scholarly journals Cloning and Characterization of Three Fatty Alcohol Oxidase Genes from Candida tropicalis Strain ATCC 20336

2004 ◽  
Vol 70 (8) ◽  
pp. 4872-4879 ◽  
Author(s):  
L. Dudley Eirich ◽  
David L. Craft ◽  
Lisa Steinberg ◽  
Afreen Asif ◽  
William H. Eschenfeldt ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Fatty Alcohol ◽  
Candida Tropicalis ◽  
Sequence Homology ◽  
Alcohol Oxidase ◽  
Dicarboxylic Acids ◽  
Hydroxy Fatty Acids ◽  
E Coli

ABSTRACT Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of ω-hydroxy fatty acids to ω-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K m values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes ω-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not ω-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K m values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.

scholarly journals A newly identified fatty alcohol oxidase gene is mainly responsible for the oxidation of long-chain ω-hydroxy fatty acids inYarrowia lipolytica

2014 ◽  
Vol 14 (6) ◽  
pp. 858-872 ◽  
Author(s):  
Michael Gatter ◽  
André Förster ◽  
Kati Bär ◽  
Miriam Winter ◽  
Christina Otto ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Alcohol ◽  
Alcohol Oxidase ◽  
Hydroxy Fatty Acids ◽  
Oxidase Gene ◽  
Long Chain

scholarly journals Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

2003 ◽  
Vol 69 (10) ◽  
pp. 5992-5999 ◽  
Author(s):  
William H. Eschenfeldt ◽  
Yeyan Zhang ◽  
Hend Samaha ◽  
Lucy Stols ◽  
L. Dudley Eirich ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Cytochrome P450 ◽  
Oleic Acid ◽  
Candida Tropicalis ◽  
Saturated Fatty Acids ◽  
Dicarboxylic Acids ◽  
Initial Step ◽  
Cytochrome P450s ◽  
Commercial Development ◽  
P450 Monooxygenase

ABSTRACT Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development.

scholarly journals Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

1971 ◽  
Vol 124 (5) ◽  
pp. 905-913 ◽  
Author(s):  
R. V. Krishna ◽  
P. R. Krishnaswamy ◽  
D. Rajagopal Rao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
E Coli ◽  
Enzymic Synthesis ◽  
Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

Dicarboxylic acids and hydroxy fatty acids in different species of fungi

2016 ◽  
Vol 71 (5) ◽  
pp. 999-1005
Author(s):  
Aleksandra Ostachowska ◽  
Piotr Stepnowski ◽  
Marek Gołębiowski
Keyword(s):  
Fatty Acids ◽  
Dicarboxylic Acids ◽  
Hydroxy Fatty Acids

EngineeringEscherichia colifor Conversion of Glucose to Medium-Chain ω-Hydroxy Fatty Acids and α,ω-Dicarboxylic Acids

2015 ◽  
Vol 5 (3) ◽  
pp. 200-206 ◽  
Author(s):  
Christopher H. Bowen ◽  
Jeff Bonin ◽  
Anna Kogler ◽  
Carlos Barba-Ostria ◽  
Fuzhong Zhang
Keyword(s):  
Fatty Acids ◽  
Dicarboxylic Acids ◽  
Hydroxy Fatty Acids ◽  
Medium Chain

scholarly journals Production of long-chain hydroxy fatty acids by Starmerella bombicola

2019 ◽  
Vol 19 (7) ◽  
Author(s):  
Marilyn De Graeve ◽  
Isabelle Van de Velde ◽  
Lien Saey ◽  
Maarten Chys ◽  
Hanne Oorts ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Dicarboxylic Acids ◽  
Strain Engineering ◽  
Microbial Production ◽  
Hydroxy Fatty Acids ◽  
Flash Chromatography ◽  
Starmerella Bombicola ◽  
Production Platform ◽  
The One ◽  
Fatty Aldehydes

ABSTRACT To decrease our dependency for the diminishing source of fossils resources, bio-based alternatives are being explored for the synthesis of commodity and high-value molecules. One example in this ecological initiative is the microbial production of the biosurfactant sophorolipids by the yeast Starmerella bombicola. Sophorolipids are surface-active molecules mainly used as household and laundry detergents. Because S. bombicola is able to produce high titers of sophorolipids, the yeast is also used to increase the portfolio of lipophilic compounds through strain engineering. Here, the one-step microbial production of hydroxy fatty acids by S. bombicola was accomplished by the selective blockage of three catabolic pathways through metabolic engineering. Successful production of 17.39 g/l (ω-1) linked hydroxy fatty acids was obtained by the successive blockage of the sophorolipid biosynthesis, the β-oxidation and the ω-oxidation pathways. Minor contamination of dicarboxylic acids and fatty aldehydes were successfully removed using flash chromatography. This way, S. bombicola was further expanded into a flexible production platform of economical relevant compounds in the chemical, food and cosmetic industries.

Substrate specificity and stereoselectivity of fatty alcohol oxidase from the yeast Candida maltosa

1992 ◽  
Vol 37 (1) ◽  
Author(s):  
Stephan Mauersberger ◽  
Hannelore Drechsler ◽  
G�nther Oehme ◽  
Hans-Georg M�ller
Keyword(s):  
Substrate Specificity ◽  
Fatty Alcohol ◽  
Alcohol Oxidase ◽  
Candida Maltosa

scholarly journals Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids

2003 ◽  
Vol 69 (10) ◽  
pp. 5983-5991 ◽  
Author(s):  
David L. Craft ◽  
Krishna M. Madduri ◽  
Mark Eshoo ◽  
C. Ron Wilson
Keyword(s):  
Fatty Acids ◽  
Cytochrome P450 ◽  
Fatty Acid ◽  
Candida Tropicalis ◽  
Strain Improvement ◽  
Dicarboxylic Acids ◽  
Fold Increase ◽  
Isolation And Characterization ◽  
Unique Genes

ABSTRACT Candida tropicalis ATCC 20336 excretes α,ω-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a β-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the ω-hydroxylase enzymes catalyzing the first step in the ω-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of ω-oxidation of n-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52A13 and CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52A17 and CYP52A18 was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was observed. These results identify the CYP52 and NCP genes as being involved in α,ω-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement.

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