Very-long-chain Fatty Acids Biosynthesis Research Articles

Overexpression of Gossypium arboreum 3-ketoacyl-CoA synthase 6 (GaKCS6) gene enhanced leaf epicuticle wax in Gossypium hirsutum L. and improved tolerance against whitefly

Cotton Leaf Curl Virus (CLCuV) is a significant threat to cotton production, as it causes Cotton Leaf Curl Disease (CLCuD). Whitefly serves as a vector for the transmission of this virus. It can be controlled by developing barriers against whitefly infestation. The leaf epicuticle wax acts as a protective barrier against whitefly attacks. Research into wax biosynthesis and the fatty acid elongation pathway has highlighted the role of the 3-ketoacyl-CoA synthase (KCS) gene family in producing very-long-chain fatty acids (VLCFAs) in plants. The 3-ketoacyl-CoA synthase 6 (GaKCS6) gene, isolated from the CLCuV-resistant FDH-170 variety of Gossypium arboreum, was cloned under the control of the CaMV35S constitutive promoter and transformed into the CLCuV-susceptible Gossypium hirsutum variety CKC-3 resulting in significantly higher leaf epicuticle wax deposition. Overexpression of GaKCS6 in the transgenic cotton plants was confirmed through quantitative real-time PCR. The transgenic plants not only exhibited average growth but also showed improvements in agronomic traits. Scanning Electron Microscope (SEM) analysis further validated the enhanced leaf epicuticle wax deposition in transgenic plants compared to non-transgenic (control). A free-choice bioassay against whiteflies demonstrated that the transgenic plants remained free of viral infection, as confirmed by real-time PCR. These findings indicate that increased leaf epicuticle wax deposition in transgenic cotton effectively prevents whitefly attacks and the transmission of CLCuV. It suggests that the GaKCS6 gene plays a crucial role in producing leaf epicuticle wax through the VLCFAs biosynthesis pathway.

BnUC1 Is a Key Regulator of Epidermal Wax Biosynthesis and Lipid Transport in Brassica napus.

The bHLH (basic helix-loop-helix) transcription factor AtCFLAP2 regulates epidermal wax accumulation, but the underlying molecular mechanism remains unknown. We obtained BnUC1mut (BnaA05g18250D homologous to AtCFLAP2) from a Brassica napus mutant with up-curling leaves (Bnuc1) and epidermal wax deficiency via map-based cloning. BnUC1mut contains a point mutation (N200S) in the conserved dimerization domain. Overexpressing BnUC1mut in ZS11 (Zhongshuang11) significantly decreased the leaf epidermal wax content, resulting in up-curled and glossy leaves. In contrast, knocking out BnUC1mut in ZS11-NIL (Zhongshuang11-near-isogenic line) restored the normal leaf phenotype (i.e., flat) and significantly increased the leaf epidermal wax content. The point mutation weakens the ability of BnUC1mut to bind to the promoters of VLCFA (very-long-chain fatty acids) synthesis-related genes, including KCS (β-ketoacyl coenzyme synthase) and LACS (long-chain acyl CoA synthetase), as well as lipid transport-related genes, including LTP (non-specific lipid transfer protein). The resulting sharp decrease in the transcription of genes affecting VLCFA biosynthesis and lipid transport disrupts the normal accumulation of leaf epidermal wax. Thus, BnUC1 influences epidermal wax formation by regulating the expression of LTP and genes associated with VLCFA biosynthesis. Our findings provide a foundation for future investigations on the mechanism mediating plant epidermal wax accumulation.

A fatty acid elongase complex regulates cell membrane integrity and septin-dependent host infection by the rice blast fungus.

Very-long-chain fatty acids (VLCFAs) regulate biophysical properties of cell membranes to determine growth and development of eukaryotes, such as the pathogenesis of the rice blast fungus Magnaporthe oryzae. The fatty acid elongase Elo1 regulates pathogenesis of M. oryzae by modulating VLCFA biosynthesis. However, it remains unknown whether and how Elo1 associates with other factors to regulate VLCFA biosynthesis in fungal pathogens. Here, we identified Ifa38, Phs1 and Tsc13 as interacting proteins of Elo1 by proximity labelling in M. oryzae. Elo1 associated with Ifa38, Phs1 and Tsc13 on the endoplasmic reticulum (ER) membrane to control VLCFA biosynthesis. Targeted gene deletion mutants Δifa38, Δphs1 and Δtsc13 were all similarly impaired as Δelo1 in vegetative growth, conidial morphology, stress responses in ER, cell wall and membrane. These deletion mutants also displayed severe damage in cell membrane integrity and failed to organize the septin ring that is essential for penetration peg formation and pathogenicity. Our study demonstrates that M. oryzae employs a fatty acid elongase complex to regulate VLCFAs for maintaining or remodelling cell membrane structure, which is important for septin-mediated host penetration.

