Identification of fungal limonene-3-hydroxylase for biotechnological menthol production

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as medical component. So far, only extraction from plant material or chemical synthesis is possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low, if compared to the highly selective L3H enzymes from the biosynthetic pathway towards menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far, was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered further fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum. Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively. Importance (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi, which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.

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Identification of fungal limonene-3-hydroxylases for biotechnological menthol production

10.1101/2020.11.25.399378 ◽

2020 ◽

Author(s):

Florence M. Schempp ◽

Ingmar Strobel ◽

Maria M. W. Etschmann ◽

Elena Bierwirth ◽

Johannes Panten ◽

...

Keyword(s):

Heterologous Gene Expression ◽

Cytochrome P450 Monooxygenases ◽

Medicinal Uses ◽

P450 Monooxygenases ◽

Naturally Occurring ◽

Alternative Approach ◽

Fragrance Compound ◽

Encoding Genes ◽

Medical Component

AbstractMore than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as medical component. So far, only extraction from plant material or chemical synthesis is possible. A sustainable alternative approach for menthol production could be a biotechnological-chemical two-step conversion, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low, if compared to the highly selective L3H enzymes from the biosynthetic pathway towards menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far, was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa.We have discovered further fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum. Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production and its additional ability to convert α-and β-pinene to verbenol and pinocarveol, respectively.Importance(−)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi, which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (−)-menthol production from renewable (+)-limonene.

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Characterization of cytochrome P450-monooxygenases of Chinese hamsters with respect to aflatoxin B1 activation

Toxicology ◽

10.1016/0300-483x(94)90076-0 ◽

1994 ◽

Vol 93 (2-3) ◽

pp. 165-173 ◽

Cited By ~ 8

Author(s):

Morio Fukuhara ◽

Eric Antignac ◽

Naomi Fukusen ◽

Kazue Kato ◽

Masanobu Kimura

Keyword(s):

Cytochrome P450 ◽

Aflatoxin B1 ◽

Cytochrome P450 Monooxygenases ◽

P450 Monooxygenases ◽

Chinese Hamsters

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Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

Archives of Microbiology ◽

10.1007/s00203-011-0753-2 ◽

2011 ◽

Vol 194 (4) ◽

pp. 243-253 ◽

Cited By ~ 50

Author(s):

Masamichi Ide ◽

Hirofumi Ichinose ◽

Hiroyuki Wariishi

Keyword(s):

Cytochrome P450 ◽

Molecular Identification ◽

Functional Characterization ◽

Brown Rot ◽

Cytochrome P450 Monooxygenases ◽

Postia Placenta ◽

P450 Monooxygenases

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Construction and Application of a Functional Library of Cytochrome P450 Monooxygenases from the Filamentous Fungus Aspergillus oryzae

Applied and Environmental Microbiology ◽

10.1128/aem.02491-10 ◽

2011 ◽

Vol 77 (9) ◽

pp. 3147-3150 ◽

Cited By ~ 21

Author(s):

K. H. M. Nazmul Hussain Nazir ◽

Hirofumi Ichinose ◽

Hiroyuki Wariishi

Keyword(s):

Cytochrome P450 ◽

Aspergillus Oryzae ◽

Cytochrome P450 Monooxygenases ◽

Functional Screening ◽

Content Type ◽

P450 Monooxygenases ◽

Rapid Characterization ◽

Catalytic Functions ◽

First Time

ABSTRACTA functional library of cytochrome P450 monooxygenases fromAspergillus oryzae(AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase inSaccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

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Characterization of Maize Cytochrome P450 Monooxygenases Induced in Response to Safeners and Bacterial Pathogens

PLANT PHYSIOLOGY ◽

10.1104/pp.125.2.1126 ◽

2001 ◽

Vol 125 (2) ◽

pp. 1126-1138 ◽

Cited By ~ 46

Author(s):

Michael W. Persans ◽

Jian Wang ◽

Mary A. Schuler

Keyword(s):

