Production Of Fatty Acid Ethyl Esters Research Articles

Production of ethyl esters from forage radish seed: An integrated sequential route using pressurized ethanol and ethyl acetate

This work investigated the production of fatty acid ethyl esters (FAEE) from forage radish seeds using a sequential process with pressurized ethanol and ethyl acetate as solvents and acyl acceptors. First, the effect of the solvent on the extraction was determined and subsequently, the sequential reaction was carried out with the material from the pressurized liquid extraction (PLE) where the effects of temperature, residence time and water concentration were investigated. Conventional extraction was carried out for comparative purposes with regards to the oil quality of the PLE and its efficiency in the reaction. The use of ethanol resulted in a high mass yield, which was attributed to the high non-lipid fraction in the extracted material, and ethyl acetate retained proteins, phenolic compounds and sugars in the defatted meal. The main fatty acids in the oil were oleic and erucic acids and high levels of minority compounds were observed in the PLE oil. In the sequential reaction, supercritical ethyl acetate at 300 °C provided the highest FAEE content (42 wt% in 25 min). The addition of water in the acyl acceptor increased the production of FAEE, reduced the thermal degradation of the esters and provided a product with low levels of acylglycerols. Oils from PLE and conventional extraction showed similar performance in the synthesis of esters. Ethyl linolenate and ethyl linoleate were the esters that showed the highest level of degradation. The highest content of FAEE (53 wt%) was reached at 300 °C with 20% (v/v) of water, resulting in a triacetin content of 0.92 wt%.

Oxidation-reduction potential affects medium-chain fatty acid ethyl ester production during wine alcohol fermentation

The medium-chain fatty acid ethyl esters (MCFAEEs) are a group of important aroma compounds generated during wine production. Wine alcohol fermentation involves several redox processes, which are affected by the oxidation–reduction potential (ORP). However, the mechanism via which ORP regulates MCFAEE production remains unclear. To investigate the effect of ORP on MCFAEE production, wine alcohol fermentation was performed using Saccharomyces cerevisiae under different ORPs. The results demonstrated that the ORPs studied (except for 90 mV) did not significantly affect cell growth, sugar consumption, and ethanol production, while the MCFAEE concentration in the simulated wines can be manipulated by ORP operation. MCFAEE levels increased till 96 h, and then decreased. The maximum MCFAEE level of 1222.97 μg/L was obtained after 96 h at 0 mV, which was 45.32% higher than that of the control. During the increase, higher relative expression of ACC1, FAS1, FAA2 and EEB1, elevated external citric acid flux, and moderate intracellular NADP+/NADPH ratio were observed at 0 mV compared to that at other ORPs. During the decrease, lowest relative expression of POX1 was detected at 0 mV. We showed for the first time the relationship between ORP operation and MCFAEE production in winemaking, which will improve the aroma quality of wine.

Fatty acid ethyl esters production from distillers corn oil by enzymatic catalysis

AbstractDistillers corn oil (DCO), a by‐product from the corn ethanol production, is an alternative source of glycerides to produce biodiesel. In the present work, this feedstock was enzymatically converted into fatty acid ethyl esters (FAEE). DCO presented relative density of 0.912 g/cm3, peroxide index of 3.05 ± 0.3 (meq/1000 g of sample) and acidity of 13.1 ± 1.96 (wt.%), with prevalence of the oleic (18:1) and linoleic (18:2) acids (sum equal to 76.9%). Three strategies were evaluated to improve the FAEE content: ethanol stepwise addition (ethanol:oil molar ratio of 4:1 (1/2 of ethanol at 0 and 1.5 h) and 3:1 (1/3 of ethanol at 0 h and 2/3 at 1.5 h or 1/3 of ethanol at 0, 1 and 2 h)); consecutive reactions (with Novozym 40086 [Rhizomucor miehei lipase] as biocatalyst); and mixture of enzymes (Novozym 40086 (6 wt.%) + Novozym 435 (Candida antarctica B lipase) (2 wt.%). The concept of combi‐lipases was evaluated for the first time using this raw material, resulting in a high ester content (>96%). The higher ester yields attained with DCO, compared to refined corn oil (33%) can be related to the better solubilization of ethanol and/or glycerol by‐product in the medium with DCO. The combi‐lipase system kept high than 80% of this initial activity after three repeated batches of reaction. The use of DCO enables the integration of corn ethanol and biodiesel production within the same general facility, which has logistic, environmental and, economic relevance.

