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Liu Y, Koh CMJ, Yap SA, Cai L, Ji L. Understanding and exploiting the fatty acid desaturation system in Rhodotorula toruloides. BIOTECHNOLOGY FOR BIOFUELS 2021; 14:73. [PMID: 33741038 PMCID: PMC7977280 DOI: 10.1186/s13068-021-01924-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Accepted: 03/06/2021] [Indexed: 05/17/2023]
Abstract
BACKGROUND Rhodotorula toruloides is a robust producer of triacylglycerol owing to its fast growth rate and strong metabolic flux under conditions of high cell density fermentation. However, the molecular basis of fatty acid biosynthesis, desaturation and regulation remains elusive. RESULTS We present the molecular characterization of four fatty acid desaturase (FAD) genes in R. toruloides. Biosynthesis of oleic acid (OA) and palmitoleic acid (POA) was conferred by a single-copy ∆9 Fad (Ole1) as targeted deletion of which abolished the biosynthesis of all unsaturated fatty acids. Conversion of OA to linoleic acid (LA) and α-linolenic acid (ALA) was predominantly catalyzed by the bifunctional ∆12/∆15 Fad2. FAD4 was found to encode a trifunctional ∆9/∆12/∆15 FAD, playing important roles in lipid and biomass production as well as stress resistance. Furthermore, an abundantly transcribed OLE1-related gene, OLE2 encoding a 149-aa protein, was shown to regulate Ole1 regioselectivity. Like other fungi, the transcription of FAD genes was controlled by nitrogen levels and fatty acids in the medium. A conserved DNA motif, (T/C)(G/A)TTGCAGA(T/C)CCCAG, was demonstrated to mediate the transcription of OLE1 by POA/OA. The applications of these FAD genes were illustrated by engineering high-level production of OA and γ-linolenic acid (GLA). CONCLUSION Our work has gained novel insights on the transcriptional regulation of FAD genes, evolution of FAD enzymes and their roles in UFA biosynthesis, membrane stress resistance and, cell mass and total fatty acid production. Our findings should illuminate fatty acid metabolic engineering in R. toruloides and beyond.
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Affiliation(s)
- Yanbin Liu
- Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore
| | - Chong Mei John Koh
- Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore
| | - Sihui Amy Yap
- Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore
| | - Lin Cai
- Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore
| | - Lianghui Ji
- Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore.
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore.
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2
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Mbuyane LL, Bauer FF, Divol B. The metabolism of lipids in yeasts and applications in oenology. Food Res Int 2021; 141:110142. [PMID: 33642009 DOI: 10.1016/j.foodres.2021.110142] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Revised: 12/26/2020] [Accepted: 01/09/2021] [Indexed: 12/14/2022]
Abstract
Lipids are valuable compounds present in all living organisms, which display an array of functions related to compartmentalization, energy storage and enzyme activation. Furthermore, these compounds are an integral part of the plasma membrane which is responsible for maintaining structure, facilitating the transport of solutes in and out of the cell and cellular signalling necessary for cell survival. The lipid composition of the yeast Saccharomyces cerevisiae has been extensively investigated and the impact of lipids on S. cerevisiae cellular functions during wine alcoholic fermentation is well documented. Although other yeast species are currently used in various industries and are receiving increasing attention in winemaking, little is known about their lipid metabolism. This review article provides an extensive and critical evaluation of our knowledge on the biosynthesis, accumulation, metabolism and regulation of fatty acids and sterols in yeasts. The implications of the yeast lipid content on stress resistance as well as performance during alcoholic fermentation are discussed and a particular emphasis is given on non-Saccharomyces yeasts. Understanding lipid requirements and metabolism in non-Saccharomyces yeasts may lead to a better management of these yeast to enhance their contributions to wine properties.
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Affiliation(s)
- Lethiwe Lynett Mbuyane
- South African Grape and Wine Research Institute, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch 7600, South Africa
| | - Florian Franz Bauer
- South African Grape and Wine Research Institute, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch 7600, South Africa
| | - Benoit Divol
- South African Grape and Wine Research Institute, Department of Viticulture and Oenology, Stellenbosch University, Stellenbosch 7600, South Africa.
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3
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Matsuzawa T, Maehara T, Kamisaka Y, Ayabe-Chujo Y, Takaku H, Yaoi K. Identification and characterization of Pseudozyma antarctica Δ12 fatty acid desaturase and its utilization for the production of polyunsaturated fatty acids. J Biosci Bioeng 2020; 130:604-609. [PMID: 32847739 DOI: 10.1016/j.jbiosc.2020.07.019] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Revised: 07/28/2020] [Accepted: 07/30/2020] [Indexed: 10/23/2022]
Abstract
Fatty acid desaturases, especially Δ12 fatty acid desaturases, are key enzymes for the production of unsaturated fatty acids in oleaginous yeasts. In this study, we identified and characterized a gene encoding Δ12 fatty acid desaturase of Pseudozyma antarctica named PaFAD2. Almost all oleic acid (C18:1) was converted to linoleic acid by the heterologous expression of the PaFAD2 gene in Saccharomyces cerevisiae and Lipomyces starkeyi oleaginous yeast. Notably, PaFad2 converted not only oleic acid to linoleic acid, but also palmitoleic acid (C16:1) to 9,12-hexadecadienoic acid (C16:2). These results indicated that the PaFAD2 gene was very useful for the production of polyunsaturated fatty acids in yeast, including oleaginous yeast.