Dimesulfazet, a novel rice paddy herbicide, is an inhibitor of very long-chain fatty acid biosynthesis.

Dimesulfazet can control annual and perennial sedges in rice paddies. Here we assessed its mode of action. We performed a phenotype assay of Arabidopsis, conducted a metabolomic analysis of Echinochloa crus-galli, and analyzed the endogenous concentration of very long-chain fatty acids (VLCFAs) in Schoenoplectiella juncoides. Dimesulfazet treatment caused curling and greening symptoms in the leaves and fiddlehead-like symptoms in the inflorescences of Arabidopsis. These symptoms were visually indistinguishable from those caused by flufenacet and benfuresate, which belong to Herbicide Resistance Action Committee (HRAC) Group 15. We performed GC-MS/MS analysis of primary metabolites and LC-MS analysis of lipids in the herbicide-treated E. crus-galli, followed by Orthogonal Partial Least Squares Discriminant Analysis clustering. The results showed that dimesulfazet belongs to the HRAC Group 15 cluster. The endogenous concentrations of C24:0, C26:0, and C28:0 decreased in dimesulfazet-treated plants as compared to those in the control. Overall, the mode of action of dimesulfazet involves the inhibition of VLCFA biosynthesis.

Integrated transcriptome and fatty acid profiling provide the insights into lipid metabolism during seed development of Acer truncatum

Owing to its numerous physiological effects, Acer truncatum seed oil (ATO) is increasingly popular as a novel source of edible oil in China. To explore the characteristics of ATO accumulation, the potential biosynthetic mechanisms of the main fatty acids (FAs) were investigated during the six different developmental stages of A. truncatum seeds (i.e., S1 - S6). The results indicated that the ATO yields significantly increased from S1 to S5, and then decreased (S6), whereby the ratio of unsaturated fatty acids reached up to 82.63–90.65% of the total ATO content. The highest content of nervonic acid was observed at S6. Dynamic transcriptome data analyses revealed that 38,932 genes were actively transcribed in the five seed developmental stages (i.e., S1-S5), 103 of which were related to lipid metabolism. Combining with the gene expression and FA profiling, the potential metabolism of the main FA was further explored. Notably, changes in C18:1-CoA transformation were observed at the later periods of A. truncatum seed development (especially from S4 to S5), which might be the key factor limiting the accumulation of very long-chain fatty acids (VLCFAs). Additionally, 24 key functional genes related to lipid metabolism were highly correlated with VLCFA biosynthesis. Finally, 11 potential transcription factors from the bHLH, GRAS, bZIP, AP2/ERF, and MADS families exhibited highly positive correlations with these key functional genes in lipid metabolism, all of which might play important roles in regulating of VLCFA accumulation.

Genome-wide identification and expression analysis of the KCS gene family in soybean (Glycine max) reveal their potential roles in response to abiotic stress.

Very long chain fatty acids (VLCFAs) are fatty acids with chain lengths of 20 or more carbon atoms, which are the building blocks of various lipids that regulate developmental processes and plant stress responses. 3-ketoacyl-CoA synthase encoded by the KCS gene is the key rate-limiting enzyme in VLCFA biosynthesis, but the KCS gene family in soybean (Glycine max) has not been adequately studied thus far. In this study, 31 KCS genes (namely GmKCS1 - GmKCS31) were identified in the soybean genome, which are unevenly distributed on 14 chromosomes. These GmKCS genes could be phylogenetically classified into seven groups. A total of 27 paralogous GmKCS gene pairs were identified with their Ka/Ks ratios indicating that they had undergone purifying selection during soybean genome expansion. Cis-acting element analysis revealed that GmKCS promoters contained multiple hormone- and stress-responsive elements, indicating that GmKCS gene expression levels may be regulated by various developmental and environmental stimuli. Expression profiles derived from RNA-seq data and qRT-PCR experiments indicated that GmKCS genes were diversely expressed in different organs/tissues, and many GmKCS genes were found to be differentially expressed in the leaves under cold, heat, salt, and drought stresses, suggesting their critical role in soybean resistance to abiotic stress. These results provide fundamental information about the soybean KCS genes and will aid in their further functional elucidation and exploitation.