Cytochrome P450 ◽

Bacterial Pathogens ◽

Cytochrome P450 Monooxygenases ◽

P450 Monooxygenases

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Cloning, Expression and Characterization of a Monooxygenase P450BM3 from Bacillus megaterium ALA2

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.518-523.5533 ◽

2012 ◽

Vol 518-523 ◽

pp. 5533-5538

Author(s):

Ting Wang ◽

Liang Liang Wang ◽

Xun Li

Keyword(s):

Escherichia Coli ◽

Cytochrome P450 ◽

Linoleic Acid ◽

Bacillus Megaterium ◽

Cytochrome P450 Monooxygenases ◽

P450 Monooxygenases ◽

Nadph Oxidation ◽

First Time ◽

Optimal Ph

Cytochrome P450 monooxygenases are enzymes which are capable of oxidising saturated and unsaturated substrates. P450BM3 from Bacillus megaterium is one of this family. For the ﬁrst time, the cyp gene for coding P450BM3 from B. megaterium ALA2 has been cloned and expressed in Escherichia coli. The recombinant enzyme is 120 kDa, containing 1049 aa. The highest activity of purified enzyme is 14.8 U/mg towards palmitic acid by monitoring the NADPH oxidation. The optimal pH and temperature were 9.0 and 40°C. The enzyme has higher activity towards linoleic acid, and 2-Methyl-7-octadecene can also be catalyzed which is a precursor of displar.

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Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

Applied and Environmental Microbiology ◽

10.1128/aem.71.11.6968-6976.2005 ◽

2005 ◽

Vol 71 (11) ◽

pp. 6968-6976 ◽

Cited By ~ 22

Author(s):

Volker Jungmann ◽

István Molnár ◽

Philip E. Hammer ◽

D. Steven Hill ◽

Ross Zirkle ◽

...

Keyword(s):

Escherichia Coli ◽

Cytochrome P450 ◽

Recombinant Proteins ◽

Oxidation Reaction ◽

Cytochrome P450 Monooxygenases ◽

Enzyme Kinetic ◽

Bacterial Strains ◽

P450 Monooxygenase ◽

P450 Monooxygenases

ABSTRACT 4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined.

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Comparative Analyses of Cytochrome P450s and Those Associated with Secondary Metabolism in Bacillus Species

International Journal of Molecular Sciences ◽

10.3390/ijms19113623 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3623 ◽

Cited By ~ 9

Author(s):

Bongumusa Mthethwa ◽

Wanping Chen ◽

Mathula Ngwenya ◽

Abidemi Kappo ◽

Puleng Syed ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

In Silico Analysis ◽

Gene Clusters ◽

Bacillus Species ◽

Cytochrome P450 Monooxygenases ◽

Enzymatic Reactions ◽

P450 Monooxygenases ◽

Stereo Selectivity

Cytochrome P450 monooxygenases (CYPs/P450s) are among the most catalytically-diverse enzymes, capable of performing enzymatic reactions with chemo-, regio-, and stereo-selectivity. Our understanding of P450s’ role in secondary metabolite biosynthesis is becoming broader. Among bacteria, Bacillus species are known to produce secondary metabolites, and recent studies have revealed the presence of secondary metabolite biosynthetic gene clusters (BGCs) in these species. However, a comprehensive comparative analysis of P450s and P450s involved in the synthesis of secondary metabolites in Bacillus species has not been reported. This study intends to address these two research gaps. In silico analysis of P450s in 128 Bacillus species revealed the presence of 507 P450s that can be grouped into 13 P450 families and 28 subfamilies. No P450 family was found to be conserved in Bacillus species. Bacillus species were found to have lower numbers of P450s, P450 families and subfamilies, and a lower P450 diversity percentage compared to mycobacterial species. This study revealed that a large number of P450s (112 P450s) are part of different secondary metabolite BGCs, and also identified an association between a specific P450 family and secondary metabolite BGCs in Bacillus species. This study opened new vistas for further characterization of secondary metabolite BGCs, especially P450s in Bacillus species.

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