Production Of Free Fatty Acids Research Articles

Effects of overexpression of STB5 in Saccharomyces cerevisiae on fatty acid biosynthesis, physiology and transcriptome.

ABSTRACTMicrobial conversion of biomass to fatty acids (FA) and products derived thereof is an attractive alternative to the traditional oleochemical production route from animal and plant lipids. This study examined if NADPH-costly FA biosynthesis could be enhanced by overexpressing the transcription factor Stb5 in Saccharomyces cerevisiae. Stb5 activates expression of multiple genes encoding enzymes within the pentose phosphate pathway (PPP) and other NADPH-producing reactions. Overexpression of STB5 led to a decreased growth rate and an increased free fatty acid (FFA) production during growth on glucose. The improved FFA synthetic ability in the glucose phase was shown to be independent of flux through the oxidative PPP. RNAseq analysis revealed that STB5 overexpression had wide-ranging effects on the transcriptome in the batch phase, and appeared to cause a counterintuitive phenotype with reduced flux through the oxidative PPP. During glucose limitation, when an increased NADPH supply is likely less harmful, an overall induction of the proposed target genes of Stb5 (eg. GND1/2, TAL1, ALD6, YEF1) was observed. Taken together, the strategy of utilizing STB5 overexpression to increase NADPH supply for reductive biosynthesis is suggested to have potential in strains engineered to have strong ability to consume excess NADPH, alleviating a potential redox imbalance.

A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae

With rapid progress in DNA synthesis and sequencing, strain engineering starts to be the rate-limiting step in synthetic biology. Here, we report a gRNA-tRNA array for CRISPR-Cas9 (GTR-CRISPR) for multiplexed engineering of Saccharomyces cerevisiae. Using reported gRNAs shown to be effective, this system enables simultaneous disruption of 8 genes with 87% efficiency. We further report an accelerated Lightning GTR-CRISPR that avoids the cloning step in Escherichia coli by directly transforming the Golden Gate reaction mix to yeast. This approach enables disruption of 6 genes in 3 days with 60% efficiency using reported gRNAs and 23% using un-optimized gRNAs. Moreover, we applied the Lightning GTR-CRISPR to simplify yeast lipid networks, resulting in a 30-fold increase in free fatty acid production in 10 days using just two-round deletions of eight previously identified genes. The GTR-CRISPR should be an invaluable addition to the toolbox of synthetic biology and automation.

Mineralocorticoid receptor blockade attenuates disrupted glutathione-dependent antioxidant defense and elevated endoglin in the hearts of pregnant rats exposed to testosterone.

Elevated testosterone during late pregnancy has been linked to cardiac dysfunction and poor pregnancy outcomes. The role of mineralocorticoid receptor (MR) in testosterone-induced cardiac dysfunction has not been fully elucidated. The study was therefore designed to investigate the role of MR on gestational excess androgen-induced cardiac disrupted glutathione-dependent antioxidant system and elevated endoglin (Eng) linking it with pregnancy outcomes. Thirty-two pregnant Wistar rats were randomly allotted into four groups (n = 8/group) receiving (sc) olive oil, testosterone propionate (0.5mg/kg) singly or in combination with non-selective MR blocker (MRB), spironolactone (0.25mg/kg; po) or selective MRB, and eplerenone (1.0mg/kg; po) in late between gestational days 14 and 19. The results showed that testosterone exposure resulted in elevated fasting blood glucose, increased cardiac mass, free fatty acid, endoglin, malonaldehyde, oxidized glutathione, uric acid, and lactate production and cardiac injury marker enzymes. On the other hand, testosterone exposure caused reduction in cardiac adenosine, nitric oxide, glutathione, glutathione peroxidase, and glucose-6-phosphate dehydrogenase activities. However, MR blockade by spironolactone and or eplerenone attenuated the effects induced by testosterone exposure. Taken together, the findings from the current study demonstrates that lategestational testosterone induces poor pregnancy outcome that is accompanied by cardiac lipotoxicity,glutathione-dependent antioxidant defense depletion, increased endoglin, lactate and uric acid productionthrough MR activation.

