Fatty acyl-CoA Synthetase Gene Research Articles

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.

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

Designing and Creating a Synthetic Omega Oxidation Pathway in Saccharomyces cerevisiae Enables Production of Medium-Chain α, ω-Dicarboxylic Acids.

Medium-chain (C8–C14) α, ω-dicarboxylic acids (α, ω-DCAs), which have numerous applications as raw materials for producing various commodities and polymers in chemical industry, are mainly produced from chemical or microbial conversion of petroleum-derived alkanes or plant-derived fatty acids at present. Recently, significant attention has been gained to microbial production of medium-chain α, ω-DCAs from simple renewable sugars. Here, we designed and created a synthetic omega oxidation pathway in Saccharomyces cerevisiae to produce C10 and C12 α, ω-DCAs from renewable sugars and fatty acids by introducing a heterogeneous cytochrome P450 CYP94C1 and cytochrome reductase ATR1. Furthermore, the deletion of fatty acyl-CoA synthetase genes FAA1 and FAA4 increased the production of medium-chain α, ω-DCAs from 4.690 ± 0.088 mg/L to 12.177 ± 0.420 mg/L and enabled the production of C14 and C16 α, ω-DCAs at low percentage. But blocking β-oxidation pathway by deleting fatty-acyl coenzyme A oxidase gene POX1 and overexpressing different thioesterase genes had no significant impact on the production and the composition of α, ω-dicarboxylic acids. Overall, our study indicated the potential of microbial production of medium-chain α, ω-DCAs from renewable feedstocks using engineered yeast.

Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

BackgroundIn vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields.ResultsWe analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain.ConclusionsFine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.

Elucidating the Pseudomonas aeruginosa Fatty Acid Degradation Pathway: Identification of Additional Fatty Acyl-CoA Synthetase Homologues

The fatty acid (FA) degradation pathway of Pseudomonas aeruginosa, an opportunistic pathogen, was recently shown to be involved in nutrient acquisition during BALB/c mouse lung infection model. The source of FA in the lung is believed to be phosphatidylcholine, the major component of lung surfactant. Previous research indicated that P. aeruginosa has more than two fatty acyl-CoA synthetase genes (fadD; PA3299 and PA3300), which are responsible for activation of FAs using ATP and coenzyme A. Through a bioinformatics approach, 11 candidate genes were identified by their homology to the Escherichia coli FadD in the present study. Four new homologues of fadD (PA1617, PA2893, PA3860, and PA3924) were functionally confirmed by their ability to complement the E. coli fadD mutant on FA-containing media. Growth phenotypes of 17 combinatorial fadD mutants on different FAs, as sole carbon sources, indicated that the four new fadD homologues are involved in FA degradation, bringing the total number of P. aeruginosa fadD genes to six. Of the four new homologues, fadD4 (PA1617) contributed the most to the degradation of different chain length FAs. Growth patterns of various fadD mutants on plant-based perfumery substances, citronellic and geranic acids, as sole carbon and energy sources indicated that fadD4 is also involved in the degradation of these plant-derived compounds. A decrease in fitness of the sextuple fadD mutant, relative to the ΔfadD1D2 mutant, was only observed during BALB/c mouse lung infection at 24 h.

Oleaginous yeast Yarrowia lipolytica mutants with a disrupted fatty acyl-CoA synthetase gene accumulate saturated fatty acid

Fatty acyl-CoA synthetases are critical enzymes involved in lipid metabolism. The oleaginous yeast Yarrowia lipolytica is currently generating interest in biofuel research due to its ability to convert raw materials into value-added end products. In this study, the putative acyl-CoA synthetase gene YAL1 in Y. lipolytica was interrupted with the copper resistance ( CRF1) gene to allow selection without antibiotics to facilitate industrial applications. Deletion of YAL1 led to reduced acyl-CoA synthetase activity. Furthermore, the fatty acid profile and lipid content of the mutant were different from the wild-type strain. The ratio of saturated to unsaturated fatty acids increased 6-fold, and the total lipid production of the mutant strain increased to 1.47-fold of the wild-type strain. The results indicate that YAL1 in Y. lipolytica is involved in fatty acid elongation and desaturation, whereas the homologous, highly conserved FAA1 gene from Saccharomyces cerevisiae was shown to be responsible for fatty acid activation. The increased ratio of saturated to unsaturated fatty acids would result in a higher combustion value and better oxidative stability for biofuel products obtained from the fatty acids from the engineered Y. lipolytica mutant.

