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Biotechnological production of methyl branched fatty aldehydes Marco Alexander Fraatz, Michael Goldmann, Thorsten Geissler, Egon Gross, Michael Backes, Jens-Michael Hilmer, Jakob P. Ley, Johanna Rost, Alexander Francke, and Holger Zorn J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04793 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 1, 2016

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Journal of Agricultural and Food Chemistry

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Biotechnological Production of Methyl-Branched Aldehydes

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Marco Alexander Fraatz,† Michael Goldmann,† Torsten Geissler,‡ Egon Gross,‡

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Michael Backes,‡ Jens-Michael Hilmer,‡ Jakob Ley,‡ Johanna Rost,† Alexander

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Francke,§ and Holger Zorn*,†

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†

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Biotechnology, Heinrich-Buff-Ring 17, 35392 Giessen, Germany

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‡

Symrise AG, Muehlenfeldstrasse 1, 37603 Holzminden, Germany

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§

Justus Liebig University Giessen, Institute of Organic Chemistry, Heinrich-Buff-Ring

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17, 35392 Giessen, Germany

Justus Liebig University Giessen, Institute of Food Chemistry and Food

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Corresponding author (Tel: +49 641 99-34900; Fax: +49 641 99-34909; E-mail:

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*

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[email protected])

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1 Environment ACS Paragon Plus


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ABSTRACT: A number of methyl-branched aldehydes impart interesting flavor

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impressions, and especially 12-methyltridecanal is a highly sought after flavoring

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compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For

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the biotechnological production of 12-methyltridecanal, the literature was screened

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for fungi forming iso-fatty acids. Suitable organisms were identified and successfully

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grown in submerged cultures. The culture medium was optimized to increase the

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yields of branched fatty acids. A recombinant carboxylic acid reductase was used to

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reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell

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catalysis was compared to that of the purified enzyme preparation. After lipase

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catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were

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converted to the corresponding aldehydes, including 12-methyltridecanal and 12-

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methyltetradecanal.

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KEYWORDS: biotransformation, carboxylic acid reductase, Conidiobolus

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heterosporus, flavor, 12-methyltridecanal, 12-methyltridecanoic acid

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INTRODUCTION

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Methyl-branched fatty acids (MBFAs) are suitable precursors for the biotechnological

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production of their corresponding aldehydes. Naturally occurring MBFAs are typically

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saturated and mono-methyl-branched. They are common, but usually minor

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constituents of lipids of bacteria and animals, and scarce in higher plants. Their

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major physiological role may be to increase the fluidity of lipids as an alternative to

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unsaturated fatty acids (FAs).1 MBFAs with a methyl branch at the penultimate

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position (isopropyl group) are called iso- and those with a methyl branch at the

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antepenultimate carbon (sec-butyl group) anteiso-fatty acids. For meat and milk of

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ruminants it is assumed that rumen bacteria are the major source of iso- and

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anteiso-FAs.2

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While extensive research has been published on the occurrence and de novo

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synthesis of branched-chain fatty acids (BCFAs) in bacteria,1 only a few studies

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report on fungal sources. Most of them date back to the late 1960s and early/mid-

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1970s and cover Conidiobolus species.3-5 Up to 73% BCFAs of total FAs have been

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reported for these Zygomycota.5 In general, the percentage of BCFAs in

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Basidiomycota is much lower.6 For Armillaria mellea, ~10% of BCFAs have been

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reported.7

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Some methyl-branched aldehydes impart interesting flavor impressions.12-

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Methyltridecanal (1) (Figure 1) especially is a highly sought after flavoring compound

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for savory foods. Its smell is reminiscent of cooked beef and tallow.8 By aroma

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extract dilution analysis (AEDA), stable isotope dilution analysis (SIDA),

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determination of the corresponding odor thresholds, and calculation of odor activity

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values (OAVs), 1 was found to be the most important contributor to the flavor of

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stewed beef.8 Additionally, it was identified as one of the character impact



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compounds of stewed beef juice odor.9 1 is also present in lower concentrations in

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other animal meat, but primarily in ruminants. For beef, concentrations of 55–150

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µg/g lipid were quantitated.8 While raw beef does not exhibit the typical smell of 1,