Microbial Production of Fatty Acid via Metabolic Engineering and Synthetic Biology

The microbial production of free fatty acids (FFAs) has gained great attention from the scientific community due to its significant environmental and economic benefits. FFAs can also be used as precursors for the production of valuable products, polymer additives and industrial chemicals by various biological or chemical reactions. Since microorganisms synthesize fatty acids (FAs) which are mainly used to form lipids for cell membrane constitution, FFAs are not normally accumulated as metabolic intermediates. FA metabolism is tightly regulated at transcriptional and post-transcriptional levels by both the transcription factor and product inhibition, meaning that FA overproduction may require extensive re-engineering of cellular metabolism. Therefore, we here present the recent efforts applied to enhance FFA production via metabolic engineering and synthetic biology with special reference to Escherichia coli.

Production of long-chain free fatty acids from metabolically engineered Rhodobacter sphaeroides heterologously producing periplasmic phospholipase A2 in dodecane-overlaid two-phase culture

BackgroundLong-chain free fatty acids (FFAs) are a type of backbone molecule that can react with alcohol to produce biodiesels. Various microorganisms have become potent producers of FFAs. Efforts have focused on increasing metabolic flux to the synthesis of either neutral fat or fatty acyl intermediates attached to acyl carrier protein (ACP), which are the source of FFAs. Membrane lipids are also a source of FFAs. As an alternative way of producing FFAs, exogenous phospholipase may be used after heterologous production and localization in the periplasmic space. In this work, we examined whether Rhodobacter sphaeroides, which forms an intracytoplasmic membrane, can be used for long-chain FFA production using phospholipase.ResultsThe recombinant R. sphaeroides strain Rs-A2, which heterologously produces Arabidopsis thaliana phospholipase A2 (PLA2) in the periplasm, excretes FFAs during growth. FFA productivity under photoheterotrophic conditions is higher than that observed under aerobic or semiaerobic conditions. When the biosynthetic enzymes for FA (β-ketoacyl-ACP synthase, FabH) and phosphatidate (1-acyl-sn-glycerol-3-phosphate acyltransferase, PlsC) were overproduced in Rs-A2, the FFA productivity of the resulting strain Rs-HCA2 was elevated, and the FFAs produced mainly consisted of long-chain FAs of cis-vaccenate, stearate, and palmitate in an approximately equimolar ratio. The high-cell-density culture of Rs-HCA2 with DMSO in two-phase culture with dodecane resulted in an increase of overall carbon substrate consumption, which subsequently leads to a large increase in FFA productivity of up to 2.0 g L−1 day−1. Overexpression of the genes encoding phosphate acyltransferase (PlsX) and glycerol-3-phosphate acyltransferase (PlsY), which catalyze the biosynthetic steps immediately upstream from PlsC, in Rs-HCA2 generated Rs-HXYCA2, which grew faster than Rs-HCA2 and showed an FFA productivity of 2.8 g L−1 day−1 with an FFA titer of 8.5 g L−1.ConclusionWe showed that long-chain FFAs can be produced from metabolically engineered R. sphaeroides heterologously producing PLA2 in the periplasm. The FFA productivity was greatly increased by high-cell-density culture in two-phase culture with dodecane. This approach provides highly competitive productivity of long-chain FFAs by R. sphaeroides compared with other bacteria. This method may be applied to FFA production by other photosynthetic bacteria with similar differentiated membrane systems.

Membrane-Located Expression of Thioesterase From Acinetobacter baylyi Enhances Free Fatty Acid Production With Decreased Toxicity in Synechocystis sp. PCC6803

