scholarly journals Quantitative in vivo evaluation of the reverse β-oxidation pathway for fatty acid production in Saccharomyces cerevisiae

2017 ◽  
Author(s):  
Paulo Gonçalves Teixeira ◽  
Verena Siewers ◽  
Jens Nielsen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Acid Production ◽  
Production Efficiency ◽  
Fatty Acid Biosynthesis ◽  
Alternative Pathway ◽  
In Vivo Evaluation ◽  
Fatty Acid Production ◽  
Oxidation Pathway

AbstractProduction of fatty acids using engineered Saccharomyces cerevisiae cells is a challenging task in part due to low efficiency of the native fatty acid biosynthesis pathway. One option for improving production efficiency relies on exploring alternative fatty acid production pathways with either improved kinetics, thermodynamics or yield properties.In this work, we explored the reverse β-oxidation pathway as an alternative pathway for free fatty acid production. Different gene combinations and analysis methods were tested for assessing pathway efficiency when expressed in the yeast Saccharomyces cerevisiae. Even though different alternatives were tested, quantitative analysis showed no improvement or major change in fatty acid production of the tested strains in our conditions. This lack of improvement suggests that the tested pathway designs and constructs are either nonfunctional in the tested conditions or the resulting strains lack a metabolic driving force that is needed for a functional pathway.We conclude that expression of the reverse β-oxidation pathway in S. cerevisiae poses many challenges when compared to expression in bacterial systems. These factors gravely hinder development efforts and success rate for producing fatty acids through this pathway.

scholarly journals A Futile Metabolic Cycle of Fatty Acyl-CoA Hydrolysis and Resynthesis in Corynebacterium glutamicum and Its Disruption Leading to Fatty Acid Production

2020 ◽  
Author(s):  
Masato Ikeda ◽  
Keisuke Takahashi ◽  
Tatsunori Ohtake ◽  
Ryosuke Imoto ◽  
Haruka Kawakami ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acids ◽  
Corynebacterium Glutamicum ◽  
Candidate Genes ◽  
Outer Layer ◽  
Acid Production ◽  
Fatty Acyl ◽  
Fatty Acid Production ◽  
Mycolic Acids

Fatty acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD) catalyze opposing reactions between acyl-CoAs and free fatty acids. Within the genome of Corynebacterium glutamicum, several candidate genes for each enzyme are present, although their functions remain unknown. Modified expressions of the candidate genes in the fatty acid producer WTΔfasR led to identification of one tes gene (tesA) and two fadD genes (fadD5 and fadD15), which functioned positively and negatively in fatty acid production, respectively. Genetic analysis showed that fadD5 and fadD15 are responsible for utilization of exogenous fatty acids and that tesA plays a role in supplying fatty acids for synthesis of the outer layer components mycolic acids. Enzyme assays and expression analysis revealed that tesA, fadD5, and fadD15 were co-expressed to create a cyclic route between acyl-CoAs and fatty acids. When fadD5 or fadD15 was disrupted in wild-type C. glutamicum, both disruptants excreted fatty acids during growth. Double disruptions of them resulted in a synergistic increase in production. Additional disruption of tesA revealed a canceling effect on production. These results indicate that the FadDs normally shunt the surplus of TesA-generated fatty acids back to acyl-CoAs for lipid biosynthesis and that interception of this shunt provokes cells to overproduce fatty acids. When this strategy was applied to a fatty acid high-producer, the resulting fadDs-disrupted and tesA-amplified strain exhibited a 72% yield increase relative to its parent and produced fatty acids, which consisted mainly of oleic acid, palmitic acid, and stearic acid, on the gram scale per liter from 1% glucose. IMPORTANCE The industrial amino acid producer Corynebacterium glutamicum has currently evolved into a potential workhorse for fatty acid production. In this organism, we obtained evidence showing the presence of a unique mechanism of lipid homeostasis, namely, a formation of a futile cycle of acyl-CoA hydrolysis and resynthesis mediated by acyl-CoA thioesterase (Tes) and acyl-CoA synthetase (FadD), respectively. The biological role of the coupling of Tes and FadD would be to supply free fatty acids for synthesis of the outer layer components mycolic acids and to recycle their surplusage to acyl-CoAs for membrane lipid synthesis. We further demonstrated that engineering of the cycle in a fatty acid high-producer led to dramatically improved production, which provides a useful engineering strategy for fatty acid production in this industrially important microorganism.

scholarly journals Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Yiming Zhang ◽  
Mo Su ◽  
Ning Qin ◽  
Jens Nielsen ◽  
Zihe Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Pyruvate Dehydrogenase ◽  
Unsaturated Fatty Acids ◽  
Pyruvate Dehydrogenase Complex ◽  
Cell Factory ◽  
Acetyl Coa ◽  
Fatty Acid Production

