purified recombinant enzyme ACS was heterologously expressed in Escherichia coli BL21 and characterised. The results showed that it converted the substrates malonate and CoA into malonyl-CoA. Under the optimal conditions, the specific activity of the purified ACS was 32.3 U·mg−1 and the conversion rate reached 98.8%. In addition, when the cell-free extracts were used as catalysts, the highest yield of malonyl-CoA was obtained after 4 h, yielding 24.2 g·L−1 with a conversion rate of 90.3%. After the product was purified and vacuum freeze-dried, a solid powder of malonyl-CoA was obtained. This study characterised and identified a new ACS and optimised the reaction conditions to efficiently synthesise pure malonyl-CoA in vitro in high yield using enzyme-mediated methods.

Combinatorial metabolic engineering and tolerance evolution of Yarrowia lipolytica for improved γ-decalactone synthesis

This study highlights an effective strategy based on the synergistic complementarity of metabolic engineering and adaptive evolution.First, the chassis strain YL013 was obtained by deleting five acyl-CoA oxidase genes to reduce the degradation of γ-decalactone(GDL). Then, adaptive laboratory evolution with increasing concentrations of γ-decalactone was employed to obtain the strain YL013–250 with increased γ-decalactone tolerance of up to 250 mg/L. Moreover, this strain produced 664.5 mg/L γ-decalactone, representing a 142 % improvement over the parental strain YL013. Furthermore, four candidate fatty acyl-CoA synthetase genes, FAA1, FAA2 FAA3 and FAT1, were modulated using the strong, constitutive TEFin promoter to investigate their roles in GDL production. The results showed that overexpression of FAA1, FAA2 and FAA3 can effectively improve the ability of Yarrowia lipolytica to synthesize GDL using ricinoleic acid as substrate, among which overexpression of FAA1 had the most significant effect on GDL yield. Finally, gene of alcohol acyltransferase (AAT1) from Prunus persica was introduced into Y. lipolytica, leading to a GDL titer of 2.2 g/L, which was a 1.32-fold increase compared with the parental strain without AAT1. Taken together, this study provides a combinatorial strategy to improve GDL production through rational regulation of pathway genes and non-rational adaptive laboratory evolution.

An improved CRISPRi system in Pichia pastoris

CRISPR interference (CRISPRi) has been developed and widely used for gene repression in various hosts. Here we report an improved CRISPRi system in Pichia pastoris by fusing dCas9 with endogenous transcriptional repressor domains. The CRISPRi system shows strong repression of eGFP, with the highest efficiency of 85%. Repression of native genes is demonstrated by targeting AOX1 promoter. AOX1 is efficiently repressed and the mutant strains show much slower growth in methanol medium. Effects of gRNA expression and processing on CRISPRi efficiency is also investigated. It is found that gRNA processing by HH/HDV ribozymes or Csy4 endoribonuclease generating clean gRNA is critical to achieve strong repression, and Csy4 cleavage shows higher repression efficiency. However, gRNA expression using native tRNA transcription and processing systems results in relatively weaker repression of eGFP. By expression of two gRNAs targeting promoters of eGFP and AOX1 in an array together with Cys4 recognition sites, both genes can be repressed simultaneously. Cys4-mediated gRNA array processing is further applied to repress fatty acyl-CoA synthetase genes (FAA1 and FAA2). Both genes are efficiently repressed, demonstrating that Cys4 endoribonuclease has the ability to cleave gRNAs array and can be can be used for multiplexed gene repression in P. pastoris.

Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation

Identifying how small molecules act to kill malaria parasites can lead to new “chemically validated” targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

Consequences of reprogramming acetyl-CoA metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the mouse liver