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Affiliation(s)
- Tomohiko Matsuzawa
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
| | - Tomoko Maehara
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
| | - Yasushi Kamisaka
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
| | - Yuko Ayabe-Chujo
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
| | - Hiroaki Takaku
- Department of Applied Life Science, Niigata University of Pharmacy and Applied Life Science, 265-1 Higashijima, Akiha-ku, Niigata 956-8603, Japan
| | - Katsuro Yaoi
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
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4
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Lamers D, Visscher B, Weusthuis RA, Francke C, Wijffels RH, Lokman C. Overexpression of delta-12 desaturase in the yeast Schwanniomyces occidentalis enhances the production of linoleic acid. BIORESOURCE TECHNOLOGY 2019; 289:121672. [PMID: 31234072 DOI: 10.1016/j.biortech.2019.121672] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/21/2019] [Revised: 06/16/2019] [Accepted: 06/17/2019] [Indexed: 06/09/2023]
Abstract
The oleaginous yeast Schwanniomyces occidentalis was previously isolated because of its excellent suitability to convert lignocellulosic hydrolysates into triacyl glycerides: it is able to use a broad range of sugars and is able to tolerate high concentrations of lignocellulosic hydrolysate inhibitors. Compared to other oleaginous yeasts S. occidentalis however produces a low content of unsaturated fatty acids. We show here that the linoleic acid content can be significantly improved by (over)expression Δ12-desaturases derived from S. occidentalis and Fusarium moniliforme. Expression was stable for the homologous expression but decreased during heterologous expression. Both homologous and heterologous expression of mCherry-Δ12-desaturase led to a 4-fold increase in linoleic acid from 0.02 g/g biomass to 0.08 g/g biomass resulting in the production of 2.23 g/L and 2.05 g/L of linoleic acid.
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Affiliation(s)
- Dennis Lamers
- HAN BioCentre, University of Applied Sciences, P.O. Box 6960, 6503 GL Nijmegen, The Netherlands; Bioprocess Engineering, Wageningen University and Research, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
| | - Bram Visscher
- HAN BioCentre, University of Applied Sciences, P.O. Box 6960, 6503 GL Nijmegen, The Netherlands.
| | - Ruud A Weusthuis
- Bioprocess Engineering, Wageningen University and Research, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
| | - Christof Francke
- HAN BioCentre, University of Applied Sciences, P.O. Box 6960, 6503 GL Nijmegen, The Netherlands.
| | - René H Wijffels
- Bioprocess Engineering, Wageningen University and Research, P.O. Box 16, 6700 AA Wageningen, The Netherlands; Faculty of Biosciences and Aquaculture, Nord University, P.O. Box 1409, 8049 Bodø, Norway.
| | - Christien Lokman
- HAN BioCentre, University of Applied Sciences, P.O. Box 6960, 6503 GL Nijmegen, The Netherlands.
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5
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Sanchez Granel ML, Cánepa C, Cid NG, Navarro JC, Monroig Ó, Verstraeten SV, Nudel CB, Nusblat AD. Gene identification and functional characterization of a Δ12 fatty acid desaturase in Tetrahymena thermophila and its influence in homeoviscous adaptation to low temperature. Biochim Biophys Acta Mol Cell Biol Lipids 2019; 1864:1644-1655. [PMID: 31421180 DOI: 10.1016/j.bbalip.2019.08.003] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Revised: 08/07/2019] [Accepted: 08/10/2019] [Indexed: 01/26/2023]
Abstract
Homeoviscous adaptation in poikilotherms is based in the regulation of the level of desaturation of fatty acids, variation in phospholipids head groups and sterol content in the membrane lipids, in order to maintain the membrane fluidity in response to changes in environmental temperature. Increased proportion of unsaturated fatty acids is thought to be the main response to low-temperature acclimation, which is mostly achieved by fatty acid desaturases. Genome analysis of the ciliate Tetrahymena thermophila and a gene knockout approach has allowed us to identify one Δ12 FAD and to study its activity in the original host and in a yeast heterologous expression system. The "PUFA index" -relative content of polyunsaturated fatty acids compared to the sum of saturated and monounsaturated fatty acid content- was ~57% lower at 15 °C and 35 °C in the Δ12 FAD gene knockout strain (KOΔ12) compared to WT strain. We characterized the role of T. thermophila Δ12 FAD on homeoviscous adaptation and analyzed its involvement in cellular growth, cold stress response, and membrane fluidity, as well as its expression pattern during temperature shifts. Although these alterations allowed normal growth in the KOΔ12 strain at 30 °C or higher temperatures, growth was impaired at temperatures of 20 °C or lower, where homeoviscous adaptation is impaired. These results stress the importance of Δ12 FAD in the regulation of cold adaptation processes, as well as the suitability of T. thermophila as a valuable model to investigate the regulation of membrane lipids and evolutionary conservation and divergence of the underlying mechanisms.
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Affiliation(s)
- Maria L Sanchez Granel
- Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Nanobiotecnología (NANOBIOTEC), Facultad de Farmacia y Bioquímica, Junín, 956, C1113AAD Ciudad Autónoma de Buenos Aires, Argentina
| | - Camila Cánepa
- Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Investigaciones Biomédicas en Retrovirus y SIDA, Facultad de Medicina, Paraguay 2155, C1121ABG Ciudad Autónoma de Buenos Aires, Argentina
| | - Nicolas G Cid
- Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Nanobiotecnología (NANOBIOTEC), Facultad de Farmacia y Bioquímica, Junín, 956, C1113AAD Ciudad Autónoma de Buenos Aires, Argentina
| | - Juan C Navarro
- Instituto de Acuicultura Torre de la Sal, CSIC (IATS-CSIC), Ribera de Cabanes, 12595, Castellón, Spain
| | - Óscar Monroig
- Instituto de Acuicultura Torre de la Sal, CSIC (IATS-CSIC), Ribera de Cabanes, 12595, Castellón, Spain
| | - Sandra V Verstraeten
- Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química y Fisicoquímica Biológicas (IQUIFIB), Facultad de Farmacia y Bioquímica, Paraguay 2155, C1121ABG Ciudad Autónoma de Buenos Aires, Argentina
| | - Clara B Nudel
- Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Nanobiotecnología (NANOBIOTEC), Facultad de Farmacia y Bioquímica, Junín, 956, C1113AAD Ciudad Autónoma de Buenos Aires, Argentina
| | - Alejandro D Nusblat
- Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Nanobiotecnología (NANOBIOTEC), Facultad de Farmacia y Bioquímica, Junín, 956, C1113AAD Ciudad Autónoma de Buenos Aires, Argentina.
