Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Campesterol is an important precursor for many sterol drugs, e.g. progesterone and hydrocortisone. In order to produce campesterol in Yarrowia lipolytica, C-22 desaturase encoding gene ERG5 was disrupted and the heterologous 7-dehydrocholesterol reductase (DHCR7) encoding gene was constitutively expressed. The codon-optimized DHCR7 from Rallus norvegicus, Oryza saliva and Xenapus laevis were explored and the strain with the gene DHCR7 from X. laevis achieved the highest titer of campesterol due to D409 in substrate binding sites. In presence of glucose as the carbon source, higher biomass conversion yield and product yield were achieved in shake flask compared to that using glycerol and sunflower seed oil. Nevertheless, better cell growth rate was observed in medium with sunflower seed oil as the sole carbon source. Through high cell density fed-batch fermentation under carbon source restriction strategy, a titer of 453±24.7 mg/L campesterol was achieved with sunflower seed oil as the carbon source, which is the highest reported microbial titer known. Our study has greatly enhanced campesterol accumulation in Y. lipolytica, providing new insight into producing complex and desired molecules in microbes.
???displayArticle.pubmedLink???
26751680
???displayArticle.pmcLink???PMC4709189 ???displayArticle.link???PLoS One
Fig 1. Overview of campesterol biosynthesis pathway and the corresponding genetic modification in Y. lipolytica.(a) Overview of campesterol biosynthesis pathway in Y. lipolytica. Campesterol was produced via expression of DHCR7 and disruption of ERG5 based on the ergosterol synthesis pathway in Y. lipolytica. Acetyl-CoA, the precursor of campesterol, is provided either by glycolysis from hydrophilic carbon sources (i.e. glucose and glycerol), or by β-oxidization from hydrophobic materials (e.g. oil). Meanwhile, acetyl-CoA from either hydrophilic or hydrophobic substrates can also generate cellular fatty acids and subsequently triacylglycerols (TAG), consisting of the de novo and the ex novo lipid accumulation process in Y. lipolytica respectively. (b) The method for expression of DHCR7 and disruption of ERG5 at the same time. The middle section of gene ERG5 was replaced by the DHCR7 expression cassette (URA3-EXP1p-DHCR7-XPR2t) in chromosome A. (c) GC-TOF/MS profile of the wild-type Y. lipolytica. ATCC 201249 and campesterol producing strain SyBE_Yl1070030. Wild-type strain (blue) showed an ergosterol peak at 21 min, while Y. lipolytica SyBE_Yl1070028 (red) showed a campesterol peak at 21.7 min.
Fig 2. Campesterol production in strains with DHCR7 from different species.Y. lipolytica strains with DHCR7 from X. laevis, O. saliva and R. norvegicus were named as SyBE_Yl1070028, SyBE_Yl1070029 and SyBE_Yl1070030 respectively. The amount of campesterol produced by these three strains was measured after 120 h shake flask culture in YPD medium.
Fig 3. Sequences and architecture differences among DHCR7 from X. laevis, O. saliva and R. norvegicus.(a) Overview the structure model of DHCR7 from X. laevis. NADPH (blue) was docked into the NADPH pocket and ergosta-5,7-dienol (yellow) modelled into the substrate binding pocket in the DHCR7 structure. The pocket for substrate binding was enlarged and present below, indicating that Y278 and D409 clamp the hydroxyl of the substrate through a hydrogen-bonding network. (b) Sequences alignment of DHCR7 from X. laevis, O. saliva and R. norvegicus. The putative cholesterol hydroxyl group binding sites and cofactor NADPH binding sites were marked by red squares and blue stars, respectively. Mutations which might attribute to the lowest activity of DHCR7 from R. norvegicus were boxed with red lines. (c) Campesterol production in Y. lipolytica strains with wild-type and mutated X. laevis DHCR7 (named as SyBE_Yl1070028 and SyBE_Yl1070031 respectively). The campesterol production for these strains was measured after 120 h shake flask culture in YPD medium.
Fig 4. Shake flask cultivation with different carbon sources.(a) Cells growth for Y. lipolytica strain SyBE_Yl1070028 with glucose, glycerol and sunflower seed oil as the carbon source. (b) Substrate (glucose, glycerol and sunflower oil) consumed by strain SyBE_Yl1070028. (c) The final campersterol titers produced by strain SyBE_Yl1070028 with glucose, glycerol and sunflower seed oil as the carbon source. (d) Cells growths specific rate (μ, h-1), biomass conversion yield (YX/S) and campesterol production yield (YP/S) for strain SyBE_Yl1070028 with different carbon source. μ is determined as ln(X/X0)/ (t-t0), where X is for total biomass (g/L) and t is for time (h). Meanwhile, YX/S represents the yield of total biomass produced per unit of substrate consumed and YP/S represents the yield of product formed per unit of substrate consumed.