It has been previously reported that photosynthetic production of extracellular free fatty acids (FFAs) in cyanobacteria was realized by thioesterases (TesA) mediated hydrolysis of fatty acyl-ACP in cytosol and excretion of the FFA outside of the cell. However, two major issues related to the genetically modified strains need to be addressed before the scale-up commercial application becomes possible: namely, the toxicity of FFAs, and the diversity of carbon lengths of fatty acids that could mimic the fossil fuel. To address those issues, we hypothesized that generating FFAs near membrane could facilitate rapid excretion of the FFA outside of the cell and thus decrease toxicity caused by intracellular FFAs in the cytosolic expression of thioesterase. To realize this, we localized a leaderless thioesterase (AcTesA) from Acinetobacter baylyi on the cytosolic side of the inner membrane of Synechocystis sp. PCC6803 using a membrane scaffolding system. The engineered strain with AcTesA on its membrane (mAcT) produced extracellular FFAs up to 171.9 ± 13.22 mg⋅L-1 compared with 40.24 ± 10.94 and 1.904 ± 0.158 mg⋅L-1 in the cytosol-expressed AcTesA (AcT) and wild-type (WT) strains, respectively. Moreover, the mAcT strain generated around 1.5 and 1.9 times less reactive oxygen species than AcT and WT, respectively. Approximately 78% of total FFAs were secreted with an average rate of 1 mg⋅L-1⋅h-1, which was higher than 0.44 mg⋅L-1⋅h-1 reported previously. In the case of mAcT strain, 60% of total secreted FFAs was monounsaturated (C18:1) which is the preferable biodiesel component. Therefore, the engineered mAcT strain shows enhanced FFAs production with less toxicity which is highly desirable for biodiesel production.

Effects of inactivation of the cyAbrB2 transcription factor together with glycogen synthesis on cellular metabolism and free fatty acid production in the cyanobacterium Synechocystis sp. PCC 6803.

Deletion of the cyAbrB2 (Sll0822) transcription factor in Synechocystis sp. PCC 6803 causes aberrant accumulation of glycogen. We previously tried to redirect the excess carbon stored as glycogen in the cyabrB2-disrupted (∆ cyabrB2) mutant by knockout of the glgC (slr1176) gene encoding glucose-1-phosphate adenylyltransferase. However, complete knockout could not be attained, suggesting that accumulation of glycogen is essential for the Δ cyabrB2 mutant. In this study, we introduced the cyabrB2 gene fused to the copper-inducible petE promoter into the ∆ cyabrB2 mutant. After complete knockout of glgC in the presence of copper, expression of P petE- cyabrB2 was turned off by copper removal to examine the effect of the double knockout of cyabrB2 and glgC. Metabolome analysis and electron microscopic observation revealed that the double knockout causes a large decrease of sugar phosphates in glycolytic and oxidative pentose phosphate pathways and an increase of organic acids in the tricarboxylic acid cycle, amino acids and storage compounds such as polyhydroxybutyrate. When the ability of production of free fatty acids was conferred, synergetic positive effects of knockout of cyabrB2 and glgC on productivity were observed by removal of both copper and nitrogen. The P petE- cyabrB2Δ glgC strain will further serve as a platform for studies on carbon allocation and metabolic engineering.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A.oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A.oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145mg/l for 5d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

Potential application of Thermomyces lanuginosus lipase (TLL) immobilized on nonporous polystyrene particles

The crescent demand for biocatalyst application, associated with the production costs and the need to reduce the dependence for the fossil fuels combined with waste recovery, which could be used as an alternative to resources, increased the interests for the enzyme immobilization. In this work, low‐cost nonporous polystyrene (PS) particles were studied as a support to Thermomyces Lanuginosus lipase (NS‐40116) immobilization. The results showed that immobilized lipase on PS matrix showed better thermal and pH stability when compared to soluble NS‐40116 lipase, with an increase of 84% in the hydrolytic activity at pH 12. Kinetic constants showed that immobilization process increased enzyme specificity by the substrate. In addition, immobilized lipase on PS matrix was effectively applied in the production of free fatty acids from commercial soybean oil demonstrating operational stability after five reuse cycles as well as potential application in the transesterification and esterification reactions by using abdominal chicken fat as substrate. © 2018 American Institute of Chemical Engineers Environ Prog, 38: 608–613, 2019

BioPS: System for screening and assessment of biofuel-production potential of cyanobacteria.