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6
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Colgrave ML, Byrne K, Caine J, Kowalczyk L, Vibhakaran Pillai S, Dong B, Lovrecz G, MacIntosh S, Scoble JA, Petrie JR, Singh S, Zhou XR. Proteomics reveals the in vitro protein digestibility of seven transmembrane enzymes from the docosahexaenoic acid biosynthesis pathway. Food Chem Toxicol 2019; 130:89-98. [PMID: 31085220 DOI: 10.1016/j.fct.2019.05.015] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Revised: 05/08/2019] [Accepted: 05/10/2019] [Indexed: 11/29/2022]
Abstract
The measurement of protein digestibility is one of the key steps in determining the safety of a genetically modified crop that has been traditionally accomplished using antibodies. Membrane proteins are often extremely difficult to express with replicated authentic tertiary structure in heterologous systems. As a result raising antibodies for use in safety assessment may not be feasible. In this study, LC-MS based proteomics was used to characterise seven transmembrane enzymes from the docosahexaenoic acid biosynthetic pathway that had been introduced into canola. The application of a two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro. Tryptic peptide markers that spanned the length of each desaturase protein were monitored and showed that these proteins were readily degraded (>95% within 5 min) and highlighted regions of the elongase enzymes that showed limited resistance to simulated gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and Δ6-elongase revealed rapid hydrolysis of the intact proteins within seconds and no fragments (>3 kDa) remained after 60 min, complementing the novel approach described herein. The LC-MS approach was sensitive, selective and did not require the use of purified proteins.
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Affiliation(s)
| | - Keren Byrne
- CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia, QLD, 4067, Australia
| | - Joanne Caine
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | - Lukasz Kowalczyk
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | | | - Bei Dong
- CSIRO Agriculture and Food, GPO Box 1700, Canberra, ACT, 2601, Australia
| | - George Lovrecz
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | - Susan MacIntosh
- Nuseed Americas, 11901 S. Austin Avenue, Alsip, IL, 60803, USA
| | - Judith A Scoble
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | - James R Petrie
- CSIRO Agriculture and Food, GPO Box 1700, Canberra, ACT, 2601, Australia
| | - Surinder Singh
- CSIRO Agriculture and Food, GPO Box 1700, Canberra, ACT, 2601, Australia
| | - Xue-Rong Zhou
- CSIRO Agriculture and Food, GPO Box 1700, Canberra, ACT, 2601, Australia
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7
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Colgrave ML, Byrne K, Pillai SV, Dong B, Leonforte A, Caine J, Kowalczyk L, Scoble JA, Petrie JR, Singh S, Zhou XR. Quantitation of seven transmembrane proteins from the DHA biosynthesis pathway in genetically engineered canola by targeted mass spectrometry. Food Chem Toxicol 2019; 126:313-321. [PMID: 30831153 DOI: 10.1016/j.fct.2019.02.035] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2018] [Revised: 02/04/2019] [Accepted: 02/25/2019] [Indexed: 12/31/2022]
Abstract
Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.
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Affiliation(s)
| | - Keren Byrne
- CSIRO Agriculture and Food, 306 Carmody Rd, St Lucia, QLD 4067, Australia
| | | | - Bei Dong
- CSIRO Agriculture and Food, GPO Box 1600, Canberra, ACT 2601, Australia
| | | | - Joanne Caine
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | - Lukasz Kowalczyk
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | - Judith A Scoble
- CSIRO Manufacturing, 343 Royal Parade, Parkville, VIC, 3052, Australia
| | - James R Petrie
- CSIRO Agriculture and Food, GPO Box 1600, Canberra, ACT 2601, Australia
| | - Surinder Singh
- CSIRO Agriculture and Food, GPO Box 1600, Canberra, ACT 2601, Australia
| | - Xue-Rong Zhou
- CSIRO Agriculture and Food, GPO Box 1600, Canberra, ACT 2601, Australia
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8
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Matsuzawa T, Maehara T, Kamisaka Y, Ara S, Takaku H, Yaoi K. Identification and characterization of Δ12 and Δ12/Δ15 bifunctional fatty acid desaturases in the oleaginous yeast Lipomyces starkeyi. Appl Microbiol Biotechnol 2018; 102:8817-8826. [PMID: 30206660 DOI: 10.1007/s00253-018-9345-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2018] [Revised: 08/07/2018] [Accepted: 08/25/2018] [Indexed: 12/16/2022]
Abstract
Fatty acid desaturases play vital roles in the synthesis of unsaturated fatty acids. In this study, Δ12 and Δ12/Δ15 fatty acid desaturases of the oleaginous yeast Lipomyces starkeyi, termed LsFad2 and LsFad3, respectively, were identified and characterized. Saccharomyces cerevisiae expressing LsFAD2 converted oleic acid (C18:1) to linoleic acid (C18:2), while a strain of LsFAD3-expressing S. cerevisiae converted oleic acid to linoleic acid, and linoleic acid to α-linolenic acid (C18:3), indicating that LsFad2 and LsFad3 were Δ12 and bifunctional Δ12/Δ15 fatty acid desaturases, respectively. The overexpression of LsFAD2 in L. starkeyi caused an accumulation of linoleic acid and a reduction in oleic acid levels. In contrast, overexpression of LsFAD3 induced the production of α-linolenic acid. Deletion of LsFAD2 and LsFAD3 induced the accumulation of oleic acid and linoleic acid, respectively. Our findings are significant for the commercial production of polyunsaturated fatty acids, such as ω-3 polyunsaturated fatty acids, in L. starkeyi.