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the methyl-branched aldehyde is released during cooking and roasting. It is assumed

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that 1 is synthesized in the ruminants’ stomachs by bacteria, and small amounts are

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incorporated into the plasmalogens after resorption and transport to the muscular

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tissues.8 This hypothesis is supported by investigations on the occurrence of 1 in

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microorganisms and physiological samples isolated from beef, in which the highest

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amounts of 1 were detected in the bacteria isolated from the rumen of bovine

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animals (475 µg/g air-dry matter) followed by protozoa (229 µg/g).10 The

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concentration of 1 in beef is correlated to the animal’s phospholipid content and

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increases with its age. Up to 810 µg/g phospholipid were reported after SIDA.11

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Additionally, the formation is effected by the feed. Pasture-fed bulls showed higher

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concentrations compared to concentrate-fed animals.12 Nevertheless, the

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concentrations of 1 in natural sources are generally very low. Even its chemical

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synthesis is comparatively complex and therefore not economic or uses

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environmental unfriendly chemicals.8,13 Albeit more convenient strategies have been

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published recently, chemically synthesized 1 may not marketed as natural.14

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Therefore, to the best of our knowledge, no natural 1 is available as a flavoring

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substance on the world market, and no biotechnological route towards 1 has been

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commercialized so far.

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For the reduction of MBFAs to their corresponding branched aldehydes, enzymes

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with carboxylic acid reduction ability are required.15 Carboxylic acid reductases

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(CAR) are oxidoreductases, which catalyze the reduction of aromatic and short-chain

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carboxylic acids.16 Recently, a CAR from Mycobacterium marinum was described


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with the ability to reduce aliphatic fatty acids up to C18.17 Concerning MBFAs, the

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reduction of 3-methylbutyrate and 3-methylpentanoate has been reported for a CAR

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from Clostridium thermoaceticum.18 No data on the reduction of longer MBFAs have

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been published so far.

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MATERIALS AND METHODS

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Chemicals. Adenosine-5'-triphosphate disodium salt (ATP), agar-agar (Kobe I), D-

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glucose, n-hexane, L-leucine, 2-mercaptoethanol, methanol, β-nicotinamide adenine

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dinucleotide phosphate tetrasodium salt (NADPH), potassium hydrogen sulfate,

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sodium chloride, and sodium sulfate were purchased from Carl Roth GmbH + Co.

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KG (Karlsruhe, Germany). tert-Butyl methyl ether (TBME), dichloromethane, diethyl

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ether, lipase from Candida rugosa (2.9 U/mg), magnesium chloride hexahydrate,

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page ruler unstained protein ladder (10–200 kDa), and yeast extract were bought

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from Fisher Scientific GmbH (Schwerte, Germany). Lithium aluminum hydride

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(LiAlH4) (for synthesis) was purchased from Merck KGaA (Darmstadt, Germany).

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Malt extract, petroleum ether (40–60 °C), and Supelco 37 component FAME mix

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were purchased from Sigma-Aldrich GmbH (Taufkirchen, Germany). 11-

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Methyltridecanoic acid, 12-methyltridecanal (1), 12-methyltridecanoic acid (2) (Figure

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1), 12-methyltetradecanal, 12-methyltetradecanoic acid, 13-methyltetradecanoic

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acid, 13-methylpentadecanoic acid, and 14-methylpentadecanoic acid were provided

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by Symrise AG (Holzminden, Germany). Boron trifluoride (20% in methanol) and

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hydrochloric acid (32%) were obtained from VWR International GmbH (Darmstadt,

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Germany).

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Microorganisms. The basidiomycetous fungus Armillaria mellea DSMZ 2941

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(honey fungus) was obtained from the German Collection of Microorganisms and



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Cell Cultures (Brunswick, Germany). The fungi Conidiobolus denaeosporus CBS

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137.57, Conidiobolus heterosporus CBS 543.63, C. heterosporus CBS 333.74, and

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C. heterosporus CBS 138.57 (ATCC 12941) were obtained from CBS-KNAW Fungal

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Biodiversity Centre (Utrecht, the Netherlands). Conidiobolus lobatus ATCC 18153

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was purchased from the American Type Culture Collection (Manassas, VA).

