An Oleate Hydroxylase from the Fungus <i>Claviceps purpurea</i>: Cloning, Functional Analysis, and Expression in Arabidopsis

Meesapyodsuk, Dauenpen; Qiu, Xiao

doi:10.1104/pp.108.117168

Cited by 43 publications

(45 citation statements)

References 38 publications

(64 reference statements)

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“…Sorghum stalks had a higher content of lignin (20%) compared with switch- ) sodium hydroxide solution, following the optimized conditions in the previous study [30]. Pretreated biomass totals of 58.6 g and 58.4 g were obtained from sorghum stalks and switchgrass, respectively.…”

Section: Sugar Recoveries From Sorghum Stalks and Switchgrassmentioning

confidence: 99%

Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures

Lee¹,

Vadlani²,

Min³

2017

JSBS

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Microbial lipids derived from oleaginous yeast could be a promising resource for biodiesel and other oleochemical materials. The objective of this study was to develop an efficient bioconversion process from lignocellulosic biomass to microbial lipids using three types of robust oleaginous yeast: T. oleaginosus, L. starkeyi, and C. albidus. Sorghum stalks and switchgrass were utilized as feedstocks for lipid production. Among oleaginous yeast strains, T. oleaginous showed better performance for lipid production using sorghum stalk hydrolysates. Lipid titers of 13.1 g•L , which was substantially higher than the value reported in literature. Assessment of overall lipid yield revealed a total of 14.3 g and 13.3 g lipids were produced by T. oleaginosus from 100 g of raw sorghum stalks and switchgrass, respectively. This study revealed that minimization of sugar loss during pretreatment and selection of appropriate yeast strains would be key factors to develop an efficient bioconversion process and improve the industrial feasibility in a lignocellulose-based biorefinery.

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Section: Sugar Recoveries From Sorghum Stalks and Switchgrassmentioning

confidence: 99%

Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures

Lee¹,

Vadlani²,

Min³

2017

JSBS

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“…For the third method, diazomethane was used as to react with unesterifi ed fatty acids (free fatty acids) in the presence of a little methanol, which catalyzes the reaction to form FAMEs ( 22 ). The FAMEs were trimethylsilylated by adding 50 l of N , O -bis(TMS)-acetamide/pyridine (1:1) and heating at 80°C for 30 min ( 11 ). Two microliter samples of total FAME-TMS derivatives were analyzed on an Agilent 6890N gas chromatograph equipped with a DB-23 column (30 m × 0.25 mm) with 0.25 µm fi lm thickness (J and W Scientifi c).…”

Section: Downloaded Frommentioning

confidence: 99%

“…Two genes encoding CpFAH, a fatty acid hydroxylase we cloned previously from the fungus Claviceps purpurea ( 11 ), and CpDGAT1, a diacylglycerol acyltransferase for the triacylglycerol (TAG) biosynthesis newly cloned from the same species, were coexpressed both in a diploid wild-type strain and in a haploid mutant strain, which resulted in the production of a high amount of ricinoleic acid mainly accumulated in the free fatty acid fraction, followed by the TAG fraction, with very little in the polar lipids. This work highlights the effectiveness of the metabolic engineering strategy and excellent capacity of the yeast for hydroxy fatty acid production.…”

Section: Fatty Acid Analysismentioning

confidence: 99%

“…Both open reading frames (ORFs) of CpFAH and CpDGAT1 were digested from pDM16 ( 11 ) and CpDGAT1/pYES2.0 by Eco RI, respectively, and ligated into pPICZ-B (Invitrogen) digested with the same restriction enzyme to generate plasmids CpFAH/ pPICZ-B and CpDGAT1/pPICZ-B respectively. To coexpress both CpFAH and CpDGAT1 , a whole cassette of CpFAH including AOX1 promoter and terminator was digested with Bgl II and cloned by blunt end ligation into CpDGAT1/pPICZ-B digested with Bam HI.…”

Section: Construction Of Pichia Expression Plasmids Of Cpfah And/or Cmentioning

confidence: 99%

“…In the fi rst method, to analyze total fatty acids including free fatty acid and O -acyl lipids in the cells, acidcatalyzed esterifi cation and transesterifi cation were used as previously described using 1% H 2 SO 4 /methanol ( 11 ). The samples were heated at 80°C for 2 h. In the second method, esterifi ed microorganisms based on fatty acid biosynthesis and assembly frameworks provides a promising solution to bioproducts from microorganisms to replace petrochemicals, as fatty acids and derivatives share many similar chemical properties with petroleum fuels and petrochemicals such as reduced state of hydrocarbons and high density of energy (12)(13)(14)(15)(16).…”