Metabolic engineering of oleaginous yeast in the lipogenic phase enhances production of nervonic acid

Insufficient biosynthesis efficiency during the lipogenic phase can be a major obstacle to engineering oleaginous yeasts to overproduce very long-chain fatty acids (VLCFAs). Taking nervonic acid (NA, C24:1) as an example, we overcame the bottleneck to overproduce NA in an engineered Rhodosporidium toruloides by improving the biosynthesis of VLCFAs during the lipogenic phase. First, evaluating the catalytic preferences of three plant-derived ketoacyl-CoA synthases (KCSs) rationally guided reconstructing an efficient NA biosynthetic pathway in R. toruloides. More importantly, a genome-wide transcriptional analysis endowed clues to strengthen the fatty acid elongation (FAE) module and identify/use lipogenic phase-activated promoter, collectively addressing the stagnation of NA accumulation during the lipogenic phase. The best-designed strain exhibited a high NA content (as the major component in total fatty acid [TFA], 46.3%) and produced a titer of 44.2 g/L in a 5 L bioreactor. The strategy developed here provides an engineering framework to establish the microbial process of producing valuable VLCFAs in oleaginous yeasts.

A 3-Ketoacyl-CoA Synthase 10 (KCS10) Homologue from Alfalfa Enhances Drought Tolerance by Regulating Cuticular Wax Biosynthesis.

Cuticular wax, forming the first line of defense against adverse environmental stresses, comprises very long-chain fatty acids (VLCFAs) and their derivatives. 3-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme for VLCFA biosynthesis. In this study, we isolated KCS10, a KCS gene from alfalfa, and analyzed the effect of gene expression on wax production and drought stress in transgenic plants. MsKCS10 overexpression increased compact platelet-like crystal deposition and promoted primary alcohol biosynthesis through acyl reduction pathways in alfalfa leaves. Overexpression of MsKCS10 induced the formation of coiled-rodlet-like crystals and increased n-alkane content through decarbonylation pathways in tobacco and tomato fruits. Overexpression of MsKCS10 enhanced drought tolerance by limiting nonstomatal water loss, improving photosynthesis, and maintaining osmotic potential under drought stress in transgenic tobacco. In summary, MsKCS10 plays an important role in wax biosynthesis, wax crystal morphology, and drought tolerance, although the mechanisms are different among the plant species. MsKCS10 can be targeted in future breeding programs to improve drought tolerance in plants.

Genome-wide identification and expression analysis of 3-ketoacyl-CoA synthase gene family in rice (Oryza sativa L.) under cadmium stress.

3-Ketoacyl-CoA synthase (KCS) is the key rate-limiting enzyme for the synthesis of very long-chain fatty acids (VLCFAs) in plants, which determines the carbon chain length of VLCFAs. However, a comprehensive study of KCSs in Oryza sativa has not been reported yet. In this study, we identified 22 OsKCS genes in rice, which are unevenly distributed on nine chromosomes. The OsKCS gene family is divided into six subclasses. Many cis-acting elements related to plant growth, light, hormone, and stress response were enriched in the promoters of OsKCS genes. Gene duplication played a crucial role in the expansion of the OsKCS gene family and underwent a strong purifying selection. Quantitative Real-time polymerase chain reaction (qRT-PCR) results revealed that most KCS genes are constitutively expressed. We also revealed that KCS genes responded differently to exogenous cadmium stress in japonica and indica background, and the KCS genes with higher expression in leaves and seeds may have functions under cadmium stress. This study provides a basis for further understanding the functions of KCS genes and the biosynthesis of VLCFA in rice.

Comparative phylogenomic insights of KCS and ELO gene families in Brassica species indicate their role in seed development and stress responsiveness