Performance of Liquid Eversa on Fatty Acid Ethyl Esters Production by Simultaneous Esterification/Transesterification of Low-to-High Acidity Feedstocks

Liquid Eversa was evaluated in hydrolysis of acylglycerols from soybean oil deodorizer distillate (SODD), as well as simultaneous esterification/transesterification of SODD with low-to-high free fatty acids (FFAs) content using ethanol as acyl acceptor. Hydrolysis of SODD at mild temperature (37 °C) and without pH control (water:SODD mass ratio of 4:1) increased its FFAs content from 17.2 wt.% to 72.5 wt.% after 48 h reaction. A cold saponification of SODD allowed a saponification phase (SODD-SP) to be recovered with 93 wt.% saponification index and 2.25 wt.% FFAs content, which was used to find the experimental conditions for simultaneous esterification/transesterification reactions by experimental design. Temperature of 35 °C, enzyme concentration of 8.36 wt.%, and molar ratio of 3.64:1 (ethanol:SODD-SP) were found as the best conditions for fatty acid ethyl esters (FAEEs) production from SODD-SP (86.56 wt.% ester yield after 23 h reaction). Under the same reaction conditions, crude SODD (17.2 wt.% FFAs) and hydrolyzed SODD (72.5 wt.% FFAs) yielded products containing around 80 wt.% FAEEs. Caustic treatment could increase the ester content to around 90 wt.% and reduce the FFAs content to less than 1 wt.%. Our results show the good performance of liquid Eversa in aqueous (hydrolysis reactions) and organic (esterification/transesterification reactions) media.

Metabolic engineering Yarrowia lipolytica for a dual biocatalytic system to produce fatty acid ethyl esters from renewable feedstock in situ and in one pot.

Given the grave concerns over increasing consumption of petroleum resources and dramatic environmental changes arising from carbon dioxide emissions worldwide, microbial biosynthesis of fatty acid ethyl ester (FAEE) biofuels as renewable and sustainable replacements for petroleum-based fuels has attracted much attention. As one of the most important microbial chassis, the nonconventional oleaginous yeast Yarrowia lipolytica has emerged as a paradigm organism for the production of several advanced biofuels and chemicals. Here, we report the engineering of Y. lipolytica for use as an efficient dual biocatalytic system for in situ and one-pot production of FAEEs from renewable feedstock. Compared to glucose with 5.7% (w/w) conversion rate to FAEEs, sunflower seed oil in the culture medium was efficiently used to generate FAEEs with 84% (w/w) conversion rate to FAEEs by the engineered Y. lipolytica strain GQY20 that demonstrates an optimized intercellular heterologous FAEE synthesis pathway. In particular, the titer of extracellular FAEEs from sunflower seed oil reached 9.9 g/L, 10.9-fold higher than that with glucose as a carbon source. An efficient dual biocatalytic system combining ex vivo and strengthened in vitro FAEE production routes was constructed by overexpression of a lipase (Lip2) variant in the background strain GQY20, which further increased FAEEs levels to 13.5 g/L. Notably, deleting the ethanol metabolism pathway had minimal impact on FAEE production. Finally, waste cooking oil, a low-cost oil-based substance, was used as a carbon source for FAEE production in the Y. lipolytica dual biocatalytic system, resulting in production of 12.5 g/L FAEEs. Thus, the developed system represents a promising green and sustainable process for efficient biodiesel production. KEY POINTS: • FAEEs were produced by engineered Yarrowia lipolytica. • A Lip2 variant was overexpressed in the yeast to create a dual biocatalytic system. • Waste cooking oil as a substrate resulted in a high titer of 12.5 g/L FAEEs.

Development and validation of a simple and reliable alternative method for process monitoring and final product quality control during fatty acid ethyl esters production

As the production of biofuels increase, there is an urgent need to easily analytically control their production at the plant level as well as to assess the quality of the final products. Especially method capable of determining fatty acid ethyl ester content of 96.5% is crucial for utilization in praxis. In this work, a refractive index method with required sensitivity was developed and validated by means of a standard reference gas chromatography method. Validation with a considerable set of real unique samples obtained at pilot scale was performed for both purposes – process monitoring at high conversions and final product quality control. The results confirmed a favourable degree of accuracy with a relative deviation lower than 3.5% from the reference value given by the gas chromatography. Moreover, application of the method for quality control of fresh and long-term stored samples revealed that the deterioration of final products during storage can be detected. The developed refractive index method is thus suitable for the simple and rapid evaluation of the quality of produced fatty acid ethyl esters and for analytical monitoring of their production process.

Engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters

BackgroundProduction of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production.ResultsWe first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing.ConclusionWe achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.

Enhancement of C6–C10 fatty acid ethyl esters production in Saccharomyces cerevisiae CA by metabolic engineering

C6–C10 fatty acid ethyl esters (FAEEs) are important aromatic substances in alcoholic beverages and include ethyl hexanoate (EH), ethyl octanoate (EO), and ethyl decanoate (ED). However, the biosynthesis capacity of these esters are limited because of poor precursor C6–C10 fatty acyl-CoA accumulation and low alcohol acyltransferase (AATase) activity levels in Saccharomyces cerevisiae. In this study, S. cerevisiae CA stain was selected as the host stain because of its relatively high C6–C10 FAEE productivity. Ester production was improved by metabolic engineering strategies. Acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1) were overexpressed to enhance the acetyl-CoA precursor accumulation. Intracellular malonyl-CoA availability was also improved by acetyl-CoA decarboxylase (ACC1Ser659Ala, Ser1157Ala) overexpression. FAS1 and FAS2 (encoding fatty acid synthase) overexpression facilitated the condensation reactions to form C6–C10 fatty acyl-CoA from malonyl-CoA and acetyl-CoA precursors. Furthermore, the strawberry alcohol acyltransferase (SAAT) was introduced into S. cerevisiae CA to catalyze the formation of C6–C10 FAEEs from C6–C10 fatty acyl-CoA and ethanol substrates. As a consequence, the newly engineered strain achieved 25.89-, 7.27-, and 9.05-fold increases in extracellular EH (7.53 mg/L), EO (13.65 mg/L) and ED (13.87 mg/L) levels, respectively, compared to the wild-type strain CA.

Evaluation of Potential Biodiesel Feedstocks: Camelina, Turnip Rape, Oil Radish and Tyfon

Background: One of the most promising alternative biofuels, competitive with regular petrol, diesel or jet fuel is biodiesel, especially derived from plant oils. Until now, various technological approaches, as well as oil sources, have been proposed for biodiesel production, but an industrially scalable technology with high end-product quality and production efficiency has not been developed and brought to the market yet. Biodiesel is produced in Europe and North America mainly from rapeseed, or canola, sunflower and soybean oil. However, other underutilized plant species could also be considered as potential oil feedstocks for biodiesel. The great perspective holds Brassicaceae family, especially such species as false flax (Camelina sativa) and Ethiopian mustard (Brassica carinata), but many other Brassicaceae crops are still out of sight. Objectives: This research has been conducted aiming to identify and compare the productivity of several Brassicaceae crops (camelina or false flax (C. sativa), turnip rape (B. campestris), oil radish (Raphanus sativus var. oleifera) and tyfon (B. rapa ssp. oleifera f. biennis × (ssp. rapifera × ssp. pekinensis)), that are suitable for biodiesel production under conditions of temperate climate regions (Northern America, Europe); and to obtain biodiesel by transesterification of fatty acids present on these species using bioethanol. Methods and Materials: Seed oil content, yield and fatty acid profiles have been studied and analysed in different genotypes of C. sativa (10), winter (6) and spring (4) B. campestris, R. sativus var. oleifera (8) and tyfon (5). The most productive crops have been identified: false flax variety ‘Evro-12’ (1620 kg of oil per hectare) and ‘Peremoha’ (1657 kg/ha); winter turnip rape variety ‘Oriana’ (1373 kg/ha), oil radish variety ‘Kyianochka’ (1445 kg/ha) and tyfon varieties ‘Fitopal’ (1730 kg/ha) and ‘Obriy’ (1860 kg/ha). According to chromatographic analysis results, oils of winter turnip rape and tyfon contain high levels (38-42,8%) of erucic (22:1) acid, while oils from spring turnip rape, false flax and oil radish possess high amounts of short-chained fatty acids (not longer than C18) – up to 85,37% in camelina breeding line FEORZhYaFD. Fatty acid ethyl esters (FAEE) were produced from oil of best genotypes and proved to comply with all main quality requirements for diesel. Results: Moreover, a new solvent-based technology of high-yield (up to 96%) FAEE production, has been firstly proposed for C. sativa oil conversion. Conclusion: Best genotypes that can be used as a plant oil source for biodiesel production have been identified for camelina, turnip rape, oil radish and tyfon species. The data obtained on seed oil content, yield and fatty acid profiles suggested that they are: false flax – breeding form FEORZhYaFD; winter turnip rape - variety ‘Oriana’; oil radish - variety ‘Rayduha’ and tyfon hybrid - variety ‘Fitopal’. Biodiesel samples obtained from these plants fit the Ukrainian standards for diesel fuel and can be used in car engines. The proposed new technological approach to produce fatty acid ethyl esters allows to reduce reaction time and to increase esters yield and quality.