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Affiliation(s)
- Tomohiko Matsuzawa
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan
| | - Tomoko Maehara
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan
| | - Yasushi Kamisaka
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan
| | - Satoshi Ara
- Department of Applied Life Science, Niigata University of Pharmacy and Applied Life Science, 265-1 Higashijima, Akiha-ku, Niigata, 956-8603, Japan
| | - Hiroaki Takaku
- Department of Applied Life Science, Niigata University of Pharmacy and Applied Life Science, 265-1 Higashijima, Akiha-ku, Niigata, 956-8603, Japan
| | - Katsuro Yaoi
- Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
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9
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Enhancement of Schizochytrium DHA synthesis by plasma mutagenesis aided with malonic acid and zeocin screening. Appl Microbiol Biotechnol 2018; 102:2351-2361. [DOI: 10.1007/s00253-018-8756-4] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2017] [Revised: 12/28/2017] [Accepted: 12/30/2017] [Indexed: 12/28/2022]
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10
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Hu Y, Zhang Y, Yu W, Hänninen H, Song L, Du X, Zhang R, Wu J. Novel Insights into the Influence of Seed Sarcotesta Photosynthesis on Accumulation of Seed Dry Matter and Oil Content in Torreya grandis cv. "Merrillii". FRONTIERS IN PLANT SCIENCE 2018; 8:2179. [PMID: 29375592 PMCID: PMC5767305 DOI: 10.3389/fpls.2017.02179] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2017] [Accepted: 12/12/2017] [Indexed: 05/10/2023]
Abstract
Seed oil content is an important trait of nut seeds, and it is affected by the import of carbon from photosynthetic sources. Although green leaves are the main photosynthetic organs, seed sarcotesta photosynthesis also supplies assimilates to seed development. Understanding the relationship between seed photosynthesis and seed development has theoretical and practical significance in the cultivation of Torreya grandis cv. "Merrillii." To assess the role of seed sarcotesta photosynthesis on the seed development, anatomical and physiological traits of sarcotesta were measured during two growing seasons in the field. Compared with the attached current-year leaves, the sarcotesta had higher gross photosynthetic rate at the first stage of seed development. At the late second stage of seed development, sarcotesta showed down-regulation of PSII activity, as indicated by significant decrease in the following chlorophyll fluorescence parameters: the maximum PSII efficiency (Fv/Fm ), the PSII quantum yield (Φ PSII ), and the photosynthetic quenching coefficient (qP). The ribulose 1, 5-bisphosphate carboxylase (Rubisco) activity, the total chlorophyll content (Chl(a+b)) and nitrogen content in the sarcotesta were also significantly decreased during that period. Treatment with DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] preventing seed photosynthesis decreased the seed dry weight and the oil content by 25.4 and 25.5%, respectively. We conclude that seed photosynthesis plays an important role in the dry matter accumulation at the first growth stage. Our results also suggest that down-regulation of seed photosynthesis is a plant response to re-balance the source-sink ratio at the second growth stage. These results suggest that seed photosynthesis is important for biomass accumulation and oil synthesis of the Torreya seeds. The results will facilitate achieving higher yields and oil contents in nut trees by selection for higher seed photosynthesis cultivars.
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Affiliation(s)
- Yuanyuan Hu
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
| | - Yongling Zhang
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
| | - Weiwu Yu
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
| | - Heikki Hänninen
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
| | - Lili Song
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
| | - Xuhua Du
- Key Laboratory of High Efficient Processing of Bamboo of Zhejiang Province, China National Bamboo Research Center, Hangzhou, China
| | - Rui Zhang
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
| | - Jiasheng Wu
- State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, China
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11
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Δ12-fatty acid desaturase is involved in growth at low temperature in yeast Yarrowia lipolytica. Biochem Biophys Res Commun 2017; 488:165-170. [DOI: 10.1016/j.bbrc.2017.05.028] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2017] [Accepted: 05/04/2017] [Indexed: 12/19/2022]
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12
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Santomartino R, Riego-Ruiz L, Bianchi MM. Three, two, one yeast fatty acid desaturases: regulation and function. World J Microbiol Biotechnol 2017; 33:89. [PMID: 28390014 DOI: 10.1007/s11274-017-2257-y] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2017] [Accepted: 03/31/2017] [Indexed: 01/01/2023]
Abstract
Fatty acid composition of biological membranes functionally adapts to environmental conditions by changing its composition through the activity of lipid biosynthetic enzymes, including the fatty acid desaturases. Three major desaturases are present in yeasts, responsible for the generation of double bonds in position C9-C10, C12-C13 and C15-C16 of the carbon backbone. In this review, we will report data addressed to define the functional role of basidiomycete and ascomycete yeast desaturase enzymes in response to various external signals and the regulation of the expression of their corresponding genes. Many yeast species have the complete set of three desaturases; however, only the Δ9 desaturase seems to be necessary and sufficient to ensure yeast viability. The evolutionary issue of this observation will be discussed.
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Affiliation(s)
- Rosa Santomartino
- Dip. di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Rome, Italy
| | - Lina Riego-Ruiz
- División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica (IPICYT), A.C., San Luis Potosí, Mexico
| | - Michele M Bianchi
- Dip. di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Rome, Italy.
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13
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De Angelis L, Rinaldi T, Cirigliano A, Bello C, Reverberi M, Amaretti A, Montanari A, Santomartino R, Raimondi S, Gonzalez A, Bianchi MM. Functional roles of the fatty acid desaturases encoded by KlOLE1, FAD2 and FAD3 in the yeast Kluyveromyces lactis. MICROBIOLOGY-SGM 2016; 162:1435-1445. [PMID: 27233577 DOI: 10.1099/mic.0.000315] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Functional properties of cell membranes depend on their composition, particularly on the relative amount of saturated, unsaturated and polyunsaturated fatty acids present in the phospholipids. The aim of this study was to investigate the effect of cell membrane composition on cell fitness, adaptation and stress response in Kluyveromyces lactis. To this purpose, we have deleted the genes FAD2 and FAD3 encoding Δ12 and ω3 desaturases in Kluyveromyces lactis, thus generating mutant strains with altered fatty acid composition of membranes. These strains were viable and able to grow in stressing conditions like hypoxia and low temperature. Deletion of the Δ9 desaturase-encoding gene KlOLE1 resulted in lethality, suggesting that this enzyme has an essential role in this yeast. Transcription of the desaturase genes KlOLE1, FAD2 and FAD3 and cellular localization of the corresponding enzymes, have been studied under hypoxia and cold stress. Our findings indicate that expression of these desaturase genes and membrane composition were modulated by hypoxia and temperature stress, although the changes induced by these and other assayed conditions did not dramatically affect the general cellular fitness.