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Escherichia coli BL21(DE3) was purchased from Merck KGaA (Darmstadt,

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Germany). The E. coli strain BL21(DE3)pET-PC2, which is able to co-express a

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carboxylic acid reductase from Mycobacterium marinum and a phosphopantetheinyl

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transferase (PPT) of Bacillus subtilis was provided by Pauli Kallio, University of

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Turku, Finland and Patrick R. Jones, Imperial College London, England.

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Cultivation. Stock cultures and precultures were maintained and grown as described

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previously.19 Yeast extract (3 g/L) and malt extract (30 g/L) were used for stock

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cultures and preculture media (YM medium). Growth periods on agar plates were: 5

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days for C. heterosporus CBS 543.63 and CBS 333.74, 14 days for CBS 138.57, C.

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denaeosporus, and C. lobatus, and 21 days for A. mellea. The standard cultivation

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period of the precultures was 3 days, and 100 mL YM medium in 250 mL Erlenmeyer

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flasks was used. Afterwards, the precultures were homogenized for 30 s at 9800 rpm

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using an Ultra Turrax homogenizer (IKA, Staufen, Germany). For the main cultures,

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20 mL of the homogenized suspension was used to inoculate 200 mL YM medium in

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500 mL Erlenmeyer flasks. The cultivations were carried out in an Ecotron incubation

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shaker (25 mm shaking diameter, 150 rpm, 24 °C) (Infors GmbH, Einsbach,

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Germany) for 5 d (if not stated otherwise). Supplementation with L-valine was done

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by adding 0.2 g/L or 2.0 g/L to the main culture medium prior to autoclaving. For

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harvesting, the fungal mycelium was separated from the culture broth by vacuum


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filtration with a Buchner funnel (110 mm) and DP 595 cellulose filter paper (Albet

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LabScience, Dassel, Germany).

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Lipid Extraction. The harvested biomass was treated with 200 mL of 4 M

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hydrochloric acid with boiling for 30 min. After addition of 100 mL hot water, filtration

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of the hydrolysis solution and neutralization, the residue was dried in a drying cabinet

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at 103 °C for 3 h. Subsequently, Soxhlet extraction was performed with 180 mL

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petroleum ether for 4 h. The solvent was evaporated by means of a rotary

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evaporator (40 °C, ≤150 mbar), and the lipid extract was dried in the drying cabinet

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at 103 °C to constant weight. After addition of 2 mL n-hexane, the lipid extract was

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stored at -20 °C until saponification and esterification.

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Preparation of fatty acid methyl esters (FAMEs). 150 mg fungal lipid extract was

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saponified in a derivatization vial with a Teflon septum by adding 4 mL 0.5 M

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methanolic sodium hydroxide solution and heating to 80 °C for 10 min. After cooling

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to room temperature, 3.5 mL of boron trifluoride (20% in methanol) was added. The

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mixture was firmly shaken and incubated for 5 min (80 °C). After cooling, 5 mL n-

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hexane was added, the mixture was heated for 1 min (80 °C), and 5 mL saturated

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sodium chloride solution was added. The organic phase was separated and dried

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over anhydrous sodium sulfate. Reference standards were directly esterified by

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addition of 1 mg fatty acid to 1 mL boron trifluoride solution. The generated FAMEs

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were analyzed by means of gas chromatography (GC).

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Protein expression. Overnight cultures were grown according to Akhtar et al. in Luria-

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Bertani (LB) medium.17 100 µL of these cultures was used for inoculation of 200 mL

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Overnight Express Instant TB Medium (Merck KGaA). Cells were incubated for 20 h

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in an incubation shaker (30 °C, 250 rpm, 25 mm shaking diameter), then separated



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by centrifugation (4 °C, 4713 x g) and stored at -20 °C. After thawing on ice, the cell

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pellets were employed for enzyme purification or biotransformation.

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Enzyme purification. The His-tagged protein was purified according to the Protino Ni-

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TED protocol (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) using 2 mg/mL

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lysozyme.17 The enzyme containing fractions were combined and 2-mercaptoethanol

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(3%, v/v) was added. 10 mL aliquots were washed seven times with phosphate

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buffer (50 mM, pH 7.5) using 30 K Macrosep Advance Centrifugal Devices (Pall Life

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Sciences, Port Washington, NY) by means of centrifugation (4 °C, 4713 x g).