Ultrasound affects the selectivity and activity of immobilized lipases applied to fatty acid ethyl ester synthesis

Hydrophobic carriers can be used to improve the activity, stability and other properties of enzymes. Physical agents, like ultrasound, may also contribute to improving the dispersion and collision of the reagent molecules, decreasing the reaction time and intensifying the catalytic process. However, its effect on the enzyme activity and reaction selectivity is still not entirely understood. Here, enzyme modulation of immobilized lipases was studied under pulsed ultrasound irradiation in fatty acid ethyl ester (FAEE) synthesis for biodiesel production. Novozym 435® and two commercial lipases from Thermomyces lanuginosus and Rhizomucor miehei, immobilized on Octadecyl-Sepabeads were used as a biocatalyst in the transesterification reaction of vegetable oils and ethanol. The use of ultrasound associated with catalysis by the Novozym 435 increased the production of FAEE by about three times (from 8.9 to 26.4%) using soybean oil and changes were observed in the profile of the products. From the sonicated reaction, ethyl-palmitate production decreased from 23.4 to 11.7%, while the ethyl-linoleate content rose from 47.5 to 59.2%. On the other hand, the T. lanuginosus lipase was less affected by sonication with the overall production of FAEE increasing from 17.2 to 24.1%, with ethyl-palmitate and ethyl-linoleate content changing from 16.2 to 17.5% and 55.0 to 47.8%, respectively. Although the changes in the production yield are not too high, the main idea here was to show that ultrasound modulates the lipase activity as well as its respective selectivity. Thus, ultrasound, is responsible for changing the ethyl ester production, which can be applied to many other biochemical processes to improve or modulate their synthesis yield.

Enhanced Production of Fatty Acid Ethyl Ester with Engineered fabHDG Operon in Escherichia coli.

Biodiesel, or fatty acid ethyl ester (FAEE), is an environmentally safe, next-generation biofuel. Conventionally, FAEE is produced by the conversion of oil/fats, obtained from plants, animals, and microorganisms, by transesterification. Recently, metabolic engineering of bacteria for ready-to-use biodiesel was developed. In Escherichia coli, it is produced by fatty acyl-carrier proteins and ethanol, with the help of thioesterase (TesB) and wax synthase (WS) enzymes. One of the foremost barriers in microbial FAEE production is the feedback inhibition of the fatty acid (FA) operon (fabHDG). Here, we studied the effect of biodiesel biosynthesis in E. coli with an engineered fabHDG operon. With a basic FAEE producing BD1 strain harboring tes and ws genes, biodiesel of 32 mg/L were produced. Optimal FAEE biosynthesis was achieved in the BD2 strain that carries an overexpressed operon (fabH, fabD, and fabG genes) and achieved up to 1291 mg/L of biodiesel, a 40-fold rise compared to the BD1 strain. The composition of FAEE obtained from the BD2 strain was 65% (C10:C2, decanoic acid ethyl ester) and 35% (C12:C2, dodecanoic acid ethyl ester). Our findings indicate that overexpression of the native FA operon, along with FAEE biosynthesis enzymes, improved biodiesel biosynthesis in E. coli.