Section: Fatty Acid Analysismentioning

confidence: 99%

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Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance

Meesapyodsuk

Chen

et al. 2015

Journal of Lipid Research

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This article is available online at http://www.jlr.org However, due to the presence of a highly potent toxin (ricin), this native oilseed plant is not considered an ideal source for hydroxy fatty acid production. Therefore, tremendous effort has recently been made in identifying genes involved in the biosynthesis of this fatty acid and using the genes to engineer oilseed crops for producing ricinoleic acid ( 3-7 ). However, to date these attempts have met with only partial success. When a hydroxylase gene from a native plant species was introduced into oilseed crops, the hydroxy fatty acid content in transgenic seeds rarely exceeded 20% of the total fatty acids ( 8-10 ). In 2008, a new hydroxylase for the biosynthesis of ricinoleic acid was identifi ed from a nonplant origin, and expression of this gene in Arabidopsis resulted in accumulation of a slightly higher level of hydroxy fatty acids in transgenic seeds ( 11 ). Very recently, coexpression of genes encoding factors involved in the networks of the hydroxylation or acyl traffi cking process along with a hydroxylase has also been attempted ( 4-7 ). Although the improved production of hydroxy fatty acids is observed, the amount in transgenic seeds still rarely exceeds 25% of the total fatty acids. It appears that selection of genes from diverse sources and addition of cofactors can make a difference for transgenic production of hydroxyl fatty acids, but production of a single hydroxy fatty acid at the commercially viable level in plants still remains as a challenging task.Microorganisms have recently emerged as promising systems for producing biofuel for transportation and platform chemicals for biopolymers because some microbes offer high output of biomass with good oil content in a short period of time and are easily used for genetic manipulation to implement a metabolic engineering strategy. Exploitation of metabolic engineering of lipid pathways in Abstract Ricinoleic acid (12-hydroxyoctadec-cis -9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system ( Pichia pastoris ) to produce ricinoleic acid using a "push" (synthesis) and "pull" (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea , was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the ⌬ 12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic ...

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Identification of a crucial amino acid implicated in the hydroxylation/desaturation ratio of CpFAH12 bifunctional hydroxylase

Robin

Guéroult

Cheikhrouhou

et al. 2019

Biotech & Bioengineering

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Claviceps purpurea bifunctional Δ12‐hydroxylase/desaturase, CpFAH12, and monofunctional desaturase CpFAD2, share 86% of sequence identity. To identify the underlying determinants of the hydroxylation/desaturation specificity, chimeras of these two enzymes were tested for their fatty acid production in an engineered Yarrowia lipolytica strain. It reveals that transmembrane helices are not involved in the hydroxylation/desaturation specificity whereas all cytosolic domains have an impact on it. Especially, replacing the CpFAH12 cytosolic part near the second histidine‐box by the corresponding CpFAD2 part annihilates all hydroxylation activity. Further mutagenesis experiments within this domain identified isoleucine 198 as the crucial element for the hydroxylation activity of CpFAH12. Monofunctional variants performing the only desaturation were obtained when this position was exchanged by the threonine of CpFAD2. Saturation mutagenesis at this position showed modulation in the hydroxylation/desaturation specificity in the different variants. The WT enzyme was demonstrated as the most efficient for ricinoleic acid production and some variants showed a better desaturation activity. A model based on the recently discovered membrane desaturase structures indicate that these changes in specificity are more likely due to modifications in the di‐iron center geometry rather than changes in the substrate binding mode.

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An Oleate Hydroxylase from the Fungus Claviceps purpurea: Cloning, Functional Analysis, and Expression in Arabidopsis

Cited by 43 publications

References 38 publications

Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures

Sustainable Production of Microbial Lipids from Lignocellulosic Biomass Using Oleaginous Yeast Cultures

Metabolic engineering of Pichia pastoris to produce ricinoleic acid, a hydroxy fatty acid of industrial importance

Identification of a crucial amino acid implicated in the hydroxylation/desaturation ratio of CpFAH12 bifunctional hydroxylase

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