Production Of Fatty Alcohols Research Articles

Production of fatty alcohols from non-edible oils by enzymatic cascade reactions

A biocatalytic cascade transforming castor oil into (R,Z)-octadec-9-en-7-ol is presented by combining a lipase catalysed hydrolysis of castor oil into ricinoleic acid followed by a photoenzymatic decarboxylation into (R,Z)-octadec-9-en-7-ol.

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate.The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60% of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150% increase in ethanol production.

Direct production of fatty alcohols from glucose using engineered strains of Yarrowia lipolytica

:Fatty alcohols are important industrial oleochemicals with broad applications and a growing market. Here, we sought to engineer Yarrowia lipolytica to serve as a renewable source of fatty alcohols (specifically hexadecanol, heptadecanol, octadecanol, and oleyl alcohol) directly from glucose. Through screening four fatty acyl-CoA reductase (FAR) enzyme variants across two engineered background strains, we identified that MhFAR enabled the highest production. Further strain engineering, fed-batch flask cultivation, and extractive fermentation improved the fatty alcohol titer to 1.5 g/L. Scale-up of this strain in a 2L bioreactor led to 5.8 g/L total fatty alcohols at an average yield of 36 mg/g glucose with a maximum productivity of 39 mg/L hr. Finally, we utilized this fatty alcohol reductase to generate a customized fatty alcohol, linolenyl alcohol, from α-linolenic acid. Overall, this work demonstrates Y. lipolytica is a robust chassis for diverse fatty alcohol production and highlights the capacity to obtain high titers and yields from a purely minimal media formulation directly from glucose without the need for complex additives.

Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker’s yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol–producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl–CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol–producing enzyme fatty acyl–CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

Investigation of fatty aldehyde and alcohol synthesis from fatty acids by αDox- or CAR-expressing Escherichia coli

Fatty aldehydes are among the most important flavor and fragrance compounds. Most biotechnological production approaches make use of the one step conversion of fatty acids from renewable sources by the enzymes α-dioxygenase (αDox) or carboxylic acid reductase (CAR). Their reaction mechanisms and cofactor dependencies are very different. In contrast to heme-containing αDox which requires only oxygen as cosubstrate, CAR needs NADPH and ATP, which is a clear argument for the application of a whole cell catalyst. Therefore we compared fatty acid biotransformations with growing Escherichia coli cells expressing αDox or CAR to investigate their suitability for fatty aldehyde and also fatty alcohol production. Our results show the main product of fatty acid conversions with αDox-expressing cells to be the expected Cn-1 aldehyde. However, 14% of the products consist of the corresponding alcohol, but in addition, 17% of the products consist of further shortened aldehydes, alcohols and acids that result from the consecutive activity of αDox and a putative endogenous fatty aldehyde dehydrogenase activity in E. coli. Conversely, CAR-expressing cells produced only the unshortened fatty aldehyde and alcohol, whereby the latter surprisingly accounts for at least 80% of the products. The considerably higher extend of aldehyde reduction of CAR-expressing cells was shown to be causally connected to the CAR-mediated fatty acid conversion. Our study provides an overview about the applicability of αDox- or CAR-based whole cell catalysts and gives a detailed description of side products as well as suggestions for tailored strain engineering.

High production of fatty alcohols in Yarrowia lipolytica by coordination with glycolysis

Fatty alcohol biosynthesis by oleaginous microbes was a promising alternative to the petroleum or other non-renewable resources-based process. However, low titer and yield hamper the further industrial and commercial applications. Here, we developed an efficient strategy to coordinate fatty alcohol with glycolysis which achieved a ‘pull-and-push’ effect to improve fatty alcohol production. Transcript profiling indicated that genes in carbohydrate metabolism were up-regulated significantly in response to high fatty alcohol production. Based on it, 11 glycolysis promoters were screening to express fatty acyl-CoA reductase (FAR) to relate the fatty alcohol production with the up-regulated carbohydrate metabolism, and the fatty alcohol production reached 557 mg/L when FAR was expressed by the promoter of PFBAin. RNA-seq and qRT-PCR analysis demonstrated that a ‘pull-and-push’ effect caused by the coordination system dynamically enhanced the product synthesis flux from top to bottom, which was also testified and intensified by doubled glucose concentration. After manipulating structural and regulatory genes of lipid metabolism, the final strain achieved up to 5.75 g/L fatty alcohol production from modified YPD medium (containing 91 g/L glucose) in shake flasks, which represented the highest titer reported to date. This work offered a feasible and effective reference for dynamic manipulation of fatty acid-derived chemicals synthesis.