Abstract Background Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. Results Overexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). Conclusions We demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.

scholarly journals Elucidation of carbon sources used for the biosynthesis of fatty acids and sterols in the trypanosomatid Leishmania mexicana

1999 ◽  
Vol 342 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Michael L. GINGER ◽  
Michael L. CHANCE ◽  
L. John GOAD
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Acid Production ◽  
Carbon Sources ◽  
Mevalonic Acid ◽  
Leishmania Mexicana ◽  
Fatty Acid Production ◽  
Quantitative Measurements ◽  
Major Sterol

Sterols are necessary for the growth of trypanosomatid protozoans; sterol biosynthesis is a potential target for the use and development of drugs to treat the diseases caused by these organisms. This study has used 14C-labelled substrates to investigate the carbon sources utilized by promastigotes and amastigotes of Leishmania mexicana for the production of sterol [mainly ergosta-5,7,24(241)-trien-3β-ol] and the fatty acid moieties of the triacylglycerol (TAG) and phospholipid (PL) of the organism. The isoprenoid precursor mevalonic acid (MVA) was incorporated into the sterols, and the sterol precursor squalene, by the promastigotes of L. mexicana. However, acetate (the precursor to MVA in most organisms) was a very poor substrate for sterol production but was readily incorporated into the fatty acids of TAG and PL. Other substrates (glucose, palmitic acid, alanine, serine and isoleucine), which are metabolized to acetyl-CoA, were also very poor precursors to sterol but were incorporated into TAG and PL and gave labelling patterns of the lipids similar to those of acetate. In contrast, the amino acid leucine was the only substrate to be incorporated efficiently into the squalene and sterol of L. mexicana promastigotes. Quantitative measurements revealed that at least 70-80% of the sterol synthesized by the promastigotes of L. mexicana is produced from carbon provided by leucine metabolism. Studies with the amastigote form of L. mexicana showed that in this case leucine was again the major sterol precursor, whereas acetate was utilized for fatty acid production.

Studies on the production of volatile fatty acids from grass by rumen liquor in an artificial rumen: III. A note on the volatile fatty acid production from crude fibre and grass cellulose

1957 ◽  
Vol 49 (2) ◽  
pp. 180-183 ◽  
Author(s):  
A. John ◽  
G. Barnett ◽  
R. L. Reid
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Acid Production ◽  
Volatile Fatty Acid ◽  
Volatile Fatty Acids ◽  
Relative Yield ◽  
Water Soluble ◽  
Cellulose Powder ◽  
Crude Fibre ◽  
Fatty Acid Production

1. The findings presented in two previous papers on the yields of volatile fatty acids, obtained by the action of rumen liquor in the artificial rumen, from fresh grass, dried grass and the water-soluble and water-insoluble separates of the latter, have been amplified by a consideration of the acids similarly obtained from specimens of chemically prepared crude fibre and cellulose, from four of the dried grass specimens.2. The proportions of different volatile fatty acids from grass crude fibre and grass cellulose resemble those obtained from cellulose powder, propionic acid being produced in greatest relative yield.3. A general review of these latter findings, in relation to those already presented, has been given.

Three Different Methods of Inhibiting Lipolysis in Human Chyme in Vitro: Efficiency and Effect on Phase Distribution of Lipids

1990 ◽  
Vol 79 (4) ◽  
pp. 349-355 ◽  
Author(s):  
D. R. Fine ◽  
P. L. Zentler-Munro ◽  
T. C. Northfield
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Acid Production ◽  
Acid Treatment ◽  
Phase Distribution ◽  
Significant Inhibition ◽  
Separate Experiment ◽  
Fatty Acid Production ◽  
Micellar Phase

1. The efficiencies of three different methods of inhibiting fatty acid production in chyme have been evaluated. The effects of each method on the phase distribution of fatty acids after ultracentrifugation have been studied. 2. Chyme fatty acid concentrations were measured during a 4 h incubation (a) without an inhibitor, (b) after acid treatment, (c) after addition of p-bromophenyl-boronic acid and (d) after heating. 3. In a separate experiment, fresh chyme was incubated to allow equilibration of lipolysis. Aliquots were treated by each inactivation method and ultracentrifuged overnight to separate the phases. Total and micellar fatty acids and glycerides were measured. 4. In the first experiment, acid treatment completely inhibited fatty acid generation producing 4 h concentrations which were 93.4 ± 5.6% (mean ± sem) of initial values compared with 398.0 ± 54.0% (P < 0.05) for uninhibited samples. p-Bromophenylboronic acid and heating gave significant but incomplete inhibition (132.9 ± 6.6% and 166.1 ± 15.2% of initial concentrations, respectively). 5. Ultracentrifugation disclosed five phases in all except the acid-treated samples, which had four. Micellar phase fatty acid concentrations were significantly higher in the acid-treated than in the untreated samples (2.6 ± 0.7 versus 1.7 ± 0.5 mmol/l, P = 0.05), as were glyceride concentrations (1.5 ± 0.4 vs 0.6 ± 0.3 mmol/l, P = 0.05). 6. It was concluded that acid treatment was the most efficient inhibitor of fatty acid production, but it disrupted the phases. p-Bromophenylboronic acid gave significant inhibition without causing phase disruption and was therefore the most useful inhibitor overall.