The evolution of the adenylate-forming protein family in beetles: Multiple luciferase gene paralogues in fireflies and glow-worms

Bioluminescence in beetles is dependent upon the enzyme luciferase. It has been hypothesised luciferase evolved from a fatty acyl-CoA synthetase gene deriving a novel bioluminescent function (neofunctionalization) after a gene duplication event. We evaluated this hypothesis within a phylogenetic framework using independent evidence obtained from the genome of Tribolium castaneum, published luciferase genes and novel luciferase and luciferase-like sequences. This phylogenetic study provides evidence for a large gene family of luciferase and luciferase-like paralogues in bioluminescent and non-bioluminescent beetles. All luciferase sequences formed a clade supporting a protoluciferase existing prior to the divergence of the Lampyridae, Elateridae and Phengodidae (Elateroidea). Multiple luciferase genes were identified from members of the Photurinae and the Luciolinae indicating complex gene duplication events within lampyrid genomes. The majority of luciferase residues were identified to be under purifying selection as opposed to positive selection. We conclude that beetle luciferase may have arisen from a process of subfunctionalization as opposed to neofunctionalization early on in the evolution of the Elateroidea.

Identification of a Fatty Acyl-CoA Synthetase Gene,lcf2+, Which Affects Viability after Entry into the Stationary Phase inSchizosaccharomyces pombe

The lcf1(+) gene, which encodes a long chain fatty acyl-CoA synthetase, is necessary for the maintenance of viability after entry into the stationary phase in Schizosaccharomyces pombe. In this study, we analyzed a paralogous gene, SPBP4H10.11c (named lcf2(+)), and we present evidence that the gene encodes a new fatty acyl-CoA synthetase. The enzyme preferentially recognized myristic acid as a substrate. A Deltalcf2 mutant showed increased viability after entry into the stationary phase in SD medium. A Deltalcf1Deltalcf2 double mutant showed a severe decrease in long-chain fatty acyl-CoA synthetase activity and a rapid loss of viability after entry into the stationary phase. These results suggest that fatty acid utilization and/or metabolism is important to determine viability in the stationary phase.

A defect in a fatty acyl-CoA synthetase gene, lcf1+, results in a decrease in viability after entry into the stationary phase in fission yeast.

An intriguing mutant was isolated in Schizosaccharomyces pombe, which is defective in the maintenance of viability after entry into the stationary phase. In the logarithmic growth phase, the mutant cells grow at the same rate as the parental cells. Upon the onset of the stationary phase, however, the mutant cells lose viability very rapidly. It was found that this phenotype was due to a mutational lesion in the lcf1+ gene, which encodes a long-chain fatty acyl-CoA synthetase. The lcf1Deltamutant shows pleiotropic phenotypes, in that they are also sensitive to high temperature (37 degrees C) and to high salt concentrations (0.9 M KCl) in the medium. Based on the fact that Lcf1 is highly homologous to Faa1 and Faa4 of Saccharomyces cerevisiae, both of which have previously been suggested to play roles in the maintenance of endogenous acyl-CoA pools, the possible function of Lcf1 in S. pombe is discussed.

Extracellular Secretion of Free Fatty Acids by Disruption of a Fatty Acyl-CoA Synthetase Gene in Saccharomyces cerevisiae

To elucidate the molecular mechanism governing fatty acid transport across the cell membrane, we first isolated a Saccharomyces cerevisiae mutant, B-1, that exhibits a reduced acyl-CoA oxidase activity and an increase in free fatty acid accumulation. Following mutagenesis of B-1, a mutant, YTS51, which secretes free fatty acids, was isolated. The concentration of free fatty acids in the YTS51 culture medium was about 17 times higher than that in B-1. The mutation that causes the fatty acid secretion phenotype occurred at a single allele, and this phenotype was suppressed by the introduction of a single copy of FAA1, a gene for acyl-CoA Synthetase, to the mutant. Although the mutation expressing this phenotype was not within FAA1 in YTS51, the disruption of FAA1 in the wild-type strain resulted in fatty acid secretion even though the level of fatty acid secretion was less than that in YTS51. We consider that YTS51 is a suitable model to elucidate the molecular basis of the fatty acid transport process.

Extracellular secretion of free fatty acids by disruption of a fatty acyl-CoA synthetase gene in Saccharomyces cerevisiae

To elucidate the molecular mechanism governing fatty acid transport across the cell membrane, we first isolated a Saccharomyces cerevisiae mutant, B-1, that exhibits a reduced acyl-CoA oxidase activity and an increase in free fatty acid accumulation. Following mutagenesis of B-1, a mutant, YTS51, which secretes free fatty acids, was isolated. The concentration of free fatty acids in the YTS51 culture medium was about 17 times higher than that in B-1. The mutation that causes the fatty acid secretion phenotype occurred at a single allele, and this phenotype was suppressed by the introduction of a single copy of FAA1, a gene for acyl-CoA Synthetase, to the mutant. Although the mutation expressing this phenotype was not within FAA1 in YTS51, the disruption of FAA1 in the wild-type strain resulted in fatty acid secretion even though the level of fatty acid secretion was less than that in YTS51. We consider that YTS51 is a suitable model to elucidate the molecular basis of the fatty acid transport process.