BackgroundCyanobacteria are one of the target groups of organisms explored for production of free fatty acids (FFAs) as biofuel precursors. Experimental evaluation of cyanobacterial potential for FFA production is costly and time consuming. Thus, computational approaches for comparing and ranking cyanobacterial strains for their potential to produce biofuel based on the characteristics of their predicted proteomes can be of great importance.ResultsTo enable such comparison and ranking, and to assist biotechnology developers and researchers in selecting strains more likely to be successfully engineered for the FFA production, we developed the Biofuel Producer Screen (BioPS) platform (http://www.cbrc.kaust.edu.sa/biops). BioPS relies on the estimation of the predicted proteome makeup of cyanobacterial strains to produce and secrete FFAs, based on the analysis of well-studied cyanobacterial strains with known FFA production profiles. The system links results back to various external repositories such as KEGG, UniProt and GOLD, making it easier for users to explore additional related information.ConclusionTo our knowledge, BioPS is the first tool that screens and evaluates cyanobacterial strains for their potential to produce and secrete FFAs based on strain’s predicted proteome characteristics, and rank strains based on that assessment. We believe that the availability of such a platform (comprising both a prediction tool and a repository of pre-evaluated stains) would be of interest to biofuel researchers. The BioPS system will be updated annually with information obtained from newly sequenced cyanobacterial genomes as they become available, as well as with new genes that impact FFA production or secretion.

Reprogramming Yeast Metabolism from Alcoholic Fermentation to Lipogenesis

Engineering microorganisms for production of fuels and chemicals often requires major re-programming of metabolism to ensure high flux toward the product of interest. This is challenging, as millions of years of evolution have resulted in establishment of tight regulation of metabolism for optimal growth in the organism's natural habitat. Here, we show through metabolic engineering that it is possible to alter the metabolism of Saccharomyces cerevisiae from traditional ethanol fermentation to a pure lipogenesis metabolism, resulting in high-level production of free fatty acids. Through metabolic engineering and process design, we altered subcellular metabolic trafficking, fine-tuned NADPH and ATP supply, and decreased carbon flux to biomass, enabling production of 33.4 g/L extracellular free fatty acids. We further demonstrate that lipogenesis metabolism can replace ethanol fermentation by deletion of pyruvate decarboxylase enzymes followed by adaptive laboratory evolution. Genome sequencing of evolved strains showed that pyruvate kinase mutations were essential for this phenotype.

Genomics-Based Insights Into the Biosynthesis and Unusually High Accumulation of Free Fatty Acids by Streptomyces sp. NP10.

Streptomyces sp. NP10 was previously shown to synthesize large amounts of free fatty acids (FFAs). In this work, we report the first insights into the biosynthesis of these fatty acids (FAs) gained after genome sequencing and identification of the genes involved. Analysis of the Streptomyces sp. NP10 draft genome revealed that it is closely related to several strains of Streptomyces griseus. Comparative analyses of secondary metabolite biosynthetic gene clusters, as well as those presumably involved in FA biosynthesis, allowed identification of an unusual cluster C12-2, which could be identified in only one other S. griseus-related streptomycete. To prove the involvement of identified cluster in FFA biosynthesis, one of its three ketosynthase genes was insertionally inactivated to generate mutant strain mNP10. Accumulation of FFAs in mNP10 was almost completely abolished, reaching less than 0.01% compared to the wild-type strain. Cloning and transfer of the C12-2 cluster to the mNP10 mutant partially restored FFA production, albeit to a low level. The discovery of this rare FFA biosynthesis cluster opens possibilities for detailed characterization of the roles of individual genes and their products in the biosynthesis of FFAs in NP10.

Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris.

The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 °C + 1%M provided the most stable viability. By contrast, the temperature of 18 °C + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.

The role of acyl-CoA thioesterase ACOT8I in mediating intracellular lipid metabolism in oleaginous fungus Mortierella alpina.

Thioesterases (TEs) play an essential role in the metabolism of fatty acids (FAs). To explore the role of TEs in mediating intracellular lipid metabolism in the oleaginous fungus Mortierella alpina, the acyl-CoA thioesterase ACOT8I was overexpressed. The contents of total fatty acids (TFAs) were the same in the recombinant strains as in the wild-type M. alpina, whilst the production of free fatty acids (FFAs) was enhanced from about 0.9% (wild-type) to 2.8% (recombinant), a roughly threefold increase. Linoleic acid content in FFA form constituted about 9% of the TFAs in the FFA fraction in the recombinant strains but only about 1.3% in the wild-type M. alpina. The gamma-linolenic acid and arachidonic acid contents in FFA form accounted for about 4 and 25%, respectively, of the TFAs in the FFA fraction in the recombinant strains, whilst neither of them in FFA form were detected in the wild-type M. alpina. Overexpression of the TE ACOT8I in the oleaginous fungus M. alpina reinforced the flux from acyl-CoAs to FFAs, improved the production of FFAs and tailored the FA profiles of the lipid species.