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Affiliation(s)
- Lorenzo De Angelis
- Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Roma, Italy
| | - Teresa Rinaldi
- Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Roma, Italy.,Pasteur Institute Cenci-Bolognetti Foundation, Viale Regina Elena 291, 00161 Roma, Italy
| | - Angela Cirigliano
- Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Roma, Italy
| | - Cristiano Bello
- Department of Environmental Biology, Sapienza Università di Roma, Roma, Italy
| | - Massimo Reverberi
- Department of Environmental Biology, Sapienza Università di Roma, Roma, Italy
| | - Alberto Amaretti
- Department of Life Sciences, Università di Modena e Reggio Emilia, Via Università, 4, 41121, Modena, Italy
| | - Arianna Montanari
- Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Roma, Italy
| | - Rosa Santomartino
- Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Roma, Italy
| | - Stefano Raimondi
- Department of Life Sciences, Università di Modena e Reggio Emilia, Via Università, 4, 41121, Modena, Italy
| | - Alicia Gonzalez
- Department of Biochemistry and Structural Biology, Universidad Nacional Autónoma de México, Mexico
| | - Michele M Bianchi
- Department of Biology and Biotechnology C. Darwin, Sapienza Università di Roma, p.le Aldo Moro 5, 00185, Roma, Italy
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14
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Hanano A, Shaban M, Almousally I, Al-Ktaifani M. Saccharomyces cerevisiae SHSY detoxifies petroleum n-alkanes by an induced CYP52A58 and an enhanced order in cell surface hydrophobicity. CHEMOSPHERE 2015; 135:418-426. [PMID: 25434275 DOI: 10.1016/j.chemosphere.2014.11.011] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/04/2014] [Revised: 11/03/2014] [Accepted: 11/05/2014] [Indexed: 06/04/2023]
Abstract
Environmental hydrocarbon contamination has a serious hazard to human health. Alkanes, the major component of hydrocarbons, can be consumed by various species of yeast. We previously identified a new strain SHSY of Saccharomyces cerevisiae with a remarkable ability to utilize the petroleum crude-oil (PCO) in aqueous solution. The current study demonstrated that the n-alkanes-assimilation activity of S. cerevisiae SHSY was related to an induced microsomal protein of 59 kDa approximately. The identified ORF encoded a protein of 517 amino acids and shared 93% sequence identity with an alkane-inducible hydroxylase CYP52A53 isolated from Scheffersomyces stipitis CBS. It was therefore referred as CYP52A58. The catalytic activity of the recombinant CYP52A58 was confirmed by the hydroxylation of n-alkanes, it showed an optimal mono-terminal hydroxylation activity toward n-hexadecane. Moreover, the ability of the yeast to use n-alkanes was accompanied with an increasing level in cell wall mannoproteins. Two differential protein bands were detected in the mannoproteins extracted from PCO-grown yeast. In parallel, a significant increase in the fatty acids content with a high degree of unsaturation was subsequently detected in the PCO-grown yeast. This study characterizes a safe and potential microorganism to remove n-alkanes from the aquatic environment.
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Affiliation(s)
- Abdulsamie Hanano
- Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), B.P. Box 6091, Damascus, Syria.
| | - Mouhnad Shaban
- Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), B.P. Box 6091, Damascus, Syria
| | - Ibrahem Almousally
- Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), B.P. Box 6091, Damascus, Syria
| | - Mahmoud Al-Ktaifani
- Department of Chemistry, Atomic Energy Commission of Syria (AECS), B.P. Box 6091, Damascus, Syria
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15
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Gomes D, Aguiar TQ, Dias O, Ferreira EC, Domingues L, Rocha I. Genome-wide metabolic re-annotation of Ashbya gossypii: new insights into its metabolism through a comparative analysis with Saccharomyces cerevisiae and Kluyveromyces lactis. BMC Genomics 2014; 15:810. [PMID: 25253284 PMCID: PMC4190384 DOI: 10.1186/1471-2164-15-810] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2014] [Accepted: 08/15/2014] [Indexed: 12/17/2022] Open
Abstract
BACKGROUND Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins. RESULTS After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG's annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii's metabolism in comparison to the one of S. cerevisiae (post-WGD - whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism. CONCLUSIONS This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii's metabolism.
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Affiliation(s)
- Daniel Gomes
- CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
| | - Tatiana Q Aguiar
- CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
| | - Oscar Dias
- CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
| | - Eugénio C Ferreira
- CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
| | - Lucília Domingues
- CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
| | - Isabel Rocha
- CEB - Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
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16
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Buček A, Matoušková P, Sychrová H, Pichová I, Hrušková-Heidingsfeldová O. Δ12-Fatty acid desaturase from Candida parapsilosis is a multifunctional desaturase producing a range of polyunsaturated and hydroxylated fatty acids. PLoS One 2014; 9:e93322. [PMID: 24681902 PMCID: PMC3969366 DOI: 10.1371/journal.pone.0093322] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Accepted: 03/03/2014] [Indexed: 11/28/2022] Open
Abstract
Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named CpFad2 and CpFad3, respectively, from the opportunistic pathogenic yeast Candida parapsilosis. Overexpression of CpFad3 in Saccharomyces cerevisiae strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, i.e., α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). CpFad2 produces a range of Δ12- and Δ15-PUFAs. The major products of CpFad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of CpFad2. These results demonstrate that CpFAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina).
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Affiliation(s)
- Aleš Buček
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Petra Matoušková
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Hana Sychrová
- Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
| | - Iva Pichová
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
- * E-mail: (IP); (OHH)
| | - Olga Hrušková-Heidingsfeldová
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
- * E-mail: (IP); (OHH)
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17
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Cloning and functional analysis of HpFAD2 and HpFAD3 genes encoding Δ12- and Δ15-fatty acid desaturases in Hansenula polymorpha. Gene 2013; 533:110-8. [PMID: 24100086 DOI: 10.1016/j.gene.2013.09.115] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2013] [Revised: 09/25/2013] [Accepted: 09/26/2013] [Indexed: 12/19/2022]
Abstract
Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha Δ12-fatty acid desaturase (HpFad2) and Δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, Δ9, Δ12) and α-linolenic acid (ALA, αC18:3, Δ9, Δ12, Δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215bp and 1239bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2Δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode Δ12- and Δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a Δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.