Utilizing a Wastewater-Based Medium for Engineered Saccharomyces cerevisiae for the Biological Production of Fatty Alcohols and Carboxylic Acids to Replace Petrochemicals

Personal care items, pharmaceutical formulations, food additives, detergents, plasticizers, and industrial solvents manufactured from artificially synthesized acids or petrochemicals can be produced with more sustainable methods. The metabolic engineering of microbial hosts, specifically the heterotrophic yeast Saccharomyces cerevisiae, using a cost-effective wastewater-based growth medium could potentially provide a solution. Upregulation of the fatty acid biosynthesis (FAS) pathway present in this species is key to increasing all fatty acid-derived products. Three plasmids containing genes responsible for the production of the enzymes acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS), and fatty acid reductase (Far1), were transformed into S. cerevisiae cells using the PEG-LiOAc method. Transformation was conducted separately and together to optimize efficiency. We hypothesized that transformed cells would display more varied fatty acid and fatty alcohol profiles, and an increased ability to grow in a modified wastewater-based medium while degrading dissolved organics. A spectrophotometric assay predicated on the oxidation of NADPH to NADP+ was performed to determine the activity of the overexpressed enzymes. High performance liquid chromatography (HPLC) analysis underscored the presence of C-16 and C-18 fatty alcohols and fatty acids present in the yeast. Finally, a gas chromatography-mass spectrometry (GC-MS) analysis portrayed a reduction in organic compounds in wastewater media that was metabolized by S. cerevisiae while evidence of ethanol production via fermentation was seen. The study adds to research on renewable energy alternatives, but more importantly, demonstrates an effective method in which S. cerevisiae can biologically produce valuable specialty and commodity chemicals to mitigate petroleum use while removing organic content from wastewater.

A facile and robust non-natural three enzyme biocatalytic cascade based on Escherichia coli surface assembly for fatty alcohol production

Fatty alcohol is considered as a promising clean fuel alternative to fossil fuel. In this study, we developed a facile and robust multiple enzyme complex of lipase (LIP), carboxylic acid reductase (CAR) and aldehyde reductase (AHR) based on cohesin-dockerin specific interaction and E. coli-ice nucleation protein (INP) surface assembly for highly efficient conversion of triacylglycerols (TAGs) to fatty alcohols. The proximity effect, customized ratio and positional arrangement of the three enzymes were readily genetically generated. The optimized cell associated three enzyme complex gave 73% of conversion yield, and showed significantly improved stability in higher temperature, acidic/alkaline environments, polar organic solvents, high concentration of substrate, and good recyclability in successive biotransformations. The three enzyme complex provides an efficient non-natural synthetic cascade route for fatty alcohol bioproduction alternative to complex de novo biosynthesis route.

Ruthenium catalyst for fatty alcohols production

Ruthenium catalyst for fatty alcohols production

A gene cluster for fatty alcohol synthesis from a Reinekea-related bacterium that accumulates fatty alcohols.

This study reports on a marine bacterium that accumulates fatty alcohols (C14,16,18 ) at more than 1% (w/w) of the dry cell weight. This unique bacterium, designated as strain 1-4, is related to the genus Reinekea. A novel gene cluster for fatty alcohol synthesis, phsAB, is identified from strain 1-4. The phsA product shows significant homology to fatty acyl-CoA reductase (51% identity), whereas the phsB product shows very low homology to lipases. Interestingly, phsA alone causes Escherichia coli to accumulate fatty alcohols at 19% (w/w) of the dry cell weight. Moreover, the phsA-containing E. coli accumulate more fatty alcohols (24%) and grow faster after phsB is introduced, indicating that phsAB could greatly assist the mass production of fatty alcohols.

Enrichment of syngas-converting mixed microbial consortia for ethanol production and thermodynamics-based design of enrichment strategies

BackgroundThe production of ethanol through the biochemical conversion of syngas, a mixture of H2, CO and CO2, has been typically studied using pure cultures. However, mixed microbial consortia may offer a series of benefits such as higher resilience and adaptive capacity, and non-sterile operation, all of which contribute to reducing the utility consumption when compared to pure culture-based processes. This work focuses on the study of strategies for the enrichment of mixed microbial consortia with high ethanologenic potential, investigating the effect of the operational conditions (pH and yeast extract addition) on both the ethanol yield and evolution of the microbial community along the enrichment process. The pH was selected as the main driver of the enrichment as it was expected to be a crucial parameter for the selection of carboxydotrophic bacteria with high ethanologenic potential. Additionally, a thermodynamic analysis of the network of biochemical reactions carried out by syngas-converting microbial consortia was performed and the potential of using thermodynamics as a basis for the selection of operational parameters favoring a specific microbial activity was evaluated.ResultsAll enriched consortia were dominated by the genus Clostridium with variable microbial diversity and species composition as a function of the enrichment conditions. The ethanologenic potential of the enriched consortia was observed to increase as the initial pH was lowered, achieving an ethanol yield of 59.2 ± 0.2% of the theoretical maximum in the enrichment at pH 5. On the other hand, yeast extract addition did not affect the ethanol yield, but triggered the production of medium-chain fatty acids and alcohols. The thermodynamic analysis of the occurring biochemical reactions allowed a qualitative prediction of the activity of microbial consortia, thus enabling a more rational design of the enrichment strategies targeting specific activities. Using this approach, an improvement of 22.5% over the maximum ethanol yield previously obtained was achieved, reaching an ethanol yield of 72.4 ± 2.1% of the theoretical maximum by increasing the initial acetate concentration in the fermentation broth.ConclusionsThis study demonstrated high product selectivity towards ethanol using mixed microbial consortia. The thermodynamic analysis carried out proved to be a valuable tool for interpreting the metabolic network of microbial consortia-driven processes and designing microbial-enrichment strategies targeting specific biotransformations.