Very long-chain fatty acids (VLCFAs) possess more than twenty carbon atoms and are the major components of seed storage oil, wax, and lipids. FAE (Fatty Acid Elongation) like genes take part in the biosynthesis of VLCFAs, growth regulation, and stress responses, and are further comprised of KCS (Ketoacyl-CoA synthase) and ELO (Elongation Defective Elongase) sub-gene families. The comparative genome-wide analysis and mode of evolution of KCS and ELO gene families have not been investigated in tetraploid Brassica carinata and its diploid progenitors. In this study, 53 KCS genes were identified in B. carinata compared to 32 and 33 KCS genes in B. nigra and B. oleracea respectively, which suggests that polyploidization might has impacted the fatty acid elongation process during Brassica evolution. Polyploidization has also increased the number of ELO genes in B. carinata (17) over its progenitors B. nigra (7) and B. oleracea (6). Based on comparative phylogenetics, KCS, and ELO proteins can be classified into eight and four major groups, respectively. The approximate date of divergence for duplicated KCS and ELO genes varied from 0.03 to 3.20 million years ago (MYA). Gene structure analysis indicated that the maximum number of genes were intron-less and remained conserved during evolution. The neutral type of selection seemed to be predominant in both KCS and ELO genes evolution. String-based protein-protein interaction analysis suggested that bZIP53, a transcription factor might be involved in the activation of transcription of ELO/KCS genes. The presence of biotic and abiotic stress-related cis-regulatory elements in the promoter region suggests that both KCS and ELO genes might also play their role in stress tolerance. The expression analysis of both gene family members reflect their preferential seed-specific expression, especially during the mature embryo development stage. Furthermore, some KCS and ELO genes were found to be specifically expressed under heat stress, phosphorus starvation, and Xanthomonas campestris infection. The current study provides a basis to understand the evolution of both KCS and ELO genes in fatty acid elongation and their role in stress tolerance.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Strigolactones act downstream of gibberellins to regulate fiber cell elongation and cell wall thickness in cotton (Gossypium hirsutum).

Strigolactones (SLs) are a class of phytohormones that regulate plant shoot branching and adventitious root development. However, little is known regarding the role of SLs in controlling the behavior of the smallest unit of the organism, the single cell. Here, taking advantage of a classic single-cell model offered by the cotton (Gossypium hirsutum) fiber cell, we show that SLs, whose biosynthesis is fine-tuned by gibberellins (GAs), positively regulate cell elongation and cell wall thickness by promoting the biosynthesis of very long-chain fatty acids (VLCFAs) and cellulose, respectively. Furthermore, we identified two layers of transcription factors (TFs) involved in the hierarchical regulation of this GA-SL crosstalk. The top-layer TF GROWTH-REGULATING FACTOR 4 (GhGRF4) directly activates expression of the SL biosynthetic gene DWARF27 (D27) to increase SL accumulation in fiber cells and GAs induce GhGRF4 expression. SLs induce the expression of four second-layer TF genes (GhNAC100-2, GhBLH51, GhGT2, and GhB9SHZ1), which transmit SL signals downstream to two ketoacyl-CoA synthase genes (KCS) and three cellulose synthase (CesA) genes by directly activating their transcription. Finally, the KCS and CesA enzymes catalyze the biosynthesis of VLCFAs and cellulose, respectively, to regulate development of high-grade cotton fibers. In addition to providing a theoretical basis for cotton fiber improvement, our results shed light on SL signaling in plant development at the single-cell level.

Genome-Wide Identification and Expression Profiling of KCS Gene Family in Passion Fruit (Passiflora edulis) Under Fusarium kyushuense and Drought Stress Conditions.

Plant and fruit surfaces are covered with cuticle wax and provide a protective barrier against biotic and abiotic stresses. Cuticle wax consists of very-long-chain fatty acids (VLCFAs) and their derivatives. β-Ketoacyl-CoA synthase (KCS) is a key enzyme in the synthesis of VLCFAs and provides a precursor for the synthesis of cuticle wax, but the KCS gene family was yet to be reported in the passion fruit (Passiflora edulis). In this study, thirty-two KCS genes were identified in the passion fruit genome and phylogenetically grouped as KCS1-like, FAE1-like, FDH-like, and CER6-like. Furthermore, thirty-one PeKCS genes were positioned on seven chromosomes, while one PeKCS was localized to the unassembled genomic scaffold. The cis-element analysis provides insight into the possible role of PeKCS genes in phytohormones and stress responses. Syntenic analysis revealed that gene duplication played a crucial role in the expansion of the PeKCS gene family and underwent a strong purifying selection. All PeKCS proteins shared similar 3D structures, and a protein–protein interaction network was predicted with known Arabidopsis proteins. There were twenty putative ped-miRNAs which were also predicted that belong to nine families targeting thirteen PeKCS genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation results were highly associated with fatty acid synthase and elongase activity, lipid metabolism, stress responses, and plant-pathogen interaction. The highly enriched transcription factors (TFs) including ERF, MYB, Dof, C2H2, TCP, LBD, NAC, and bHLH were predicted in PeKCS genes. qRT-PCR expression analysis revealed that most PeKCS genes were highly upregulated in leaves including PeKCS2, PeKCS4, PeKCS8, PeKCS13, and PeKCS9 but not in stem and roots tissues under drought stress conditions compared with controls. Notably, most PeKCS genes were upregulated at 9th dpi under Fusarium kyushuense biotic stress condition compared to controls. This study provides a basis for further understanding the functions of KCS genes, improving wax and VLCFA biosynthesis, and improvement of passion fruit resistance.