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental contaminant that induces the progression of steatosis to steatohepatitis with fibrosis in mice. Furthermore, TCDD reprograms hepatic metabolism by redirecting glycolytic intermediates while inhibiting lipid metabolism. Here, we examined the effect of TCDD on hepatic acetyl-coenzyme A (acetyl-CoA) and β-hydroxybutyrate levels as well as protein acetylation and β-hydroxybutyrylation. Acetyl-CoA is not only a central metabolite in multiple anabolic and catabolic pathways, but also a substrate used for posttranslational modification of proteins and a surrogate indicator of cellular energy status. Targeted metabolomic analysis revealed a dose-dependent decrease in hepatic acetyl-CoA levels coincident with the phosphorylation of pyruvate dehydrogenase (E1), and the induction of pyruvate dehydrogenase kinase 4 and pyruvate dehydrogenase phosphatase, while repressing ATP citrate lyase and short-chain acyl-CoA synthetase gene expression. In addition, TCDD dose-dependently reduced the levels of hepatic β-hydroxybutyrate and repressed ketone body biosynthesis gene expression. Moreover, levels of total hepatic protein acetylation and β-hydroxybutyrylation were reduced. AMPK phosphorylation was induced consistent with acetyl-CoA serving as a cellular energy status surrogate, yet subsequent targets associated with re-establishing energy homeostasis were not activated. Collectively, TCDD reduced hepatic acetyl-CoA and β-hydroxybutyrate levels eliciting starvation-like conditions despite normal levels of food intake.

Overexpression the BnLACS9 could increase the chlorophyll and oil content in Brassica napus

BackgroundChlorophyll is a very important pigment involved in photosynthesis, while plant acyl-CoA biosynthesis is derived from plastid-localized fatty acids (FAs). Until now, the regulation of the acyl-CoA pathway for chlorophyll biosynthesis is still unknown.ResultsHere, we identified a long-chain acyl-CoA synthetase (LACS) gene BnLACS9 from Brassica napus. BnLACS9 complemented a LACS-deficient yeast strain YB525, which indicated that BnLACS9 has the LACS function. BnLACS9 was localized in the chloroplast envelope membrane, while mainly expressed in young leaves and flowers. Overexpression of BnLACS9 in Nicotiana benthamiana resulted in an increase in total CoA and MGDG content. In B. napus with overexpression of BnLACS9, the number of chloroplast grana lamellae and the chlorophyll content, as well as the MGDG and DGDG contents, increased compared to wild type. The net photosynthetic rate, dry weight of the entire plant and oil content of seeds increased significantly, accompanied by an increase in chlorophyll content. Transcriptome analysis revealed that overexpression of BnLACS9 improved the pathway of acyl-CoA biosynthesis and further improved the enzymes in the glycolipid synthesis pathway, while acyl-CoA was the substrate for glycolipid synthesis. The increased glycolipids, especially MGDG and DGDG, accelerated the formation of the chloroplast grana lamellae, which increased the number of chloroplast thylakoid grana lamella and further lead to increased chlorophyll content.ConclusionsIn the present study, we demonstrated that BnLACS9 played a crucial role in glycolipids and chlorophyll biosynthesis in B. napus. The results also provide a new direction and theoretical basis for the improvement of the agronomic traits of plants.

Evolution, characterization, and immune response function of long-chain acyl-CoA synthetase genes in rainbow trout (Oncorhynchus mykiss) under hypoxic stress.

Long-chain acyl-CoA synthetases (Acsls), members of the acyl-activating enzyme superfamily, haves been systematically characterized in mammals and certain fishes, but the research on their involvement in reproductive development and hypoxic stress response in rainbow trout remains limited. In this study, we investigated the acsl gene structure and physical and chemical characteristics and the evolutionary relationship among acsl genes using the NCBI/Ensembl database. Using hypoxia treatment experiment, acsl gene expression in various organs and its regulation were investigated. A total of 11 acsl genes were identified in rainbow trout. Phylogenetic analyses found that acsl genes in rainbow trout were clustered into two clades: acsl3/4 and acsl1/2/5/6, and the additional gene duplication observed resulted from the third round of genome duplication unique to teleosts. Multiple sequence alignment and conserved motif analyses showed that the sequence of acsl proteins was highly conserved. Real-time quantitative PCR (RT-qPCR) showed that the acsl genes were highly expressed in immune tissues (liver and head kidney). Under hypoxia, the expression of acsl genes was upregulated, suggesting that they enhance the tolerance to hypoxia and are involved in the immune response in rainbow trout. Our study provides valuable insights into teleost evolution and effects of hypoxia on biological immunity and form a basis for further research on the functional characteristics of acsl genes.

Third Generation Genome Sequencing Reveals That Endobacteria in Nematophagous Fungi Esteya vermicola Contain Multiple Genes Encoding for Nematicidal Proteins.