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18
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Synthesis and production of unsaturated and polyunsaturated fatty acids in yeast: current state and perspectives. Appl Microbiol Biotechnol 2012; 95:1-12. [DOI: 10.1007/s00253-012-4105-1] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2012] [Revised: 04/12/2012] [Accepted: 04/12/2012] [Indexed: 10/28/2022]
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19
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Blacklock BJ, Scheffler BE, Shepard MR, Jayasuriya N, Minto RE. Functional diversity in fungal fatty acid synthesis: the first acetylenase from the Pacific golden chanterelle, Cantharellus formosus. J Biol Chem 2010; 285:28442-9. [PMID: 20606235 PMCID: PMC2937869 DOI: 10.1074/jbc.m110.151498] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2010] [Revised: 07/03/2010] [Indexed: 01/12/2023] Open
Abstract
Acetylenic specialized metabolites containing one or more carbon-carbon triple bonds are widespread, being found in fungi, vascular and lower plants, marine sponges and algae, and insects. Plants, moss, and most recently, insects, have been shown to employ an energetically difficult, sequential dehydrogenation mechanism for acetylenic bond formation. Here, we describe the cloning and heterologous expression in yeast of a linoleoyl 12-desaturase (acetylenase) and a bifunctional desaturase with Delta(12)-/Delta(14)-regiospecificity from the Pacific golden chanterelle. The acetylenase gene, which is the first identified from a fungus, is phylogenetically distinct from known plant and fungal desaturases. Together, the bifunctional desaturase and the acetylenase provide the enzymatic activities required to drive oleate through linoleate to crepenynate and the conjugated enyne (14Z)-dehydrocrepenynate, the branchpoint precursors to a major class of acetylenic natural products.
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Affiliation(s)
- Brenda J. Blacklock
- From the
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202 and
| | - Brian E. Scheffler
- the
Genomics and Bioinformatics Research Unit, Jamie Whitten Delta States Research Center, Stoneville, Mississippi 38776
| | - Michael R. Shepard
- From the
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202 and
| | - Naomi Jayasuriya
- From the
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202 and
| | - Robert E. Minto
- From the
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, Indiana 46202 and
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20
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Cao Y, Xian M, Yang J, Xu X, Liu W, Li L. Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli. Protein Expr Purif 2009; 69:209-14. [PMID: 19716420 DOI: 10.1016/j.pep.2009.08.011] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2009] [Revised: 08/16/2009] [Accepted: 08/24/2009] [Indexed: 11/30/2022]
Abstract
Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS-PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1Delta9) and cis-vaccenate (18:1Delta11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1Delta9) was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase.
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Affiliation(s)
- Yujin Cao
- Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, 266101 Qingdao, China
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21
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Yazawa H, Iwahashi H, Kamisaka Y, Kimura K, Uemura H. Production of polyunsaturated fatty acids in yeastSaccharomyces cerevisiaeand its relation to alkaline pH tolerance. Yeast 2009; 26:167-84. [DOI: 10.1002/yea.1659] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022] Open
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22
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Evolution-related amino acids play important role in determining regioselectivity of fatty acid desaturase from Pichia pastoris. Mol Biol Rep 2008; 36:567-73. [DOI: 10.1007/s11033-008-9215-6] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2007] [Accepted: 02/11/2008] [Indexed: 10/22/2022]
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23
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Gostincar C, Turk M, Trbuha T, Vaupotic T, Plemenitas A, Gunde-Cimerman N. Expression of fatty-acid-modifying enzymes in the halotolerant black yeast Aureobasidium pullulans (de Bary) G. Arnaud under salt stress. Stud Mycol 2008; 61:51-9. [PMID: 19287526 PMCID: PMC2610307 DOI: 10.3114/sim.2008.61.04] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Multiple tolerance to stressful environmental conditions of the black, yeast-like fungus Aureobasidium pullulans is achieved through different adaptations, among which there is the restructuring of the lipid composition of their membranes. Here, we describe three novel genes encoding fatty-acid-modifying enzymes in A. pullulans, along with the levels of their mRNAs under different salinity conditions. High levels of Delta(9)-desaturase and Delta(12)-desaturase mRNAs were seen at high salinities, which were consistent with an increased desaturation of the fatty acids in the cell membranes. Elevated levels of elongase mRNA were also detected. Surprisingly, increases in the levels of these mRNAs were also seen following hypo-osmotic shock, while hyperosmotic shock had exactly the opposite effect, demonstrating that data that are obtained from up-shift and down-shift salinity studies should be interpreted with caution.
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Affiliation(s)
- C Gostincar
- Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, SI-1000 Ljubljana, Slovenia
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24
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Yazawa H, Iwahashi H, Kamisaka Y, Kimura K, Aki T, Ono K, Uemura H. Heterologous production of dihomo-gamma-linolenic acid in Saccharomyces cerevisiae. Appl Environ Microbiol 2007; 73:6965-71. [PMID: 17873077 PMCID: PMC2074983 DOI: 10.1128/aem.01008-07] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
To make dihomo-gamma-linolenic acid (DGLA) (20:3n-6) in Saccharomyces cerevisiae, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase, rat Delta6 fatty acid desaturase, and rat elongase genes. Because Fad2p is able to convert the endogenous oleic acid to linoleic acid, this allowed DGLA biosynthesis without the need to supply exogenous fatty acids on the media. Medium composition, cultivation temperature, and incubation time were examined to improve the yield of DGLA. Fatty acid content was increased by changing the medium from a standard synthetic dropout medium to a nitrogen-limited minimal medium (NSD). Production of DGLA was higher in the cells grown at 15 degrees C than in those grown at 20 degrees C, and no DGLA production was observed in the cells grown at 30 degrees C. In NSD at 15 degrees C, fatty acid content increased up until day 7 and decreased after day 10. When the cells were grown in NSD for 7 days at 15 degrees C, the yield of DGLA reached 2.19 microg/mg of cells (dry weight) and the composition of DGLA to total fatty acids was 2.74%. To our knowledge, this is the first report describing the production of polyunsaturated fatty acids in S. cerevisiae without supplying the exogenous fatty acids.