The features of the fatty acid composition of Pyrus L. total lipids are determined by mountain ecosystem conditions

The composition of fatty acids (FAs) of total lipids of pericarp, seeds, and leaves of Pyrus caucasica Fed. and Pyrus communis L. growing in mountain ecosystems at different altitudes (300, 700 and 1200 m) was studied. It was found that the greatest differences in the relative content of FAs within a species, depending on the altitudes above sea level, were characteristic of the outer tissues of the pericarp (peel) and leaves, which were in direct contact with the external environment. Pericarp parenchyma to a lesser extent, and seeds practically did not differ in FA composition at different heights. At altitudes with increased UV radiation, conjugated octadecadienoates: rumenic acid (9,11–18:2) and 10,12–18:2 were registered in the pericarp and leaf of Purys L., the functions of which in plants were practically not studied. The wild P. caucasica at all growing altitudes was characterized by more very-long-chain FAs (VLCFAs) than the P. communis cultivar. At 700 m, most likely when exposed to fungal infections, the relative number of VLCFAs increased significantly, and new species of individual odd-chaine FAs appeared in their composition in both representatives. It was especially worth noting the appearance in peel and leaf melissic acid (30:0), which was rarely recorded in the plant. A characteristic feature of only P. communis at an altitude of 700 m was the large number of unsaturated individual VLCFAs. Based on the data obtained, a scheme of possible pathways for VLCFA biosynthesis in P. communis were proposed.

Characterization of uptake and metabolism of very long-chain fatty acids in peroxisome-deficient CHO cells

Fatty acids (FAs) longer than C20 are classified as very long-chain fatty acids (VLCFAs). Although biosynthesis and degradation of VLCFAs are important for the development and integrity of the myelin sheath, knowledge on the incorporation of extracellular VLCFAs into the cells is limited due to the experimental difficulty of solubilizing them. In this study, we found that a small amount of isopropanol solubilized VLCFAs in aqueous medium by facilitating the formation of the VLCFA/albumin complex. Using this solubilizing technique, we examined the role of the peroxisome in the uptake and metabolism of VLCFAs in Chinese hamster ovary (CHO) cells. When wild-type CHO cells were incubated with saturated VLCFAs (S-VLCFAs), such as C23:0 FA, C24:0 FA, and C26:0 FA, extensive uptake was observed. Most of the incorporated S-VLCFAs were oxidatively degraded without acylation into cellular lipids. In contrast, in peroxisome-deficient CHO cells uptake of S-VLCFAs was marginal and oxidative metabolism was not observed. Extensive uptake and acylation of monounsaturated (MU)-VLCFAs, such as C24:1 FA and C22:1 FA, were observed in both types of CHO cells. However, oxidative metabolism was evident only in wild-type cells. Similar manners of uptake and metabolism of S-VLCFAs and MU-VLCFAs were observed in IFRS1, a Schwan cell-derived cell line. These results indicate that peroxisome-deficient cells limit intracellular S-VLCFAs at a low level by halting uptake, and as a result, peroxisome-deficient cells almost completely lose the clearance ability of S-VLCFAs accumulated outside of the cells.

AP2/ERF transcription factors orchestrate very long chain fatty acid biosynthesis during Arabidopsis lateral root development

AP2/ERF transcription factors orchestrate very long chain fatty acid biosynthesis during Arabidopsis lateral root development

The role of very long chain fatty acids in yeast physiology and human diseases.