The effect of the lipid extraction method used in biodiesel production on the integrated recovery of biodiesel and biogas from Nannochloropsis gaditana, Isochrysis galbana and Arthrospira platensis

The integrated production of biodiesel and biogas has been explored by using N. gaditana, I. galbana and A. platensis as substrate. Three fatty acid ethyl ester (FAEE) production approaches were assessed, namely an indirect process with previously extracted lipids (IPPEL), an indirect process with extracted lipids as free fatty acids (IPELFFA) and a direct process (DP) without a previous lipid extraction stage. Besides de FAEE production, these approaches are considered as pre-treatments of the microbial biomasses evaluated in this study with the objective of increasing the methane yield during the anaerobic digestion. Biogas yields of the residues generated under the three FAEE production methodologies were compared to the biogas yield of the raw feedstock. In all cases, the biodiesel and biogas yields were higher after the FAEE production process from the previously extracted lipids as free fatty acids (IPELFFA). Therefore, this constitutes an interesting and promising route in the joint production of biodiesel and biogas.

Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil

Fatty acid ethyl esters (FAEEs) can potentially be used as biodiesel, which provides a renewable alternative to petroleum-derived diesel. FAEEs are primarily produced via transesterification of vegetable oil with an alcohol catalyzed by a strong base, which raises safety concerns. Microbial production presents a more environmentally sustainable method for FAEE production, and by harnessing the ability of oleaginous yeast Yarrowia lipolytica to degrade and assimilate hydrophobic substrates, FAEE production could be coupled to food waste bioremediation. In this study, we engineered Y.lipolytica to produce FAEEs from dextrose as well as from vegetable cooking oil as a model food waste. Firstly, we introduced pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) from Zymomonas mobilis to reconstitute the heterologous pathway for ethanol production. Second, we introduced and compared two heterologous wax ester synthases ws2 and maqu_0168 from Marinobacter sp. for FAEE biosynthesis. Next, we disrupted competitive pathways to increase fatty acyl-CoA pool, and optimized carbon sources and cell density for shake-flask fermentation. The engineered strain showed a 24-fold improvement in FAEE production titer over the starting strain. Moreover, we explored the potential of the engineered strain for FAEE production from the model food waste by supplementing vegetable cooking oil to the culture medium. To the best of our knowledge, this is the first report on FAEE production with the supplementation of vegetable cooking oil in Y.lipolytica. These findings provide valuable insights into the engineering of Y.lipolytica for high-level production of FAEEs and its utilization in food waste bioremediation.

Overproduction of Fatty Acid Ethyl Esters by the Oleaginous Yeast Yarrowia lipolytica through Metabolic Engineering and Process Optimization.

Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y.lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y.lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

Gate‐to‐gate life cycle assessment of biosurfactants and bioplasticizers production via biotechnological exploitation of fats and waste oils

AbstractBACKGROUNDThis study investigated the biotransformation of fats and waste oils to glycolipid biosurfactants and bioplasticizers. The ecological performance and environmental impacts of the bioprocesses were evaluated aiming to assess their present environmental status and thus suggest future improvements using life cycle assessment (LCA) methodology.RESULTSBiosurfactants, namely rhamnolipids and sophorolipids, were obtained via fermentation. Bioplasticizers, fatty acid ethyl esters (FAEE) and monoglycerides (MAG), were developed via enzymatic catalysis with selected enzymes in mesophilic temperatures via ethanolysis and glycerolysis, respectively. The study revealed that air emissions, electricity and thermal energy requirements are the key contributors to the potential environmental impacts in the life cycle impact assessment (LCIA). More specifically, rhamnolipids production has less energetic needs compared with sophorolipids manufacturing, resulting in lower environmental impacts. The increased thermal requirements of the MAG production phase is the main contributor to their negative environmental performance, with the overall energy consumption for MAG production being 3‐fold higher than for the FAEE formation phase.CONCLUSIONSThe assessment identified that among the biosurfactant production processes, sophorolipids production resulted in 22.7% higher environmental impact compared with rhamnolipids. Similarly, FAEE production can be classified as a more environment‐ friendly process compared with MAG, resulting in 67% lower environmental impact based on the environmental indicators assessed. © 2018 Society of Chemical Industry

Biochar as porous media for thermally-induced non-catalytic transesterification to synthesize fatty acid ethyl esters from coconut oil

This study put great emphasis on evaluating biochar as porous media for the thermally-induced non-catalytic transesterification reaction to synthesize fatty acid ethyl esters (FAEE) from coconut oil. Thermogravimetric analysis (TGA) of coconut oil experimentally justified that the bond dissociation of fatty acid from the backbone of triglycerides (TGs) could be achieved, which finding could be applied to the non-catalytic transesterification reaction. To use biochar as porous medium, the surficial morphology of maize residue biochar (MRB) was characterized, revealing that biochar possessed the wider pore size distribution ranging from meso- to macro-pores than SiO2. The highest yield of FAEE from non-catalytic transesterification of coconut oil in the presence of MRB was 87% at 380°C. To further enhance the FAEE yield, further studies associated with the production of FAEE with biochar made from different biomasses and various pyrolytic conditions should be performed.