Anaerobic production of medium-chain fatty alcohols via a β-reduction pathway

In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse β-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and β-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse β-oxidation pathway produced n-alcohols at titers of 1.8 g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

Investigation on CO2 bio-mitigation using Halomonas stevensii in laboratory scale bioreactor: Design of downstream process and its economic feasibility analysis

In the present study, H. stevensii was cultivated on a semi-continuous mode in a laboratory scale bio-reactor using CO2(g) [15% (v/v)] as carbon source and thiosulfate (S2O32−) as an energy source for the total duration of three days. Approximately, 100% CO2(g) removal from gaseous phase was achieved. Leachate obtained was subjected to different downstream bio-processing strategies. Biomass harvesting using filtration and recovery of metabolites without cell disruption using solvent extraction from wet biomass was observed as the best downstream processing strategy. Qualitative analysis of products was carried out using gas chromatography and mass spectroscopy (GC–MS) and their results have indicated fatty alcohols (C8–C27) as primary metabolites. Fourier transform infrared spectroscopy (FTIR) analysis, approximate material balance and thermodynamic analysis have confirmed the intracellular assimilation of CO2(g) as HCO3− and its metabolization into fatty alcohols. Economic feasibility of the process has suggested that the developed downstream process has the capability to replace coconut oil based process for the production of fatty alcohols (C12–C14) and it can be utilized for the production of dodecanol as compared to the tetradecanol.

Optimization of C16 and C18 fatty alcohol production by an engineered strain of Lipomyces starkeyi.

The oleaginous yeast Lipomyces starkeyi was engineered for the production of long-chain fatty alcohols by expressing a fatty acyl-CoA reductase, mFAR1, from Mus musculus. The optimal conditions for production of fatty alcohols by this strain were investigated. Increased carbon-to-nitrogen ratios led to efficient C16 and C18 fatty alcohol production from glucose, xylose and glycerol. Batch cultivation resulted in a titer of 1.7g/L fatty alcohol from glucose which represents a yield of 28mg of fatty alcohols per gram of glucose. This relatively high level of production with minimal genetic modification indicates that L. starkeyi may be an excellent host for the bioconversion of carbon-rich waste streams, particularly lignocellulosic waste, to C16 and C18 fatty alcohols.

Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX

Fatty alcohol production in Synechocystis sp. PCC 6803 was achieved through heterologous expression of the fatty acyl-CoA/ACP reductase Maqu2220 from the bacteria Marinobacter aquaeolei VT8 and the fatty acyl-ACP reductase DPW from the rice Oryza sativa. These platform strains became models for testing multiplex CRISPR-interference (CRISPRi) metabolic engineering strategies to both improve fatty alcohol production and to study membrane homeostasis. CRISPRi allowed partial repression of up to six genes simultaneously, each encoding enzymes of acyl-ACP-consuming pathways. We identified the essential phosphate acyltransferase enzyme PlsX (slr1510) as a key node in C18 fatty acyl-ACP consumption, repression of slr1510 increased octadecanol productivity threefold over the base strain and gave the highest specific titers reported for this host, 10.3mgg−1 DCW. PlsX catalyzes the first committed step of phosphatidic acid synthesis, and has not been characterized in Synechocystis previously. We found that accumulation of fatty alcohols impaired growth, altered the membrane composition, and caused a build-up of reactive oxygen species.

Heterologous transporter expression for improved fatty alcohol secretion in yeast

The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.

Modular pathway engineering for the microbial production of branched-chain fatty alcohols

The intrinsic structural properties of branched long-chain fatty alcohols (BLFLs) in the range of C12 to C18 make them more suitable as diesel fuel replacements and for other industrial applications than their straight-chain counterparts. While microbial production of straight long-chain fatty alcohols has been achieved, biosynthesis of BLFLs has never been reported. In this work, we engineered four different biosynthetic pathways in Escherichia coli to produce BLFLs. We then employed a modular engineering approach to optimize the supply of α-keto acid precursors and produced either odd-chain or even-chain BLFLs with high selectivity, reaching 70 and 75% of total fatty alcohols, respectively. The acyl-ACP and alcohol-producing modules were also extensively optimized to balance enzyme expression level and ratio, resulting in a 6.5-fold improvement in BLFL titers. The best performing strain overexpressed 14 genes from 6 engineered operons and produced 350 mg/L of BLFLs in fed-batch fermenter. The modular engineering strategy successfully facilitated microbial production of BLFLs and allowed us to quickly optimize new BLFL pathway with high titers and product specificity. More generally, this work provides pathways and knowledge for the production of BLFLs and BLFL-related, industry-relevant chemicals in high titers and yields.