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Determination of protein concentration. The protein concentration was measured by

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means of a Roti Nanoquant-Kit K880 (Carl Roth GmbH + Co. KG) according to the

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manufacturer’s protocol. The purified CAR enzyme solution was diluted with 50 mM

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phosphate buffer (pH 7.5) to a final protein concentration of 100 µg/mL and used for

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in vitro bioconversion experiments.

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and

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Coomassie staining. Discontinuous SDS-PAGE was performed according to

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Laemmli.20 The gels were stained overnight at room temperature with Coomassie

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brilliant blue G250 solution.21

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Biotransformation – whole cell catalysis. Twenty-five mg cells were suspended in 1

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mL phosphate buffer (100 mM, pH 7.5) in a 10 mL reaction vial and 0.5 mM 2 (in n-

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hexane) and 100 mM D-glucose were added. If not stated otherwise, the vials were

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incubated for 24 h in an incubation shaker at 30 °C and 250 rpm. Prior to solvent

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extraction with 1 mL dichloromethane, the pH was adjusted to pH 2 with hydrochloric

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acid (18%, w/w). As controls, analogous transformations with BL21(DE3) E. coli

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cultures and an empty plasmid were performed with 0.25 mM 1 and 0.5 mM 2 as

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substrates. For additional negative controls, 25 mg E. coli BL21(DE3)pET-PC2 cell


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pellets were heated for 30 min at 95 °C in a water bath prior to the addition of the

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substrate and D-glucose.

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In vitro conversion with purified enzymes. For the conversion of 2, ATP (1 mM),

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NADPH (1 mM), MgCl2 (10 mM), and 2 (0.5 mM) were added to 0.9 mL CAR-

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solution (100 µg/mL in 50 mM phosphate buffer, pH 7.5) in a total reaction volume of

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1011 µL. If not stated otherwise, the reaction mixture was incubated for 24 h at 30 °C

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and subsequently extracted as described above. Alternatively, 150 mg lipid extract of

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C. heterosporus CBS 333.74 was suspended in phosphate buffer containing ATP (1

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mM), NADPH (1 mM), MgCl2 (10 mM), 2 (0.5 mM), 0.9 mL CAR-solution (100 µg/mL

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in 50 mM phosphate buffer, pH 7.5), and 200 µL lipase solution (Candida rugosa; 12

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mg/mL, 7 U in phosphate buffer) was added. The final volume of the reaction mixture

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was 1210 µL. The mixture was incubated for 2 h at 30 °C and subsequently

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extracted as described above. For negative controls, the CAR solutions were heated

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for 30 min at 95 °C prior to the reaction. In additional controls, the enzyme solution

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was substituted by the same volume of 50 mM phosphate buffer.

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Gas chromatography. GC analyses were performed according to Kleofas et al.22 For

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compound identification, GC-mass spectrometry (MS) analyses on two columns of

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different polarity and calculation of the corresponding retention indices (RIs) were

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performed. For quantitation, response factors were determined by means of a GC

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equipped with a flame ionization detector (FID). In general, 1 µL sample was injected

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with a split of 10:1 for GC-MS and 2:1 for GC-FID. The RIs of the methyl esters of

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the MBFAs were as follows: methyl 11-methyltridecanoate 1977 (HP-Innowax) and

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1761 (DB-5), methyl 12-methyltridecanoate 1962 and 1754, methyl 12-

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methyltetradecanoate 2080 and 1865, methyl 13-methyltetradecanoate 2065 and



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1857, methyl 13-methylpentadecanoate 2210 and 1969, and methyl 14-

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methylpentadecanoate 2196 and 1961.

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Synthesis of 12-methyltridecanol. For the synthesis of 12-methyltridecanol (3)

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(Figure 1) 2.00 g 2 dissolved in 10 mL diethyl ether was added dropwise to 665 mg

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of LiAlH4 in 20 mL diethyl ether, and the mixture was stirred overnight. The reaction

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was stopped by addition of ice-water and the crude product was extracted with 50

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mL diethyl ether. After drying with sodium sulfate and removal of the solvent by

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distillation, the crude product was purified by column-chromatography (MTBE:n-

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hexane 1:3, v/v).