Abstract P3-07-10: Modulation of FASN under obese conditions

Abstract Introduction: Obesity is known to be associated with a worse breast cancer prognosis, in part through altering metabolism in cells of the tumor microenvironment. In particular, changes in metabolism associated with fatty acid utilization have been noted in not only breast cancer, but also several other cancer types. This includes changes to both expression and activity of the Fatty Acid Synthase enzyme (FASN), which is responsible for production of long chain fatty acids, including palmitate. These changes in long chain fatty acid production can modulate tumor behavior through modulation of energy utilization such as beta-oxidation, as well as plasma membrane modulation with phospholipids. Our previous studies have demonstrated that exposure to obese conditions induces significant changes in breast cancer cell proliferation. Additionally, obesity modulates activity of other cells within the tumor microenvironment, including adipocytes, which might influence the cancer cell itself. We hypothesize one particular mechanism that supports these changes is obesity-induced upregulation of FASN and that FASN may be a viable target to limit obesity-induced progression. Methods and Results: FASN has been shown to promote cancer cell proliferation through generating fatty acid precursors required for cell proliferation, altering membrane fluidity, and activating oncogenic signaling pathways. To determine if modulation of FASN is an important mechanism by which obesity promotes disease progression, MCF-7 breast cancer cells and human pre-adipocyte cells (ASC) were exposed to 2% sera from obese postmenopausal women and 2% sera from non-obese (control) women. Preliminary quantitative PCR results demonstrated that exposure to the obese sera resulted in increased expression of FASN in both the cancer cells as well as the ASC. Current studies are on-going to determine if 1) FASN up-regulation results in increased long-chain and free fatty acid production in both the cancer and adipocyte cells, 2) whether changes in long chain and free fatty acid production results in altered metabolism and plasma membrane status and 3) whether targeting FASN with a new generation of FASN inhibitors currently being investigated in the clinic can modulate obesity-induced disease progression. Conclusions: Our findings indicate that obesity promotes upregulation of FASN in several cells within the tumor microenvironment, including adipocytes and the cancer cell itself. We have also found that using a FASN inhibitor is effective in limiting cancer cell viability and proliferation. Our on-going studies will confirm if this is an important mechanism by which obesity promotes disease progression. Since FASN inhibitors are currently being investigated in the clinic, the results of these studies will provide a better understanding of how obesity alters the biology of the disease, and may identify a novel target for improving patient outcomes. Citation Format: Pham T, Oberman A, Kim I, Lee G, Quach D, Galván G, Jolly C, Cavazos D, Brenner A, deGraffenried L. Modulation of FASN under obese conditions [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-07-10.

Visceral Fat Is Increased in Individuals With Crohnʼs Disease: A Comparative Analysis With Healthy Controls