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Affiliation(s)
- Hisashi Yazawa
- National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
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25
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Murayama SY, Negishi Y, Umeyama T, Kaneko A, Oura T, Niimi M, Ubukata K, Kajiwara S. Construction and functional analysis of fatty acid desaturase gene disruptants in Candida albicans. MICROBIOLOGY-SGM 2006; 152:1551-1558. [PMID: 16622072 DOI: 10.1099/mic.0.28751-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Polyunsaturated fatty acids (PUFAs), including linoleic acid (C18 : 2) and alpha-linolenic acid (C18 : 3), are major components of membranes. PUFAs are produced from monounsaturated fatty acids by several fatty acid desaturases (FADs) in many fungi, but Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans do not have these enzymes. Although the fungal pathogen Candida albicans produces C18 : 2 and C18 : 3, the enzymes that synthesize them have not yet been investigated. In this report, two ORFs, CaFAD2 and CaFAD3, were identified based on their homology to other yeast FADs, and CaFAD2 and CaFAD3 gene disruptants were constructed. Cafad2Delta and Cafad3Delta lost their ability to produce C18 : 2 and C18 : 3, respectively. Furthermore, S. cerevisiae cells expressing CaFad2p converted palmitoleic acid (C16 : 1) and C18 : 1 to hexadecadienoic acid (C16 : 2) and C18 : 2, respectively, and CaFad3p-expressing cells converted C18 : 2 to C18 : 3. These results strongly supported that CaFAD2 encodes the Delta12 FAD and that CaFAD3 encodes the omega3 FAD. However, phenotypic analysis demonstrated that the presence of these PUFAs did not affect the virulence to mice, or morphogenesis in the culture media used to induce morphological change of C. albicans.
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Affiliation(s)
- Somay Yamagata Murayama
- Laboratory of Infectious Agents Surveillance, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
| | - Yumiko Negishi
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
- Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
- Laboratory of Infectious Agents Surveillance, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
| | - Takashi Umeyama
- Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
| | - Aki Kaneko
- Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
| | - Takahiro Oura
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
| | - Masakazu Niimi
- Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
| | - Kimiko Ubukata
- Laboratory of Infectious Agents Surveillance, Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
| | - Susumu Kajiwara
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
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26
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Li MC, Li H, Wei DS, Xing LJ. Cloning and molecular characterization of Δ 12-fatty acid desaturase gene from Mortierella isabellina. World J Gastroenterol 2006; 12:3373-9. [PMID: 16733854 PMCID: PMC4087868 DOI: 10.3748/wjg.v12.i21.3373] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To clone Δ12 -fatty acid desaturase gene of Mortierella isabellina and to functionally characterize this gene in vitro and in vivo.
METHODS: Reverse transcriptional polymerase chain reaction (RT-PCR) was used to clone the open reading frame of Δ12-fatty acid desaturase gene (D12D) of Mortierella isabellina. Plasmids pEMICL12 and pYMICL12 were constructed with it. pEMICL12 was transformed into Escherichia coli (E.coli) strain BL21 using CaCl2 method for expression after induction with IPTG. pTMICL12 was transformed into Saccharomyces cerevisiae strain INVSc1 using lithium acetate method for expression under the induction of galactose. Northern blotting method was used to investigate the effect of temperature on the transcriptional level of this gene in S.cerevisiae strain INVSc1.
RESULTS: Recombinant plasmids pEMICL12 and pTMICL12 were successfully constructed and transformed into E.coli and S.cerevisiae separately with appropriate method. After induction with IPTG and galactose, it was found that expression of Δ12-fatty acid desaturase genes in E.coli and S. cerevisiae under appropriate conditions led to the production of active Δ12-fatty acid desaturase, which could convert 17.876% and 17.604% of oleic acid respectively to linoleic acid by GC-MS detection in vitro and in vivo.
CONCLUSION: Cloning and expression of M.isabellina D12D gene in E.coli and S.cerevisiae is successfully completed.
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MESH Headings
- Blotting, Northern
- Cloning, Molecular
- DNA, Complementary/analysis
- DNA, Complementary/genetics
- DNA, Fungal/analysis
- DNA, Fungal/genetics
- Electrophoresis, Polyacrylamide Gel
- Escherichia coli/enzymology
- Escherichia coli/genetics
- Fatty Acid Desaturases/genetics
- Fatty Acid Desaturases/metabolism
- Fungal Proteins/genetics
- Fungal Proteins/metabolism
- Gene Expression Regulation, Fungal
- Genes, Fungal/genetics
- Genetic Vectors
- Mortierella/enzymology
- Mortierella/genetics
- Plasmids/genetics
- RNA, Fungal/analysis
- RNA, Fungal/genetics
- RNA, Messenger/analysis
- RNA, Messenger/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Saccharomyces cerevisiae/enzymology
- Saccharomyces cerevisiae/genetics
- Temperature
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Affiliation(s)
- Ming-Chun Li
- Department of Microbiology, The College of Life Science, Nankai University, Tianjin, China
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Oura T, Kajiwara S. Cloning and functional characterization of a fatty acid synthase component FAS2 gene from Saccharomyces kluyveri. Curr Genet 2006; 49:393-402. [PMID: 16479401 DOI: 10.1007/s00294-006-0063-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2005] [Revised: 01/25/2006] [Accepted: 01/28/2006] [Indexed: 10/25/2022]
Abstract
A gene coding the alpha subunit of fatty acid synthase (FAS2) was isolated from the budding yeast Saccharomyces kluyveri. Nucleotide sequence analysis indicated that this gene, termed Sk-FAS2, coded a protein having an amino acid sequence 83% identical to the FAS2 protein of S. cerevisiae (Sc-FAS2). The Sk-FAS2 gene was able to functionally complement an S. cerevisiae fas2 disruptant. This Sk-FAS2-expressing strain was found to produce larger amounts of C18 than C16, in contrast to the Sc-FAS2-expressing fas2 strain. In addition, fusion genes of Sk-FAS2 and Sc-FAS2 were transformed into a fas2-disrupted strain of S. cerevisiae, and fatty acid analysis of these transformants suggested that the region containing the acyl carrier protein and beta-ketoacyl reductase domains of yeast FAS2 protein play an important role in determining carbon chain length of fatty acids.
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Affiliation(s)
- Takahiro Oura
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B5 Nagatsuta, 226-8501, Yokohama, Kanagawa, Japan.