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1.68 g (90%) of a clear oil was isolated. EI-MS: 69 (100), 55 (100), 43 (90), 41 (88),

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83 (78), 97 (54), 111 (26), 140 (10), 168 (7), 125 (6); RI: 2118 (HP-Innowax), 1647

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(DB-5)

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Statistics. Analyses were performed at least in duplicate, and results are reported as

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means ± half range or standard deviations, respectively.

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RESULTS AND DISCUSSION

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Production of methyl-branched fatty acids. After screening of the literature for

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MBFAs-producing fungi, six different fungi were chosen for further investigations.

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Five fungi were Conidiobolus species, three of them C. heterosporus strains from

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different origins. All of them grew well in submerged cultures, and yeast extract / malt

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extract medium was found to be an appropriate standard medium. After harvesting

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and lipid extraction, the fatty acid profiles of the fungi were analyzed. BCFAs were

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detected in all lipid samples, but in varying quantities (Table 1). The C. heterosporus

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strains showed the highest share of iso- and anteiso-FAs. More than 55% of MBFAs

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were detected in the lipid extracts of C. heterosporus CBS 543.63. In the literature,


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~40% 2 have been reported for C. heterosporus ATCC 12941 (CBS 138.57) and for

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Conidiobolus lobatus.4,5 In our studies, up to 40% 2 was observed with C.

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heterosporus CBS 543.63 and CBS 333.74, while C. heterosporus CBS 138.57 and

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C. lobatus only formed ~ 20% 2. The observed differences might be related to

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different media used or culture parameters applied.

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According to Řezanka and Mareš the basidiomycetous fungus Armillaria mellea

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(honey fungus) contains ~10% of BCFA, including small amounts of the odd-

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numbered iso-FA 13-methyltetradecanoic acid (1.8%).7 Odd-numbered iso-FAs have

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not been reported for other fungal species. Therefore, the fatty acid profile of A.

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mellea was analyzed as well. However, all identified iso-FAs were even-carbon-

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numbered while the anteiso-FAs were odd-carbon-numbered (Table 1). For the

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Conidiobolus species, this was in accordance with Tyrrell and Weatherston.5

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C. heterosporus CBS 333.74 was chosen for all further experiments due to its overall

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better ability to be cultivated in liquid media compared to other C. heterosporus

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strains. Its growth in submerged cultures was investigated over a period of two

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weeks. Both, the biomass and the lipid yield increased steadily over time (Figure 2).

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The lipid content of the dry biomass was 25–30%. The amounts of saturated fatty

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acids decreased until day 14 by about 10%, whereas the share of unsaturated fatty

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acids increased slightly (data not shown). On day 14, the highest levels of 2 were

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observed.

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For certain bacteria and some Conidiobolus species it is known that supplementation

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of the culture medium with the amino acid L-valine may increase the formation of

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even numbered iso-FAs, i.e. 2 or 14-methylpentadecanoic acid.5,23,24 Based on these

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observations, the culture media of C. heterosporus CBS 333.74 were supplemented

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with 0.2 g/L and 2.0 g/L L-valine, respectively. For 2, an increase of 10% (0.2 g/L)



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and 38% (2.0 g/L) was observed. For 14-methylpentadecanoic acid, the increase

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was less pronounced with 4% and 10%.

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Biotransformation to methyl branched aldehydes. Initially, several enzymes were

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considered for the reduction of MBFAs to their corresponding aldehydes. Various

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enzymes have been described to reduce aromatic carboxylic acids. The well

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investigated CAR from Nocardia species are known especially to reduce acids with

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aromatic residues, but successful reduction of aliphatic acids has not been reported

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for them yet.25,26 However, for the CAR from M. marinum, the reduction of aliphatic

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fatty acids, with a range from C8–C18, was published recently.17 Because of the

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structural similarities, the CAR from M. marinum was chosen for the

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biotransformation of MBFAs. The CAR was heterologously expressed and purified to

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electrophoretic homogeneity. The molecular weight of the CAR was determined to

