The Studies in Constructing Yeast Cell Factories for the Production of Fatty Acid Alkyl Esters

Fatty acid alkyl esters have broad applications in biofuels, lubricant formulas, paints, coatings, and cosmetics. Traditionally, these esters are mostly produced through unsustainable and energy-intensive processes. In contrast, microbial production of esters from renewable and sustainable feedstocks may provide a promising alternative and has attracted widespread attention in recent years. At present, yeasts are used as ideal hosts for producing such esters, due to their availability for high-density fermentation, resistance to phage infection, and tolerance against toxic inhibitors. Here, we summarize recent development on the biosynthesis of alkyl esters, including fatty acid ethyl esters (FAEEs), fatty acid short-branched chain alkyl esters (FASBEs), and wax esters (WEs) by various yeast cell factories. We focus mainly on the enzyme engineering strategies of critical wax ester synthases, and the pathway engineering strategies employed for the biosynthesis of various ester products. The bottlenecks that limit productivity and their potential solutions are also discussed in this review.

Plastidial wax ester biosynthesis as a tool to synthesize shorter and more saturated wax esters

Background Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings, and cosmetics. They can be produced chemically from fossil fuel or plant-derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, tightly controlled and tailor-made WE synthesis in plants would be a sustainable, beneficial, and valuable commodity. Here, we report the expression of ten combinations of WE producing transgenes in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and composition in the transgenic plants. Results The transgenes consisted of different combinations of a FATTY ACYL-COA/ACP REDUCTASE (FAR) and two WAX SYNTHASES/ACYL-COA:DIACYLGLYCEROL O-ACYLTRANSFERASES (WSD), namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access distinct alcohol and acyl substrate pools within A. thaliana cells. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis. Conclusions A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE that are mainly derived from mid- and long-chain saturated and monounsaturated substrates.

Lipidomic, Transcriptomic, and BSA-660K Single Nucleotide Polymorphisms Profiling Reveal Characteristics of the Cuticular Wax in Wheat

Plant epidermal wax helps protect plants from adverse environmental conditions, maintains the function of tissues and organs, and ensures normal plant development. However, the constituents of epidermal wax and the regulatory mechanism of their biosynthesis in wheat have not been fully understood. Wheat varieties with different wax content, Jinmai47 and Jinmai84, were selected to comparatively analyze their waxy components and genetic characteristics, using a combination of lipidomic, transcriptomic, and BSA-Wheat 660K chip analysis. Through lipidomic analysis, 1287 lipid molecules were identified representing 31 lipid subclasses. Among these, Diacylglycerols (DG), (O-acyl)-ω-hydroxy fatty acids (OAHFA), wax ester (WE), Triacylglycerols (TG), and Monoradylglycerols (MG) accounted for 96.4% of the total lipids in Jinmai84 and 94.5% in Jinmai47. DG, OAHFA, and WE were higher in Jinmai84 than in Jinmai47 with the content of OAHFA 2.88-fold greater and DG 1.66-fold greater. Transcriptome sequence and bioinformatics analysis revealed 63 differentially expressed genes related to wax biosynthesis. Differentially expressed genes (DEGs) were found to be involved with the OAHFA, DG, and MG of synthesis pathways, which enriched the wax metabolism pathway. Non-glaucous and glaucous bulks from a mapping population were used to identify single nucleotide polymorphisms (SNP) via 660K chip analysis. Two loci centered on chromosomes 2D and 4B were detected and the locus on 4B is likely novel. These data improve understanding of complex lipid metabolism for cuticular wax biosynthesis in wheat and lay the foundation for future detailed investigation of mechanisms regulating wax metabolism.

Genome-wide association study (GWAS) of leaf wax components of apple

The wax layer of apple leaves plays an important role in improving stress resistance, but relatively little is known about the mechanisms of wax synthesis and transport in apple leaves. In this study, 17 wax components, including alcohols, alkanes, fatty acids and terpenes, were analyzed by gas chromatography-tandem mass spectrometry (GC-MS) from the leaves of 123 apple germplasms. Whole-genome sequencing of these apple accessions yielded 5.9 million high-quality single nucleotide polymorphisms (SNPs). We performed a genome-wide association study (GWAS) on 17 wax components and identified several genes related to wax synthesis and transport, including MdSHN1 (SHINE1), MdLTP4 (LIPID TRANSFER PROTEIN4), MdWSD1 (WAX ESTER SYNTHASE/ACYL-COA DIAC-YLGLYCEROL ACYLTRANSFERASE1), MdRDR1 (RNA-DEPENDENT RNA POLYMERASE1), MdACBP6 (ACYL-COA-BINDING PROTEIN6), MdNLE (NOTCHLESS) and MdABCG21 (ATP-BINDING CASSETTE G21). Moreover, we identified some prominent SNPs that may affect gene expression and protein function. These results provide insights into mechanisms of wax synthesis and transport in apple leaves and broaden the genetic resources and basis for facilitating resistance breeding.

Effect of Biodegradable Hydrophilic and Hydrophobic Emulsifiers on the Oleogels Containing Sunflower Wax and Sunflower Oil

The use of an appropriate oleogelator in the structuring of vegetable oil is a crucial point of consideration. Sunflower wax (SFW) is used as an oleogelator and displays an excellent potential to bind vegetable oils. The current study aimed to look for the effects of hydrophobic (SPAN-80) and hydrophilic (TWEEN-80) emulsifiers on the oleogels prepared using SFW and sunflower oil (SO). The biodegradability and all formulations showed globular crystals on their surface that varied in size and number. Wax ester, being the most abundant component of SFW, was found to produce fibrous and needle-like entanglements capable of binding more than 99% of SO. The formulations containing 3 mg of liquid emulsifiers in 20 g of oleogels showed better mechanical properties such as spreadability and lower firmness than the other tested concentrations. Although the FTIR spectra of all the formulations were similar, which indicated not much variation in the molecular interactions, XRD diffractograms confirmed the presence of β′ form of fat crystals. Further, the mentioned formulations also showed larger average crystallite sizes, which was supported by slow gelation kinetics. A characteristic melting point (Tm~60 °C) of triglyceride was visualized through DSC thermograms. However, a higher melting point in the case of few formulations suggests the possibility of even a stable β polymorph. The formed oleogels indicated the significant contribution of diffusion for curcumin release. Altogether, the use of SFW and SO oleogels with modified properties using biodegradable emulsifiers can be beneficial in replacing saturated fats and fat-derived products.

Plastidial Wax Ester Biosynthesis As A Tool To Synthesize Shorter And More Saturated Wax Esters

Background: Wax esters (WE) are neutral lipids that consist of a fatty alcohol esterified to a fatty acid. WE are valuable feedstocks in industry for producing lubricants, coatings and cosmetics. They can be produced chemically from fossil fuel or plant derived triacylglycerol. As fossil fuel resources are finite, the synthesis of WE in transgenic plants may serve as an alternative source. As chain length and desaturation degree of the alcohol and acyl moieties determine the physicochemical properties of WE and their field of application, a tightly controlled tailor-made WE synthesis in plants is aimed for. Here, we report the generation of ten combinations of WE producing transgenes expressed in Arabidopsis thaliana. In order to study their suitability for WE production in planta, we analyzed WE amount and synthesized WE species in the transgenic plants.Results: The transgenes consisted of different combinations of a fatty acyl-CoA/ACP reductase (FAR) and two wax synthases/acyl-CoA:diacylglycerol O-acyltransferases (WSD) namely WSD2 and WSD5 from the bacterium Marinobacter aquaeoleoi. We generated constructs with and without plastidial transit peptides to access diverse alcohol and acyl substrate pools within A. thaliana. We observed WE formation with plastid and cytosol-localized FAR and WSD in seeds. A comparative WE analysis revealed the production of shorter and more saturated WE by plastid-localized WE biosynthesis compared to cytosolic WE synthesis.Conclusions: A shift of WE formation into seed plastids is a suitable approach for tailor-made WE production and can be used to synthesize WE mainly derived from mid and long chain saturated and monounsaturated substrates.

Evolution and Characterization of Acetyl Coenzyme A: Diacylglycerol Acyltransferase Genes in Cotton Identify the Roles of GhDGAT3D in Oil Biosynthesis and Fatty Acid Composition

Cottonseed oil is rich in unsaturated fatty acids (UFAs) and serves as an edible oil in human nutrition. Reports suggest that acyl-coenzyme A: diacylglycerol acyltransferases (DGAT) and wax ester synthase/DGAT (WSD1) genes encode a key group of enzymes that catalyze the final step for triacylglycerol biosynthesis and enable an important rate-limiting process. However, their roles in oil biosynthesis and the fatty acid profile of cotton seed are poorly understood. Therefore, the aim of this study was to identify and characterize DGAT and WSD1 genes in cotton plants and examine their roles in oil biosynthesis, the fatty acid profile of cotton seeds, and abiotic stress responses. In this study, 36 GhDGAT and GhWSD1 genes were identified in upland cotton (G. hirsutum) and found to be clustered into four groups: GhDGAT1, GhDGAT2, GhDGAT3, and GhWSD1. Gene structure and domain analyses showed that the GhDGAT and GhWSD1 genes in each group are highly conserved. Gene synteny analysis indicated that segmental and tandem duplication events occurred frequently during cotton evolution. Expression analysis revealed that GhDGAT and GhWSD1 genes function widely in cotton development and stress responses; moreover, several environmental stress and hormone response-related cis-elements were detected in the GhDGAT and GhWSD1 promoter regions. The predicted target transcription factors and miRNAs imply an extensive role of GhDGAT and GhWSD1 genes in stress responses. Increases in GhDGAT3 gene expression with increases in cottonseed oil accumulation were observed. Transformation study results showed that there was an increase in C18:1 content and a decrease in C18:2 and C18:3 contents in seeds of Arabidopsis transgenic plants overexpressing GhDGAT3D compared with that of control plants. Overall, these findings contributed to the understanding of the functions of GhDGAT and GhWSD1 genes in upland cotton, providing basic information for further research.

The Application of a Rotating Packed Bed Reactor for the Synthesis of High Viscosity Wax Ester

Wax esters are high value-added products and widely used in a broad range of commercial fields. The enzymatic synthesis of wax ester from plant oil is more attractive than the traditional chemical method due to being environmental-friendly and limiting the use of hazardous chemicals. However, the high mass transfer resistance from the high viscous substrate leads to a low conversion and long reaction time in the continues stirred tank reactor (CSTR). The rotating packed bed reactor (RPBR) offers high mass transfer and can be used to enhance the enzymatic wax ester synthesis process. From the results, it could be concluded that the mass transfer was enhanced with the optimization of the centrifugal factor of the RPBR. Under the optimal process conditions, the wax ester yield of 96.4% was obtained after 4 hours reaction, and the half-life of the catalyst corresponded with 64 hours, while nine batches achieved of yields above 90%. The investigation proved that the RPBR is an attractive and effective reactor for heterogeneous bio-catalysis in high viscosity of 7.39~12.27 Cst at 40 °C.

Increased Cuticle Waxes by Overexpression of WSD1 Improves Osmotic Stress Tolerance in Arabidopsis thaliana and Camelina sativa

To ensure global food security under the changing climate, there is a strong need for developing ‘climate resilient crops’ that can thrive and produce better yields under extreme environmental conditions such as drought, salinity, and high temperature. To enhance plant productivity under the adverse conditions, we constitutively overexpressed a bifunctional wax synthase/acyl-CoA:diacylglycerol acyltransferase (WSD1) gene, which plays a critical role in wax ester synthesis in Arabidopsis stem and leaf tissues. The qRT-PCR analysis showed a strong upregulation of WSD1 transcripts by mannitol, NaCl, and abscisic acid (ABA) treatments, particularly in Arabidopsis thaliana shoots. Gas chromatography and electron microscopy analyses of Arabidopsis seedlings overexpressing WSD1 showed higher deposition of epicuticular wax crystals and increased leaf and stem wax loading in WSD1 transgenics compared to wildtype (WT) plants. WSD1 transgenics exhibited enhanced tolerance to ABA, mannitol, drought and salinity, which suggested new physiological roles for WSD1 in stress response aside from its wax synthase activity. Transgenic plants were able to recover from drought and salinity better than the WT plants. Furthermore, transgenics showed reduced cuticular transpirational rates and cuticle permeability, as well as less chlorophyll leaching than the WT. The knowledge from Arabidopsis was translated to the oilseed crop Camelina sativa (L.) Crantz. Similar to Arabidopsis, transgenic Camelina lines overexpressing WSD1 also showed enhanced tolerance to drought stress. Our results clearly show that the manipulation of cuticular waxes will be advantageous for enhancing plant productivity under a changing climate.