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28
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Kainou K, Kamisaka Y, Kimura K, Uemura H. Isolation of Δ12 and ω3-fatty acid desaturase genes from the yeastKluyveromyces lactis and their heterologous expression to produce linoleic and α-linolenic acids inSaccharomyces cerevisiae. Yeast 2006; 23:605-12. [PMID: 16823888 DOI: 10.1002/yea.1378] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Two clones with homology to known fatty acid desaturase genes were isolated from the yeast Kluyveromyces lactis. The first gene, which we designate KlFAD2, consists of 411 amino acids with an overall identity of 73.0% to FAD2 from Saccharomyces kluyveri. It exhibited Delta12 fatty acid desaturase activity when expressed in S. cerevisiae under the control of ADH1 promoter and produced endogenous linoleic acid. The second clone, which we designate KlFAD3, consists of 415 amino acids with an overall identity of 79.3% to FAD3 from S. kluyveri. It exhibited omega3 fatty acid desaturase activity in S. cerevisiae when expressed under the control of ADH1 promoter in the presence of the exogenous substrate linoleic acid and produced alpha-linolenic acid. Co-expression of KlFAD2 and KlFAD3 resulted in the endogenous production of both linoleic and alpha-linolenic acids. The yield of alpha-linolenic acid reached 0.8% of total fatty acids and its production was not increased by adding exogenous oleic acid; alpha-linolenic acid reached 8.7% when exogenous linoleic acid was available.
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Affiliation(s)
- Kumiko Kainou
- National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
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29
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Sakai H, Kajiwara S. Cloning and functional characterization of a Delta12 fatty acid desaturase gene from the basidiomycete Lentinula edodes. Mol Genet Genomics 2005; 273:336-41. [PMID: 15838640 DOI: 10.1007/s00438-005-1138-5] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2004] [Accepted: 03/03/2005] [Indexed: 12/26/2022]
Abstract
In the basidiomycete Lentinula edodes, a famous edible mushroom (shiitake), the fatty acyl composition of total lipids has previously been shown to change during cell differentiation. In the present study, we succeeded in cloning a gene for a Delta12 fatty acid desaturase from L. edodes. The ORF of this gene (named Le-FAD2) consists of 1308 bp and codes for 435 amino acids. The deduced Le-FAD2 protein shows 40-45% identity to Delta12 fatty acid desaturases from other fungi, and the three histidine clusters typical of the catalytic domain of such enzymes are conserved. Expression of the Le-FAD2 gene in the budding yeast Saccharomyces cerevisiae indicated that its product was able to synthesize linoleic acid (C18:2). Analysis of Le-FAD2 expression in L. edodes revealed that levels of transcription were higher in fruiting body primordia and in mature fruiting bodies, the two differentiated tissues, than in mycelium, and reduction of the growth temperature from 25 to 18 degrees C had no effect on the level of the Le-FAD2 transcript. Thus, although Le-FAD2 expression is correlated with the alteration in the complement of unsaturated fatty acids (UFAs) observed during fruiting body formation, the gene does not respond to a downshift in temperature to 18 degrees C.
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Affiliation(s)
- Hiromichi Sakai
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-B5, Nagatsuta, Midori-ku, Yokohama, Kanagawa, 226-8501, Japan
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30
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Current awareness on yeast. Yeast 2004; 21:1133-40. [PMID: 15529464 DOI: 10.1002/yea.1095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
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31
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Wei D, Li M, Zhang X, Ren Y, Xing L. Identification and characterization of a novel delta12-fatty acid desaturase gene from Rhizopus arrhizus. FEBS Lett 2004; 573:45-50. [PMID: 15327973 DOI: 10.1016/j.febslet.2004.06.100] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2004] [Revised: 06/20/2004] [Accepted: 06/28/2004] [Indexed: 11/20/2022]
Abstract
Based on the sequence information of Delta12-fatty acid desaturase genes (from Mucor circinelloides, Mortierella alpina, Mucor rouxii and Aspergillus nidulans), which were involved in the conversion from C18:1 to C18:2, a cDNA sequence putatively encoding a Delta12-fatty acid desaturase was isolated from Rhizopus arrhizus using the combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Sequence analysis indicated that it had an open reading frame (ORF) of 1170 bp, coding for 389 amino acid residues of 45 kDa, pI of the deduced protein was 7.01. The deduced amino acid sequence of this cloned cDNA showed high identity to those filamentous fungal Delta12-desaturases mentioned above, including three conserved histidine-rich motifs and two hydrophobic domains. Functional identification was done heterologously in Saccharomyces cerevisiae strain INVScl. The result demonstrated that the deduced amino acid sequence exhibited Delta12-fatty acid desaturase activity, suggesting that this gene encoded for a membrane-bound desaturase, Delta12-fatty acid desaturase.
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Affiliation(s)
- Dongsheng Wei
- Department of Microbiology, Nankai University, Tianjin 300071, PR China
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32
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Oura T, Kajiwara S. Saccharomyces kluyveri FAD3 encodes an ω3 fatty acid desaturase. Microbiology (Reading) 2004; 150:1983-1990. [PMID: 15184584 DOI: 10.1099/mic.0.27049-0] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Fungi, like plants, are capable of producing the 18-carbon polyunsaturated fatty acids linoleic acid andα-linolenic acid. These fatty acids are synthesized by catalytic reactions of Δ12 andω3 fatty acid desaturases. This paper describes the first cloning and functional characterization of a yeastω3 fatty acid desaturase gene. The deduced protein encoded by theSaccharomyces kluyveri FAD3gene (Sk-FAD3) consists of 419 amino acids, and shows 30–60 % identity with Δ12 fatty acid desaturases of several eukaryotic organisms and 29–31 % identity withω3 fatty acid desaturases of animals and plants. DuringSk-FAD3expression inSaccharomyces cerevisiae,α-linolenic acid accumulated only when linoleic acid was added to the culture medium. The disruption ofSk-FAD3led to the disappearance ofα-linolenic acid inS. kluyveri. These findings suggest thatSk-FAD3is the onlyω3 fatty acid desaturase gene in this yeast. Furthermore, transcriptional expression ofSk-FAD3appears to be regulated by low-temperature stress in a manner different from the other fatty acid desaturase genes inS. kluyveri.
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Affiliation(s)
- Takahiro Oura
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
| | - Susumu Kajiwara
- Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
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