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be 127 kDa (Figure 3) which was in agreement with the results of Akhtar et al.17

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For whole cell biotransformation, 2 was incubated for 24 h with different amounts of

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E. coli cell pellets expressing CAR. The formation of up to 28.0 ± 1.8 mol% 3 was

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observed, but no 1 was detected. To further elucidate the formation of 1, shorter

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biotransformation periods were checked. Here, 1 was identified, with the highest

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amounts (3.1 ± 0.6 mol%) after 2 h (Figure 4A). The concentration of 3 steadily

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increased over the first 6 h (20 ± 3.4 mol%). In contrast, in none of the negative

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controls (without CAR, with heat inactivated CAR, with BL21(DE3) and empty

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plasmid) a reduction of 2 to 1 or 3 was detected. On the other hand, the E. coli strain

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BL21(DE3) with empty plasmid converted 1 to 3 (data not shown).

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To prevent the accumulation of 3 and thus to increase the amounts of 1, the in vitro

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conversion of 2 was further investigated with the purified CAR enzyme. Therefore,

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the recombinant polyhistidine-tagged CAR was purified by means of immobilized


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metal ion affinity chromatography, and 2 was incubated with the isolated CAR in the

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presence of the required cofactors for 2–8 h. After 2 h, 22.1 ± 0.53 mol% 1 were

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detected, and the highest conversion yield was found with 28 ± 7.8 mol% after 8 h

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(Figure 4B). In all samples, only trace amounts of 3 were determined. This led us to

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the conclusion, that the CAR is essential for the initial reduction of 2 to 1, whereas

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native E. coli enzymes (most probably alcohol dehydrogenases) are responsible for

285

the reduction of the formed aldehyde to its corresponding alcohol.

286

Next, the fatty acids produced by C. heterosporus CBS 333.74 were directly

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employed. In addition to the CAR, a lipase from Candida rugosa was added to the

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reaction mixture to release esterified fatty acids. Besides 12-methyltridecanal and

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12-methyltetradecanal, several linear aldehydes, like dodecanal, tridecanal,

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tetradecanal, pentadecanal, and hexadecanal were generated (Figure 5).

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In conclusion, the enzymatic reduction of methyl-branched fatty acids to their

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corresponding aldehydes has been shown in this study for the first time. 2 was

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converted enzymatically to the highly sought after flavor compound 12-

294

methyltridecanal. Even though the initial concentrations obtained with the

295

investigated one-pot reaction were rather low, the general feasibility was clearly

296

shown. Future studies will concentrate on aspects of increasing the yields of 1 by

297

direct enzymatic conversion of fungal lipids.

298 299

ABBREVIATIONS

300

AEDA, aroma extract dilution analysis; BCFA, branched-chain fatty acid; MBFA,

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methyl-branched fatty acid; OAV, odor activity value; PPT, phosphopantetheinyl

302

transferase; SIDA, stable isotope dilution analysis; YM, yeast extract-malt extract.

303



304

ACKNOWLEDGMENT

305

We are very grateful to Pauli Kallio, University of Turku, and Patrik R. Jones,

306

Imperial College London, who generously provided the BL21(DE3)pET-PC2 E. coli

307

strain.

308 309

ASSOCIATED CONTENT

310

Supporting Information

311

GC-MS chromatogram of a C. denaeosporus lipid extract after transesterification to

312

the corresponding fatty acid methyl esters and mass spectrum of methyl 12-

313

methyltridecanoate identified in the C. denaeosporus lipid extract. This material is

314

available free of charge via the Internet at http://pubs.acs.org.

315 316

Funding

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The authors MAF and HZ are grateful for financial support by the excellence initiative

318

of the Hessian Ministry of Science and Art which encompasses a generous grant for

319

the LOEWE Center for Insect Biotechnology & Bioresources.

320 321

Notes

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The authors declare no competing financial interest. Torsten Geissler, Egon Gross,

323

Jens-Michael Hilmer, and Jakob Ley are members of Symrise AG holding intellectual

324

property on 12-methyltridecanal.

325 326 327 328

REFERENCES (1)

Kaneda, T. Iso- and anteiso-fatty acids in bacteria: Biosynthesis, function,

and taxonomic significance. Microbiol. Rev. 1991, 55, 288–302.