Fatty acids (FAs) are a highly diverse class of molecules that can have variable chain length, number of double bonds and hydroxylation sites. FAs with 22 or more carbon atoms are described as very long chain fatty acids (VLCFAs). VLCFAs are synthesized in the endoplasmic reticulum (ER) through a four-step elongation cycle by membrane embedded enzymes. VLCFAs are precursors forthe synthesis of sphingolipids (SLs) and glycerophospholipids. Besides their role as lipid constituents, VLCFAs are also found as precursors of lipid mediators. Mis-regulation of VLCFA metabolism can result in a variety of inherited diseases ranging from ichthyosis, to myopathies and demyelination. The enzymes for VLCFA biosynthesis are evolutionary conserved and many of the pioneering studies were performed in the model organism Saccharomyces cerevisiae. A growing body of evidence suggests that VLCFA metabolism is intricately regulated to maintain lipid homeostasis. In this review we will describe the metabolism of VLCFAs, how they are synthesized, transported and degraded and how these processes are regulated, focusing on budding yeast. We will review how lipid metabolism and membrane properties are affected by VLCFAs and which impact mutations in the biosynthetic genes have on physiology. We will also briefly describe diseases caused by mis-regulation of VLCFAs in human cells.

Discovery of broad-spectrum fungicides that block septin-dependent infection processes of pathogenic fungi.

Many pathogenic fungi depend on the development of specialized infection structures called appressoria to invade their hosts and cause disease. Impairing the function of fungal infection structures therefore provides a potential means by which diseases could be prevented. In spite of this extraordinary potential, however, relatively few anti-penetrant drugs have been developed to control fungal diseases, of either plants or animals. In the present study, we report the identification of compounds that act specifically to prevent fungal infection. We found that the organization of septin GTPases, which are essential for appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae, requires very-long-chain fatty acids (VLCFAs), which act as mediators of septin organization at membrane interfaces. VLCFAs promote septin recruitment to curved plasma membranes and depletion of VLCFAs prevents septin assembly and host penetration by M. oryzae. We observed that VLCFA biosynthesis inhibitors not only prevent rice blast disease, but also show effective, broad-spectrum fungicidal activity against a wide range of fungal pathogens of maize, wheat and locusts, without affecting their respective hosts. Our findings reveal a mechanism underlying septin-mediated infection structure formation in fungi and provide a class of fungicides to control diverse diseases of plants and animals.

Yarrowia lipolytica as a Metabolic Engineering Platform for the Production of Very-Long-Chain Wax Esters.

The oleaginous yeast Yarrowia lipolytica is an attractive cell factory platform strain and can be used for sustainable production of high-value oleochemical products. Wax esters (WEs) have a good lubricating property and are usually used as a base for the production of advanced lubricants and emollient oils. In this study, we reported the metabolic engineering of Y. lipolytica to heterologously biosynthesize high-content very-long-chain fatty acids (VLCFAs) and fatty alcohols and efficiently esterify them to obtain very-long-chain WEs. Co-expression of fatty acid elongases from different sources in Y. lipolytica could yield VLCFAs with carbon chain lengths up to 24. Combining with optimization of the central metabolic modules could further enhance the biosynthesis of VLCFAs. Furthermore, through the screening of heterologous fatty acyl reductases (FARs), we enabled high-level production of fatty alcohols. Genomic integration and heterologous expression of wax synthase (WS) and FAR in a VLCFA-producing Y. lipolytica strain yielded 95-650 mg/L WEs with carbon chain lengths from 32 to 44. Scaled-up fermentation in 5 L laboratory bioreactors significantly increased the production of WEs to 2.0 g/L, the highest content so far in yeasts. This study contributes to the further efficient biosynthesis of VLCFAs and their derivatives.

AKR2A interacts with KCS1 to improve VLCFAs contents and chilling tolerance of Arabidopsis thaliana.

Summary Arabidopsis thaliana AKR2A plays an important role in plant responses to cold stress. However, its exact function in plant resistance to cold stress remains unclear. In the present study, we found that the contents of very long‐chain fatty acids (VLCFAs) in akr2a mutants were decreased, and the expression level of KCS1 was also reduced. Overexpression of KCS1 in the akr2a mutants could enhance VLCFAs contents and chilling tolerance. Yeast‐2‐hybrid and bimolecular fluorescence complementation (BIFC) results showed that the transmembrane motif of KCS1 interacts with the PEST motif of AKR2A both in vitro and in vivo. Overexpression of KCS1 in akr2a mutants rescued akr2a mutant phenotypes, including chilling sensitivity and a decrease of VLCFAs contents. Moreover, the transgenic plants co‐overexpressing AKR2A and KCS1 exhibited a greater chilling tolerance than the plants overexpressing AKR2A or KCS1 alone, as well as the wild‐type. AKR2A knockdown and kcs1 knockout mutants showed the worst performance under chilling conditions. These results indicate that AKR2A is involved in chilling tolerance via an interaction with KCS1 to affect VLCFA biosynthesis in Arabidopsis.