Toward solar biodiesel production from CO2 using engineered cyanobacteria.

Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to various biochemicals including fatty acid-derived biodiesel. Recently, Synechococcus elongatus PCC 7942, a model cyanobacterium, has been engineered to convert CO2 to fatty acid ethyl esters (FAEEs) as biodiesel. Modular pathway has been constructed for FAEE production. Several metabolic engineering strategies were discussed to improve the production levels of FAEEs, including host engineering by improving CO2 fixation rate and photosynthetic efficiency. In addition, protein engineering of key enzyme in S. elongatus PCC 7942 was implemented to address issues on FAEE secretions toward sustainable FAEE production from CO2. Finally, advanced metabolic engineering will promote developing biosolar cell factories to convert CO2 to feasible amount of FAEEs toward solar biodiesel.

Simultaneous Enzymatic Transesterification and Esterification of an Acid Oil Using Fermented Solid as Biocatalyst

AbstractThis work studied the enzymatic synthesis of fatty acid ethyl esters (FAEE) for potential use as biodiesel via simultaneous esterification and transesterification of acid oil from macaúba in a solvent‐free system. A fermented and dry babassu cake with lipase activity from Rhizomucor miehei was used as biocatalyst. FAEE content above 85% was achieved after 96 h of reaction with enzyme loading of 13 U per g of oil, 120 mmol of hydrous ethanol (95% ethanol and 5% water)/20 mmol of oil (molar ratio ethanol:oil of 6:1), at 40 °C. After two consecutive enzymatic reactions, 90.8 wt% FAEE content was obtained. These results demonstrate a promising transesterification/esterification method for FAEE production from an acid and low‐cost oil and the process has potential to decrease the costs of enzymatic biodiesel production.

Photosynthetic CO2 Conversion to Fatty Acid Ethyl Esters (FAEEs) Using Engineered Cyanobacteria.

Metabolic engineering of cyanobacteria has received attention as a sustainable strategy to convert carbon dioxide to fatty acid-derived chemicals that are widely used in the food and chemical industries. Herein, Synechococcus elongatus PCC 7942, a model cyanobacterium, was engineered for the first time to produce fatty acid ethyl esters (FAEEs) from CO2. Due to the lack of an endogenous ethanol production pathway and wax ester synthase (AftA) activity in the wild-type cyanobacterium, we metabolically engineered S.elongatus PCC 7942 by expressing heterologous AftA and introducing the ethanol pathway, resulting in detectable peaks of FAEEs. To enhance FAEE production, a heterologous phosphoketolase pathway was introduced in the FAEE-producing strain to supply acetyl-CoA. Subsequent optimization of the cyanobacterial culture with a hexadecane overlay resulted in engineered S.elongatus PCC 7942 that produced photosynthetic FAEEs (10.0 ± 0.7 mg/L/OD730) from CO2. This paper is the first report of photosynthetic production of FAEEs from CO2 in cyanobacteria.

Production of FAME biodiesel in E. coli by direct methylation with an insect enzyme.

Most biodiesel currently in use consists of fatty acid methyl esters (FAMEs) produced by transesterification of plant oils with methanol. To reduce competition with food supplies, it would be desirable to directly produce biodiesel in microorganisms. To date, the most effective pathway for the production of biodiesel in bacteria yields fatty acid ethyl esters (FAEEs) at up to ~1.5 g/L. A much simpler route to biodiesel produces FAMEs by direct S-adenosyl-L-methionine (SAM) dependent methylation of free fatty acids, but FAME production by this route has been limited to only ~16 mg/L. Here we employ an alternative, broad spectrum methyltransferase, Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). By introducing DmJHAMT in E. coli engineered to produce medium chain fatty acids and overproduce SAM, we obtain medium chain FAMEs at titers of 0.56 g/L, a 35-fold increase over titers previously achieved. Although considerable improvements will be needed for viable bacterial production of FAMEs and FAEEs for biofuels, it may be easier to optimize and transport the FAME production pathway to other microorganisms because it involves fewer enzymes.