Fig 5. Bioreactor fermentation with different carbon sources.Campesterol producing strain SyBE_Yl1070028 was fed-batch cultured in glucose (a) or in sunflower seed oil (b) with carbon source restriction strategy. YX/S (yield of total biomass produced per unit of substrate consumed) and YP/S (yield of product formed per unit of substrate consumed) for fed-batch bioreactor cultures were compared with those from the shake-flask experiments (c).
Fig 6. Lipid accumulation in Y. lipolytica strain SyBE_Yl1070028 cultured with different carbon sources.(a) The morphology and the lipid body (indicated by arrows) of the engineered Y. lipolytica strain in shake flask cultivation with glucose, glycerol and sunflower seed oil as the carbon source. (b) The lipid accumulation on the dry cell weight (DCW) basis for the shake flask trials.
Aggelis,
Microbial fatty acid specificity.
1997,
Pubmed
Athenstaedt,
Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source.
2006,
Pubmed
Beopoulos,
Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation.
2009,
Pubmed
Beopoulos,
Yarrowia lipolytica as a model for bio-oil production.
2009,
Pubmed
Blazeck,
Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach.
2011,
Pubmed
Ding,
Biosynthesis of Taxadiene in Saccharomyces cerevisiae : selection of geranylgeranyl diphosphate synthase directed by a computer-aided docking strategy.
2014,
Pubmed
Dulermo,
Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: new insights into the role of intracellular lipases and lipid body organisation.
2013,
Pubmed
Duport,
Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast.
1998,
Pubmed
Fickers,
Hydrophobic substrate utilisation by the yeast Yarrowia lipolytica, and its potential applications.
2005,
Pubmed
FOLCH,
A simple method for the isolation and purification of total lipides from animal tissues.
1957,
Pubmed
Gao,
One-step integration of multiple genes into the oleaginous yeast Yarrowia lipolytica.
2014,
Pubmed
Holzschu,
Evaluation of industrial yeasts for pathogenicity.
1979,
Pubmed
Jacquier,
Mechanisms of sterol uptake and transport in yeast.
2012,
Pubmed
Leonard,
Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control.
2010,
Pubmed
Li,
Structure of an integral membrane sterol reductase from Methylomicrobium alcaliphilum.
2015,
Pubmed
Lin,
RADOM, an efficient in vivo method for assembling designed DNA fragments up to 10 kb long in Saccharomyces cerevisiae.
2015,
Pubmed
Makri,
Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures.
2010,
Pubmed
Matoba,
Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica.
1988,
Pubmed
Matthäus,
Production of lycopene in the non-carotenoid-producing yeast Yarrowia lipolytica.
2014,
Pubmed
Mlícková,
Lipid accumulation, lipid body formation, and acyl coenzyme A oxidases of the yeast Yarrowia lipolytica.
2004,
Pubmed
Morin,
Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica.
2011,
Pubmed
Naruse,
Alterations of plant sterols, lathosterol, oxidative stress and inflammatory markers after the combination therapy of ezetimibe and statin drugs in type 2 diabetic patients.
2015,
Pubmed
Papanikolaou,
Influence of glucose and saturated free-fatty acid mixtures on citric acid and lipid production by Yarrowia lipolytica.
2006,
Pubmed
Papanikolaou,
Yarrowia lipolytica as a potential producer of citric acid from raw glycerol.
2002,
Pubmed
Papanikolaou,
Kinetic profile of the cellular lipid composition in an oleaginous Yarrowia lipolytica capable of producing a cocoa-butter substitute from industrial fats.
2001,
Pubmed
Rywińska,
Comparison of citric acid production from glycerol and glucose by different strains of Yarrowia lipolytica.
2010,
Pubmed
Sestric,
Growth and neutral lipid synthesis by Yarrowia lipolytica on various carbon substrates under nutrient-sufficient and nutrient-limited conditions.
2014,
Pubmed
Shao,
DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways.
2009,
Pubmed
Sitepu,
Manipulation of culture conditions alters lipid content and fatty acid profiles of a wide variety of known and new oleaginous yeast species.
2013,
Pubmed
Souza,
A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance.
2011,
Pubmed
Su,
Alleviating Redox Imbalance Enhances 7-Dehydrocholesterol Production in Engineered Saccharomyces cerevisiae.
2015,
Pubmed
Szczebara,
Total biosynthesis of hydrocortisone from a simple carbon source in yeast.
2003,
Pubmed
Trott,
AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading.
2010,
Pubmed
Wang,
Characterization of a new GlnR binding box in the promoter of amtB in Streptomyces coelicolor inferred a PhoP/GlnR competitive binding mechanism for transcriptional regulation of amtB.
2012,
Pubmed
Wang,
High-level production of the industrial product lycopene by the photosynthetic bacterium Rhodospirillum rubrum.
2012,
Pubmed
Witsch-Baumgartner,
Mutations in the human DHCR7 gene.
2001,
Pubmed
Wu,
Biological variation of β-sitosterol, campesterol, and lathosterol as cholesterol absorption and synthesis biomarkers.
2014,
Pubmed
Xue,
Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica.
2013,
Pubmed
Zou,
7-Dehydrocholesterol reductase activity is independent of cytochrome P450 reductase.
2011,
Pubmed