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(2)

Vlaeminck, B.; Fievez, V.; Cabrita, A. R. J.; Fonseca, A. J. M.; Dewhurst, R.

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J. Factors affecting odd- and branched-chain fatty acids in milk: A review. Anim.

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Feed Sci. Technol. 2006, 131, 389–417.

332

(3)

Tyrrell, D. The fatty acid composition of some Entomophthoraceae. II. The

333

occurrence of branched-chain fatty acids in Conidiobolus denaesporus Drechsl.

334

Lipids 1968, 3, 368–372.

335 336 337

(4)

Tyrrell, D. The fatty acid composition of some Entomophthoraceae. III. Can.

J. Microbiol. 1971, 17, 1115–1118. (5)

Tyrrell, D.; Weatherston, J. The fatty acid composition of some

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403



404


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405

FIGURE CAPTIONS

406

Figure 1. Structures of 12-methyltridecanal (1), 12-methyltridecanoic acid (2), and

407

12-methyltridecanol (3).

408 409

Figure 2. Dry biomass, lipids, and relative lipid content of C. heterosporus CBS

410

333.74 grown in submerged cultures over a cultivation period of 14 d.

411 412

Figure 3. SDS-PAGE of the CAR enzyme preparation after heterologous expression

413

and purification according to the procedure described by Akhtar et al.;17 left lane

414

shows a molecular mass marker (kDa), right lane shows the isolated CAR.

415 416

Figure 4. Kinetics of the whole cell catalyzed reduction of 2 with CAR from M.

417

marinum co-expressed with PPT in E. coli for 1–6 h (A) and by the recombinant

418

CAR, purified by means of immobilized metal ion affinity chromatography, for 2–8 h

419

(B).

420 421

Figure 5. Section of a GC-MS chromatogram (total icon current, scan mode, m/z 33–

422

300) recorded after injection of an organic solvent extract of a reaction mixture of the

423

enzymatic hydrolysis of a fungal lipid and reduction of the thereby generated free

424

fatty acids by CAR.

425



Page 20 of 27

TABLES Table 1. Relative Fatty Acid (FA) Compositions (%) of A. mellea, C. denaeosporus, C. heterosporus CBS 543.63, CBS 333.74, CBS 138.57, and C. lobatus (CLO) after Lipid Extraction and Transesterification to the Corresponding Fatty Acid Methyl Esters.

fatty acid

C.

C. heterosporus

C. heterosporus

C. heterosporus

denaeosporus

CBS 543.63

CBS 333.74

CBS 138.57

C. lobatus

A. mellea

a14:0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

i14:0

5.0

8.6

40.1

34.5

20.1

18.8

a15:0

1.4

11.1

13.6

12.0

12.7

7.8

i15:0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

a16:0

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

i16:0

0.7

0.4

4.2

7.0

2.1

2.1

∑ unassigned FAs

28.3

28.6

19.6

21.4

7.7

7.8

∑ saturated FAs*

31.2

48.3

21.1

20.7

47.6

44.0

∑ unsaturated

33.4

3.0

1.4

4.5

9.8

19.5

20 ACS Paragon Plus Environment

Page 21 of 27


FAs

a14:0 11-methyltridanoic acid, i14:0 12-methyltridanoic acid, a:15:0 12-methyltetradecanoic acid, i15:0 13-methyltetradecanoic acid, a16:0 13-methylpentadecanoic acid, i16:0 14-methylpentadecanoic acid, n.d. not detected, * excluding listed methyl-branched fatty acids



FIGURES

Figure 1


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12

30

10

25

8

20

6

15

4

10

2

5

0

0 2

4

6

8

10

cultivation time [d] dry biomass

lipids

lipids / dry biomass


12

14

lipids / dry biomass [%]

dry biomass & lipids [g L-1]

Figure 2


Figure 3


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Figure 4



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tetradecanal

tridecanal

150000

dodecanal

abundance

200000

100000

50000

hexadecanal

250000

pentadecanal

300000

12-methyltetradecanal

12-methyltridecanal

Figure 5

0 30

31

32

33

34

35

36

37

38

39

40

41

[min]


42

43

44

45

46

Page 27 of 27


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