Esteya vermicola is the first recorded endoparasitic nematophagous fungus with high infectivity capacity, attacking the pinewood nematode Bursaphelenchus xylophilus which causes pine wilt disease. Endosymbionts are found in the cytoplasm of E. vermicola from various geographical areas. We sequenced the genome of endobacteria residing in E. vermicola to discover possible biological functions of these widespread endobacteria. Multilocus phylogenetic analyses showed that the endobacteria form a previously unidentified lineage sister to Phyllobacterium myrsinacearum species. The number of genes in the endobacterium was 4542, with 87.8% of the proteins having a known function. It contained a high proportion of repetitive sequences, as well as more Acyl-CoA synthetase genes and genes encoding the electron transport chain, compared with compared with plant-associated P. zundukense Tri 48 and P. myrsinacearum DSM 5893. Thus, this symbiotic bacterium is likely to be more efficient in regulating gene expression and energy release. Furthermore, the endobacteria in nematophagous fungi Esteya vermicola contained multiple nematicidal subtilase/subtilisin encoding genes, so it is likely that endobacteria cooperate with the host to kill nematodes.

Firefly genomes illuminate the evolution of beetle bioluminescent systems.

Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome assemblies of six species of bioluminescent beetles have recently been published. These studies have focused on the evolution of novelties; luciferase, and the biosynthesis of luciferin and defensive chemicals. For example, clustering of the luciferase gene with acyl-CoA synthetase genes on a chromosome in luminous beetle genomes suggests the involvement of tandem gene duplications and neofunctionalization during the evolution of beetle bioluminescence. Several candidate genes for critical roles in beetle bioluminescence have been identified, but their functional analyses are still ongoing. The establishment of a long-term mass-rearing system and strain will be the key for the post-genome research on bioluminescent beetles. Lastly, the application of contemporary chromosome-scale genome assembly techniques to luminous beetles will help resolve outstanding evolutionary questions, such as how many times bioluminescence evolved in this clade.

Glycolytic and oxidative muscles under acute glucose supplementation differ in their metabolic responses to fatty acyl-CoA synthetase gene suppression

Glycolytic and oxidative muscles under acute glucose supplementation differ in their metabolic responses to fatty acyl-CoA synthetase gene suppression

A tandem duplication in Drosophila melanogaster shows enhanced expression beyond the gene copy number.

Tandem duplicated genes are common features of genomes, but the phenotypic consequences of their origins are not well understood. It is not known whether a simple doubling of gene expression should be expected, or else some other expression outcome. This study describes an experimental framework using engineered deletions to assess any contribution of locally acting cis- and globally acting trans-regulatory factors to expression interactions of particular tandem duplicated genes. Acsx1L (CG6300) and Acsx1R (CG11659) are tandem duplicates of a putative acyl-CoA synthetase gene found in Drosophila melanogaster. Experimental deletions of the duplicated segments were used to investigate whether the presence of 1 tandem duplicated block influences the expression of its neighbor. Acsx1L, the gene in the left block, shows much higher expression than either its duplicate Acsx1R or the single Acsx1 in Drosophila simulans. Acsx1L expression decreases drastically upon deleting the right-hand duplicated block. Crosses among wildtype and deletion strains show that high tandem expression is primarily due to cis-acting interactions between the duplicated blocks. No effect of these genes on cuticular hydrocarbons was detected. Sequence and phylogenetic analysis suggest that the duplication rose to fixation in D. melanogaster and has been subject to extensive gene conversion. Some strains actually carry 3 tandem copies, yet strains with 3 Acsx1s do not have higher expression levels than strains with 2. Surveys of tandem duplicate expression have typically not found the expected 2-fold increase in expression. This study suggests that cis-regulatory interactions between duplicated blocks could be responsible for this trend.

A transcriptional journey from sucrose to endosperm oil bodies in triple transgene oily wheat grain