scholarly journals Gene Transformation by Chitosan Nanoparticle to Enhance Fatty Acid Production in Zea mays (L.)

2021 ◽  
Vol 26 (5) ◽  
pp. 2971-2978
Author(s):  
EMAN TAWFIK HUSSIEN ◽  
◽  
MOHAMMED IBRAHIM DAHAB ◽  
KAREEM MOHAMMED ABD-ELATTY ◽  
ISLAM HAMDY EL-SHENAWY ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Zea Mays ◽  
Fatty Acid ◽  
Acid Production ◽  
Chitosan Nanoparticles ◽  
Gene Transformation ◽  
Gas Chromatography Mass Spectrometry ◽  
Acetyl Coa ◽  
Fatty Acid Production ◽  
Transgenic Lines

Zea mays is an important crop and an essential source of fatty acids. Hence, increasing and adding new fatty acids led to the enhancement of these properties. Transformation of external Acetyl-CoA gene (Aco) can enhance fatty acid components, as ACo is expressed into Acetyl-CoA carboxylase (ACCase) enzyme, which is the first essential step in the fatty acid production process. Chitosan nanoparticles are safe and fast polymer nanoparticles that are applied for gene transformation. Conventional PCR was performed for the detection of the ACo gene in both transgenic and nontransgenic maize lines. The results confirm the presence of the gene in the transgenic lines and absence in non-transgenic lines. The Gas chromatography-mass spectrometry (GC-MS) analysis for fatty acid contents in transgenic and non-transgenic maize lines showed an increase in fatty acid contents in transgenic lines compared to non-transgenic ones. Besides, the transgenic maize’s lines produced extra new fatty acids not found in non-transgenic ones.

scholarly journals The role of fluconazole in the regulation of fatty acid and unsaponifiable matter biosynthesis in Schizochytrium sp. MYA 1381

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Jun Li ◽  
Hao Zhou ◽  
Xueshan Pan ◽  
Zhipeng Li ◽  
Yinghua Lu ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Cell Growth ◽  
Unsaponifiable Matter ◽  
Fatty Acid Biosynthesis ◽  
Mevalonate Pathway ◽  
Reducing Power ◽  
Fatty Acid Production ◽  
Membrane Function ◽  
Β Carotene

Abstract Background Schizochytrium has been widely used in industry for synthesizing polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acid (DHA). However, unclear biosynthesis pathway of PUFAs inhibits further production of the Schizochytrium. Unsaponifiable matter (UM) from mevalonate pathway is crucial to cell growth and intracellular metabolism in all higher eukaryotes and microalgae. Therefore, regulation of UM biosynthesis in Schizochytrium may have important effects on fatty acids synthesis. Moreover, it is well known that UMs, such as squalene and β-carotene, are of great commercial value. Thus, regulating UM biosynthesis may also allow for an increased valuation of Schizochytrium. Results To investigate the correlation of UM biosynthesis with fatty acids accumulation in Schizochytrium, fluconazole was used to block the sterols pathway. The addition of 60 mg/L fluconazole at 48 h increased the total lipids (TLs) at 96 h by 16% without affecting cell growth, which was accompanied by remarkable changes in UMs and NADPH. Cholesterol content was reduced by 8%, and the squalene content improved by 45% at 72 h, which demonstrated fluconazole’s role in inhibiting squalene flow to cholesterol. As another typical UM with antioxidant capacity, the β-carotene production was increased by 53% at 96 h. The increase of squalene and β-carotene could boost intracellular oxidation resistance to protect fatty acids from oxidation. The NADPH was found to be 33% higher than that of the control at 96 h, which meant that the cells had more reducing power for fatty acid synthesis. Metabolic analysis further confirmed that regulation of sterols was closely related to glucose absorption, pigment biosynthesis and fatty acid production in Schizochytrium. Conclusion This work first reported the effect of UM biosynthesis on fatty acid accumulation in Schizochytrium. The UM was found to affect fatty acid biosynthesis by changing cell membrane function, intracellular antioxidation and reducing power. We believe that this work provides valuable insights in improving PUFA and other valuable matters in microalgae.

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