BACKGROUND: The prevalences of overweight (20-40%) and obesity (15-30%) among individuals with inflammatory bowel disease (IBD) are similar to those observed in the general population. The accumulation of visceral fat is a potentially inflammatory factor because it increases the production of inflammation mediators and free fatty acids. It also may be linked to an increased endotoxinemia associated with the augmentation in the intestinal permeability. It is known that obesity is associated with a chronic inflammatory state, but few studies have evaluated the visceral fat content and its role in individuals with Crohn's disease (CD). Aim: To compare the nutritional status, body composition and proportion of visceral fat between CD individuals and a control group (CG). METHODS: A cross-sectional study that enrolled individuals with CD and a CG comprised of healthy volunteers. The stratification according to the nutritional status was carried out by means of the body mass index (BMI). The percentage of body fat percentage (%BF) and the visceral fat (VF) were estimated by means of DEXA (dual energy X-ray absorption). The visceral fat proportion was evaluated by means of the VF/BMI and VF/%BF ratios. RESULTS: A total of 78 individuals were included. The control group was comprised of 28 healthy subjects aged 35.39±10 years old; 60.7% were women; mean BMI: 23.94±3.34 kg/m2; mean VF: 511.82±448.68 grams(g); mean CRP: 0.81±1.78 ng/mL. The CD group was comprised of 50 patients; 11 (22%) were underweight (BMI:18.20±1.97 kg/m2; %BF: 24.46±10.01; VF: 217.18±218.95g; CRP: 4,12±4,84); 18 (36%) presented normal weight (BMI: 22.43±1.48kg/m2; %BF: 30.92±6.63; VF: 542.00±425.47 grams and CRP:4.40±1.78); 21(42%) were overweight or obese (BMI: 29.48±3.78kg/m2; %BF: 39.91±7.33; VF: 1525.23±672.76 grams and CRP: 1.33±2.06). The VF/BMI ratio was higher in the CD group when compared to CG (32.41 ± 24.63 vs. 20.01±16.23 grams per BMI point; P=0.02); likewise, the VF/%BF was also higher in the CD group (35.21±23.33 vs. 15.60±12.55 grams per percentage point; P<0.001). It means that, in CD subjects, there is a disproportion of visceral fat distribution when compared with CG group. CONCLUSION(S): Among individuals with CD, BMI presents a direct correlation with the visceral fat content. The fat distribution in the CD Group was disproportionate when compared to the CG. These results indicate the presence of an adiposopathy in CD subjects, which is evidenced by a higher visceral fat tissue volume.

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.

RECOVERY OF BY-PRODUCTS FROM THE OLIVE OIL PRODUCTION AND THE VEGETABLE OIL REFINING FOR BIODIESEL PRODUCTION

The by-products acid oil from soapstock of vegetable oil refining and olive pomace oil were evaluated for biodiesel production. Enzymatic hydroesterification was studied to convert the acid oil ( 34 wt.% free fatty acids) into methyl esters; due to the low free fatty acid content of the fresh olive pomace oil ( 2 wt.%), alkaline transesterification was conducted. The results from the enzymatic hydrolysis (35˚C, 24 h, 200 rpm) showed a clear influence of enzyme concentration (0.1 – 5 wt.%, relative to oil) and water:oil ratio (1:0.25 and 1:0.5 w:w) towards free fatty acid production. After applying the best established conditions (3 wt.% of enzyme and 1:0.5 water: oil ratio, w:w), enzymatic esterification was performed (35˚C, 7 h, 200 rpm, 2 wt.% of enzyme and 2:1 molar ratio of methanol to acid). Hydroesterification led to a product with a methyl esters content of about 84 wt.% whereas the esterification alone allowed reaching only around 65 wt.%. The olive pomace oil was obtained from chemical extraction of fresh olive pomace ( 18 wt.% of oil). By performing direct alkaline transesterification (65˚C, 1 wt.% NaOH, 1 h and 6:1 molar ratio of methanol to oil) a product with a purity of 90 wt.% was obtained. The olive pomace storage in the air during 2 weeks led to an increase in the oil free fatty acid content of almost 2 fold showing the relevance of developing storage and conservation strategies to ensure a sustainable recovery of this by-product. Both by-products showed potential for biodiesel production.

Increasing Extracellular Free Fatty Acid Production in Escherichia coli by Disrupting Membrane Transport Systems.

Transposon mutagenesis was used to identify three mutants of E. coli that exhibited increased free fatty acid (FFA) production, which resulted from the disruption of genes related to membrane transport. Deletion of envR, gusC, and mdlA individually in a recombinant E. coli strain resulted in 1.4-, 1.8-, and 1.2-fold increases in total FFA production, respectively. In particular, deletion of envR increased the percentage of extracellular FFA to 46%, compared with 29% for the control strain. Multiple deletion of envR, gusC, mdlA, ompF, and fadL had a synergistic effect on FFA production, resulting in high extracellular FFA production, comprising up to 50% of total FFA production. This study has identified new membrane proteins involved in FFA production and showed that genetic engineering targeting these membrane transporters is important to increase both total FFA and extracellular FFA production.

A metabolic engineering strategy for producing free fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metabolism.

In recent years, bio-based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L-1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L-1 . Next, an isolated oleate-resistant strain produced 382.8 mg L-1 of free fatty acids. Finely, acyl-CoA carboxylase gene (ACC1) over-expression resulted to production of 1436.7 mg L-1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L-1 during test tube cultivation.