Transgenic wheat expressing Wrinkled1 (Wri1), diacylglycerol acyltransferase 1 (DGAT1), and Oleosin genes with endosperm active promoters, in the companion paper were found to accumulate 5–10 times the quantity of triacylglyceride (TAG) in the endosperm compared to the control. In this study we track the pleiotropic effects on transcription at four grain development time points, of genes whose products are involved in the various pathways from sugar import to oil body formation, as well as other major metabolic pathways in the developing grain. The transcriptional impacts were substantial with early increases in genes for sucrose transport, hexose phosphate formation, cytoplasmic glycolysis, and ADP-glucose transport to the plastid. Corresponding to the late expression of the Wrinkled1 transgene, there was a late up-regulation of the entire suite of genes in the plastidial fatty acid synthesis pathway. Transport of fatty acid precursors to the ER was likely facilitated by early up-regulation of FAX1 (Fatty acid export) and LACS8 (long-chain acyl-CoA synthetase) genes. With the exception of the transgenic DGAT1, there were relatively few and moderate changes to transcription of other genes involved in TAG assembly in the ER, although the direct use of the acyl-PC pool appears to be down-regulated. A large up-regulation was observed at the three earliest time points of many genes associated with the formation of lipid droplets, including endogenous oleosins, caleosins, stereoleosins, seipins, and a variety of other lipid body associated proteins. Also unanticipated was the early down-regulation of a number of genes whose products are involved in lipid body turnover and peroxisomal β-oxidation of released fatty acids. Despite the substantial increase in TAG accumulation, the transgenic grains were not reduced in starch or protein and the transcriptional analysis demonstrated an early up-regulation of the entire suite of starch synthesis genes and protein storage genes.

Improvement of Free Fatty Acid Secretory Productivity in Aspergillus oryzae by Comprehensive Analysis on Time-Series Gene Expression.

Aspergillus oryzae is a filamentous fungus that has historically been utilized in the fermentation of food products. In recent times, it has also been introduced as a component in the industrial biosynthesis of consumable compounds, including free fatty acids (FFAs), which are valuable and versatile products that can be utilized as feedstocks in the production of other commodities, such as pharmaceuticals and dietary supplements. To improve the FFA secretory productivity of A. oryzae in the presence of Triton X-100, we analyzed the gene expression of a wild-type control strain and a disruptant strain of an acyl-CoA synthetase gene, faaA, in a time-series experiment. We employed a comprehensive analysis strategy using the baySeq, DESeq2, and edgeR algorithms to clarify the vital pathways for FFA secretory productivity and select genes for gene modification. We found that the transport and metabolism of inorganic ions are crucial in the initial stages of FFA production and revealed 16 candidate genes to be modified in conjunction with the faaA disruption. These genes were verified through the construction of overexpression strains, and showed that the manipulation of reactions closer to the FFA biosynthesis step led to a higher increase in FFA secretory productivity. This resulted in the most successful overexpression strains to have an FFA secretory productivity more than two folds higher than that of the original faaA disruptant. Our study provides guidance for further gene modification for FFA biosynthesis in A. oryzae and for enhancing the productivity of other metabolites in other microorganisms through metabolic engineering.

Polymorphisms of the ACSL1 Gene Influence Milk Production Traits and Somatic Cell Score in Chinese Holstein Cows.

Simple SummaryMilk production traits of cows are important economic indicators of the livestock industry. Many dairy farms strive to improve the quality of their milk. Long-chain acyl-CoA synthetase 1 (ACSL1) is a gene related to lipid metabolism. It is widely found in various organisms and can affect fat content and protein content in milk. Single nucleotide polymorphisms (SNP) refers to the polymorphism of DNA sequence caused by a single nucleotide variation at the gene level, which plays a vital function in the genetic study of milk production traits in dairy cows. Our study identified six SNPs of the ACSL1 gene in Chinese Holstein cows, which were related to milk yield, milk fat content, milk protein content and somatic cell score (SCS) to some extent. In summary, the pleiotropic effects of bovine ACSL1 for milk production traits were found in this paper, which will provide a reference for Chinese Holstein cow breeding selection and high economic benefits.Improving the quality of milk is a challenge for zootechnicians and dairy farms across the globe. Long-chain acyl-CoA synthetase 1 (ACSL1) is a significant member of the long-chain acyl-CoA synthetase gene family. It is widely found in various organisms and influences the lactation performance of cows, including fat percentage, milk protein percentage etc. Our study was aimed to investigate the genetic effects of single nucleotide polymorphisms (SNPs) in ACSL1 on milk production traits. Twenty Chinese Holstein cows were randomly selected to extract DNA from their blood samples for PCR amplification and sequencing to identify SNPs of the bovine ACSL1 gene, and six SNPs (5’UTR-g.20523C>G, g.35446C>T, g.35651G>A, g.35827C>T, g.35941G>A and g.51472C>T) were discovered. Then, Holstein cow genotyping (n = 992) was performed by Sequenom MassARRAY based on former SNP information. Associations between SNPs and milk production traits and somatic cell score (SCS) were analyzed by the least-squares method. The results showed that SNP g.35827C>T was in high linkage disequilibrium with g.35941G>A. Significant associations were found between SNPs and test-day milk yield (TDMY), fat content (FC), protein content (PC) and SCS (p < 0.05). Among these SNPs, SNP 5’UTR-g.20523C>G showed an extremely significant effect on PC and SCS (p < 0.01). The SNP g.35446C>T showed a statistically significant effect on FC, PC, and SCS (p < 0.01), and also TDMY (p < 0.05). The SNP g.35651G>A had a statistically significant effect on PC (p < 0.01). The SNP g.35827C>T showed a highly significant effect on TDMY, FC, and SCS (p < 0.01) and significantly influenced PC (p < 0.05). Lastly, SNP g.51472C>T was significantly associated with TDMY, FC, and SCS (p < 0.05). In summary, the pleiotropic effects of bovine ACSL1 for milk production traits were found in this paper, but further investigation will be required on the intrinsic correlation to provide a theoretical basis for the research on molecular genetics of milk quality traits of Holstein cows.