Rewiring neutral lipids production for the de novo synthesis of wax esters in Rhodococcus opacus PD630

Rhodococcus opacus PD630 accumulates significant amounts of triacylglycerols (TAG), but is not able to de novo synthesize wax esters (WE) from structural unrelated carbon sources, such as gluconate. In this study, strain PD630 was engineered to produce WE by heterologous expression of maqu_2220 gene, which encodes a fatty acyl-CoA reductase for the production of fatty alcohols in Marinobacter hydrocarbonoclasticus. Recombinant cells produced ca. 46% of WE and 54% of TAG (of total WE+TAG) from gluconate compared with the wild type, which produced 100% of TAG. Cell growth was not affected by the heterologous expression of MAQU_2220. Several saturated and monounsaturated WE species were produced by cells, with C18:C16, C16:C16 and C16:C18 as main species. The fatty acid composition of WE fraction in PD630maqu_2220 was enriched with C16:0, C18:0, whereas C16:0, C18:0 and C18:1 predominated in the TAG fraction. Significant amounts of WE and TAG were accumulated by PD630maqu_2220 from whey, an inexpensive waste material from dairy industries, without affecting cell biomass production. This is the first report on WE synthesis by R. opacus from gluconate, which demonstrates that lipid metabolism of this bacterium is flexible enough to assimilate heterologous components for the production of new lipid derivatives with industrial interest.

Transcriptome analysis of Helicoverpa armigera male hairpencils: Alcohol biosynthesis and requirement for mating success

Many female animals use different strategies to assess male quality to increase their own reproductive fitness. In moths, females usually use chemical signals (sex pheromones) to attract males from a distance. Once males approach a female, they release close range pheromones from hairpencils to facilitate female acceptance. However, detailed mechanisms involved in male sex pheromone biosynthesis and its action in promoting female acceptance have not yet been fully characterized. This study screened a series of candidate genes via a transcriptome analysis of the male hairpencil of Helicoverpa armigera. Using pharmacological inhibitor and RNAi-mediated knockdown assays, we demonstrated that Ca2+ and cyclic-AMP were involved in pheromone biosynthesis activating neuropeptide (PBAN)-induced male sex pheromone biosynthesis. The functional analysis of candidate enzymes involved in the male sex pheromone biosynthesis pathway demonstrated that a decreased mRNA levels of acetyl-CoA carboxylase, Δ11-desaturase, and fatty-acyl reductase 2 by RNAi-mediated knockdown led to a significant decrease in the production of fatty acyl alcohols and the efficacy of female acceptance. Our results demonstrated the important role of the fatty acyl alcohol biosynthetic pathway in a PBAN-induced male sex pheromone biosynthesis and the importance of hairpencil compounds in female mating acceptance.

Engineering Saccharomyces cerevisiae for Efficient Biosynthesis of Fatty Alcohols Based on Enhanced Supply of Free Fatty Acids.

In recent years, production of fatty acid derivatives has attracted much attention because of their wide range of applications in renewable oleochemicals. Microorganisms such as Saccharomyces cerevisiae provided an ideal cell factory for such chemical synthesis. In this study, an efficient strategy for the synthesis of fatty alcohols based on enhanced supply of free fatty acids (FFAs) was constructed. The FAA1 and FAA4 genes encoding two acyl-CoA synthetases in S. cerevisiae were deleted, resulting in the accumulation of FFAs with carbon chain length from C8 to C18. The coexpression of the carboxylic acid reductase gene (car) from Mycobacterium marinum and the phosphopantetheinyl transferase gene (sfp) from Bacillus subtilis successfully converted the accumulated FFAs into fatty alcohols. The concentration of the total fatty alcohols reached 24.3 mg/L, which is in agreement with that of the accumulated FFAs. To further increase the supply of FFAs, the DGAI encoding the acyl-CoA:diacylglycerol acyltransferase involved in the rate-limiting step of triacylglycerols storage was codeleted with FAA1 and FAA4, and the acyl-CoA thioesterase gene (acot) was expressed together with car and sfp, resulting in an enhanced production of fatty alcohols, the content of which increased to 31.2 mg/L. The results herein demonstrated the efficiency of the engineered pathway for the production of fatty acid derivatives using FFAs as precursors.