Whole‐cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio‐alkane has gained attention as an ideal drop‐in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof‐of‐principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α‐dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C_12–18 free fatty acids to C_11–17 aldehydes. Co‐expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole‐cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232–237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

The ‘LipoYeasts’ project: using the oleaginous yeast Yarrowia lipolytica in combination with specific bacterial genes for the bioconversion of lipids,fats and oils into high‐value products

The oleochemical industry is currently still dominated by conventional chemistry, with biotechnology only starting to play a more prominent role, primarily with respect to the biosurfactants or lipases, e.g. as detergents, or for biofuel production. A major bottleneck for all further biotechnological applications is the problem of the initial mobilization of cheap and vastly available lipid and oil substrates, which are then to be transformed into high‐value biotechnological, nutritional or pharmacological products. Under the EU‐sponsored LipoYeasts project we are developing the oleaginous yeast Yarrowia lipolytica into a versatile and high‐throughput microbial factory that, by use of specific enzymatic pathways from hydrocarbonoclastic bacteria, efficiently mobilizes lipids by directing its versatile lipid metabolism towards the production of industrially valuable lipid‐derived compounds like wax esters (WE), isoprenoid‐derived compounds (carotenoids, polyenic carotenoid ester), polyhydroxyalkanoates (PHAs) and free hydroxylated fatty acids (HFAs). Different lipid stocks (petroleum, alkane, vegetable oil, fatty acid) and combinations thereof are being assessed as substrates in combination with different mutant and recombinant strains of Y. lipolytica, in order to modulate the composition and yields of the produced added‐value products.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

Systems metabolic engineering design: Fatty acid production as an emerging case study

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C₆ to C₁₆ are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel‐like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high‐yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high‐yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain‐length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain‐lengths and functionalities. Biotechnol. Biotechnol. Bioeng. 2014;111: 849–857. © 2014 Wiley Periodicals, Inc.     

Efficient free fatty acid production in engineered Escherichia coli strains using soybean oligosaccharides as feedstock

To be competitive with current petrochemicals, microbial synthesis of free fatty acids can be made to rely on a variety of renewable resources rather than on food carbon sources, which increase its attraction for governments and companies. Industrial waste soybean meal is an inexpensive feedstock, which contains soluble sugars such as stachyose, raffinose, sucrose, glucose, galactose, and fructose. Free fatty acids were produced in this report by introducing an acyl‐ACP carrier protein thioesterase and (3R)‐hydroxyacyl‐ACP dehydratase into E. coli. Plasmid pRU600 bearing genes involved in raffinose and sucrose metabolism was also transformed into engineered E. coli strains, which allowed more efficient utilization of these two kinds of specific oligosaccharide present in the soybean meal extract. Strain ML103 (pRU600, pXZ18Z) produced ～1.60 and 2.66 g/L of free fatty acids on sucrose and raffinose, respectively. A higher level of 2.92 g/L fatty acids was obtained on sugar mixture. The fatty acid production using hydrolysate obtained from acid or enzyme based hydrolysis was evaluated. Engineered strains just produced ～0.21 g/L of free fatty acids with soybean meal acid hydrolysate. However, a fatty acid production of 2.61 g/L with a high yield of 0.19 g/g total sugar was observed on an enzymatic hydrolysate. The results suggest that complex mixtures of oligosaccharides derived from soybean meal can serve as viable feedstock to produce free fatty acids. Enzymatic hydrolysis acts as a much more efficient treatment than acid hydrolysis to facilitate the transformation of industrial waste from soybean processing to high value added chemicals. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:686–694, 2015     

Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium‐chain‐specific thioesterase and acyl carrier protein

Biofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil‐producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium‐chain‐specific thioesterase (TE) is expected to effectively alter algae to produce medium‐chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12‐specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14‐specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG‐containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0‐containing TAGs ranged from 1.25‐ to 1.58‐fold, indicating the ability of medium‐chain‐specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.     

Engineering Escherichia coli to produce branched-chain fatty acids in high percentages

Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, Escherichia coli strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. E. coli expressing an optimized lipoylation pathway produced 276 mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181 mg/L and 72% of total FFA. While E. coli can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.     

Variation in the Fatty‐Acid Content in Seeds of Various Black,Red, and White Currant Varieties

Currant seeds, a by‐product of juice production, are recognized as a valuable source of oil rich in polyunsaturated fatty acids. We have evaluated 28 currant varieties for their oil content and fatty‐acid composition. The oil content in the seeds ranged from 18.2–27.7%, and no statistical difference between varieties of different fruit color were recorded. Furthermore, the estimated oil yields in the field production ranged from 26.4–212.4 kg/ha. The GC and GC/MS chemical profiles of the seed oils extracted from all examined varieties were common for currants. Linoleic acid (LA) was the major component, with contents ranging from 32.7–46.9% of total fatty acids, followed by α‐linolenic acid (ALA; 2.9–32.0 %), oleic acid (OA; 9.8–19.9%), γ‐linolenic acid (GLA; 3.3–18.5%), palmitic acid (PA; 4.4–8.1%), stearidonic acid (SDA; 2.2–4.7%), and stearic acid (SA; 1.2–2.4%). Quantitative differences in the fatty‐acid profiles between varieties of different fruit color were observed. Blackcurrant varieties showed significantly higher contents of LA, GLA, and PA than red and white currant varieties, whereas significantly higher amounts of ALA and OL were detected in the red and white varieties. Cluster analysis based on the chemical oil profiles joined the blackcurrants in one group, while most of the red and white cultivars joined in a second group at the same linkage distance.     

Oil content,fatty acid composition and biodiesel properties among natural provenances of Siberian apricot (Prunus sibirica L.) from China

Siberian apricot (Prunus sibirica L.), an excellent woody oil plant unique to Asia, is well known for its ability to produce high‐oil seeds for use as a promising feedstock of biodiesel. Based on the investigation of natural Siberian apricot resources in China in the early stage, seeds of Siberian apricot from 74 geographic provenances which can fully reflect the overall information were collected. In this research, seeds oil content, fatty acid composition and biodiesel properties were evaluated, and the key environmental factors that caused the variation of these in different geographic provenance were analyzed. The oil content of Siberian apricot seeds is 45.48%–61.07%, and the average was 50.95% for all provenances. The characteristics of oil can identify and quantify eight fatty acids. The most abundant fatty acids were oleic acid (C18:1; 54.02%–76.54%), followed by linoleic acid (C18:2; 16.78%–38.49%) and erucic acid (C16:0; from 3.27% to 6.12%). Monounsaturated fatty acids are the most abundant in 54.75%–77.03% compared with saturated fatty acids and polyunsaturated fatty acids. The biodiesel properties of most provenance seeds meet the standards of the ASTM D6751 and GB/T 20828, and a few meet the standards of the EN14214. Through the clustering of oil content and fatty acid composition and the analysis of biodiesel properties indexes, it is concluded that KSK provenance is the most suitable for biodiesel production. The XBZ, HHE, AES, ZLQ and LD provenances may be preserved as potential biodiesel. RDA and VPA showed that the effects of environmental factors on the oil properties of Siberian apricot were ranked as terrain factor > climate factor > soil factor, among which longitude, latitude and altitude are the main terrain indicators. These evaluations can provide reference for the effective utilization and further development of Siberian apricot as a bioenergy feedstock.     

The role of Δ6-desaturase acyl-carrier specificity in the efficient synthesis of long-chain polyunsaturated fatty acids in transgenic plants

The role of acyl‐CoA‐dependent Δ6‐desaturation in the heterologous synthesis of omega‐3 long‐chain polyunsaturated fatty acids was systematically evaluated in transgenic yeast and Arabidopsis thaliana. The acyl‐CoA Δ6‐desaturase from the picoalga Ostreococcus tauri and orthologous activities from mouse (Mus musculus) and salmon (Salmo salar) were shown to generate substantial levels of Δ6‐desaturated acyl‐CoAs, in contrast to the phospholipid‐dependent Δ6‐desaturases from higher plants that failed to modify this metabolic pool. Transgenic plants expressing the acyl‐CoA Δ6‐desaturases from either O. tauri or salmon, in conjunction with the two additional activities required for the synthesis of C20 polyunsaturated fatty acids, contained higher levels of eicosapentaenoic acid compared with plants expressing the borage phospholipid‐dependent Δ6‐desaturase. The use of acyl‐CoA‐dependent Δ6‐desaturases almost completely abolished the accumulation of unwanted biosynthetic intermediates such as γ‐linolenic acid in total seed lipids. Expression of acyl‐CoA Δ6‐desaturases resulted in increased distribution of long‐chain polyunsaturated fatty acids in the polar lipids of transgenic plants, reflecting the larger substrate pool available for acylation by enzymes of the Kennedy pathway. Expression of the O. tauriΔ6‐desaturase in transgenic Camelina sativa plants also resulted in the accumulation of high levels of Δ6‐desaturated fatty acids. This study provides evidence for the efficacy of using acyl‐CoA‐dependent Δ6‐desaturases in the efficient metabolic engineering of transgenic plants with high value traits such as the synthesis of omega‐3 LC‐PUFAs.     

Die Gefriertrocknung von Rinderkot

Fatty acids in fish can arise from two sources: synthesis de novo from non‐lipid carbon sources within the animal, or directly from dietary lipid. Acetyl‐CoA derived mainly from protein can be converted to saturated fatty acids via the combined action of acetyl‐CoA carboxylase and fatty acid synthetase. The actual rate of fatty acid synthesis de novo is inversely related to the level of lipid in the diet. Freshwater fish can de‐saturate endogenously‐synthesized fatty acids to monounsaturated fatty acids via a A9 desaturase but lack the necessary enzymes for complete de novo synthesis of polyunsaturated fatty acids which must therefore be obtained preformed from the diet. Most freshwater fish species can desaturate and elongate 18:2(n‐6) and 18:3(n‐3) to their C20 and C22 homologues but the pathways involved remain ill‐defined. Cyclooxygenase and lipoxygenase enzymes can convert C20 polyunsaturated fatty acids to a variety of eicosanoid products. The dietary ratio of (n‐3) to (n‐6) polyunsaturated fatty acids influences the pattern of eicosanoids formed. The ß‐oxidation of fatty acids can occur in both mitochondria and peroxisomes but mi‐tochondrial ß‐oxidation is quantitatively more important and can utilise a wide range of fatty acid substrates.     

Dietary polyunsaturated fatty acids affect volume and metabolism of Drosophila melanogaster sperm

Dietary fatty acids can accumulate in sperm and affect their function in vertebrates. As Drosophila melanogaster shares several pathways of lipid metabolism and shows similar lipid‐dependent phenotypes but lacks some hormones that in vertebrates regulate lipid metabolism, there is currently no clear prediction as to how dietary fatty acids affect the sperm of D. melanogaster. We manipulated the amount and identity of dietary polyunsaturated fatty acids (PUFA) in the food of D. melanogaster males (a treatment known to affect membrane fluidity) and measured changes in sperm parameters. We found that (a) males reared on food containing PUFA‐rich, plant‐derived lipids showed a slower increase in sperm volume over male age compared to males reared on yeast‐derived lipid food which is richer in saturated fatty acids. (b) The resistance of sperm membrane integrity to osmotic stress was not altered by dietary lipid treatment, but (c) food containing yeast‐derived lipids induced a 46% higher in situ rate of production of reactive oxygen species in sperm cells. These findings show that dietary lipids have similar effects on sperm parameters in Drosophila as in vertebrates, affect some, but not all, sperm parameters and modulate male reproductive ageing. In concert with recent findings of sex‐specific seasonal variation of diet choice in the wild, our results suggest a substantial dietary impact on the dynamics of male reproduction in the wild.     

Fused dimerization increases expression,solubility, and activity of bacterial dehydratase enzymes

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ‐FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.     

Fatty‐acid profiles of juvenile lake trout reflect experimental diets consisting of natural prey

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PEROXIREDOXIN Q stimulates the activity of the chloroplast 16:1Δ3trans FATTY ACID DESATURASE4

Thylakoid membrane lipids, comprised of glycolipids and the phospholipid phosphatidylglycerol (PG), are essential for normal plant growth and development. Unlike other lipid classes, chloroplast PG in nearly all plants contains a substantial fraction of the unusual trans fatty acid 16:1^Δ3trans or 16:1t. We determined that, in Arabidopsis thaliana, 16:1t biosynthesis requires both FATTY ACID DESATURASE4 (FAD4) and a thylakoid‐associated redox protein, PEROXIREDOXIN Q (PRXQ), to produce wild‐type levels of 16:1t. The FAD4–PRXQ biochemical relationship appears to be very specific in planta, as other fatty acids (FA) desaturases do not require peroxiredoxins for their activity, nor does FAD4 require other chloroplast peroxiredoxins under standard growth conditions. Although most of chloroplast PG assembly occurs at the inner envelope membrane, FAD4 was primarily associated with the thylakoid membranes facing the stroma. Furthermore, co‐production of PRXQ with FAD4 was required to produce Δ3‐desaturated FAs in yeast. Alteration of the redox state of FAD4 or PRXQ through site‐directed mutagenesis of conserved cysteine residues impaired Δ3 FA production. However, these mutations did not appear to directly alter disulfide status of FAD4. These results collectively demonstrate that the production of 16:1t is linked to the redox status of the chloroplast through PRXQ associated with the thylakoids.