Acyl-CoA synthetases, Aal4 and Aal7, are involved in the utilization of exogenous fatty acids in Yarrowia lipolytica.

The yeast Yarrowia lipolytica assimilates hydrophobic compounds, such as n-alkanes and fatty acids, as sole carbon and energy sources. It has been shown that the acyl-CoA synthetase (ACS) genes, FAT1 and FAA1, are involved in the activation of fatty acids produced during the metabolism of n-alkanes, but the ACS genes that are involved in the metabolism of fatty acids from the culture medium remains to be identified. In this paper, we have identified the ACS genes involved in the utilization of exogenous fatty acids. RNA-seq analysis and qRT-PCR revealed that the transcript levels of the peroxisomal ACS-like protein-encoding genes AAL4 and AAL7 were increased in the presence of oleic acid. The single deletion mutant of AAL4 or AAL7 and double deletion mutant of AAL4 and AAL7 did not show any defects in the growth on the medium containing glucose, glycerol, n-alkanes, or fatty acids. In contrast, the mutant with deletion of seven genes, FAA1, FAT1-FAT4, AAL4, and AAL7, showed severe growth defects on the medium containing dodecanoic acid or oleic acid. These results suggest that Aal4p and Aal7p play important roles in the metabolism of exogenous fatty acids in collaboration with Faa1p and Fat1p-Fat4p.

Enhancement of the productivity of free dihomo-γ-linolenic acid via co-overexpression of elongase and two desaturase genes in Aspergillus oryzae

Free dihomo-γ-linolenic acid (DGLA), a polyunsaturated free fatty acid (FFA), is a precursor of the eicosanoid prostaglandin E1 and is expected to be a source material for artificial production. We previously constructed the Aspergillus oryzae mutant strain ARA1 that produced free DGLA from the disruptant of faaA, an acyl-CoA synthetase gene, where FFA productivity increased by 9.2-fold compared with that of the wild-type strain. Here, we aimed to achieve enhancement of free DGLA productivity. Because saturated FFAs, such as palmitic acid and stearic acid, accounted for about 45% and 25% of total FFAs produced by ARA1, respectively, we used a strategy to facilitate elongation and desaturation of these FFAs to oleic acid and linoleic acid by overexpressing genes encoding elongase, Δ9-desaturase, and Δ12-desaturase originally expressed in A.oryzae. Ten genes were predicted to encode desaturases, and their overexpression DNA constructs were introduced into ARA1. AO090001000224 and AO090011000488 facilitated Δ12-desaturation and Δ9-desaturation most, respectively, following overexpression. Next, ARA1 strain was modified to DGLA1cre strain for producing free DGLA as a final product, and co-overexpression of these two desaturase genes was then introduced to DGLA1cre. Moreover, single overexpression of two genes predicted to encode elongases was additionally introduced, and only AO090003000572 facilitated elongation. Consequently, in the co-overexpression mutant of AO090001000224, AO090011000488, and AO090003000572, free DGLA content ratio increased by 1.8-fold from ARA1 to 14.5%, and the productivity also increased by 1.8-fold to 0.086mmol/g-dry-cell-weight. The yield was 284mg/L. These findings provided insights into strategies for improving microbial production of polyunsaturated FFAs.

Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity.

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity.