Biotechnological Production of Methyl-Branched Aldehydes

Article abstract of DOI:10.1021/acs.jafc.6b04793

A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.

Full text of DOI:10.1021/acs.jafc.6b04793

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Article
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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Biotechnological Production of MethylꢀBranched Aldehydes
Marco Alexander Fraatz,† Michael Goldmann,† Torsten Geissler,‡ Egon Gross,‡
Michael Backes,‡ JensꢀMichael Hilmer,‡ Jakob Ley,‡ Johanna Rost,† Alexander
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Francke,§ and Holger Zorn ,
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† Justus Liebig University Giessen, Institute of Food Chemistry and Food
Biotechnology, HeinrichꢀBuffꢀRing 17, 35392 Giessen, Germany
‡ Symrise AG, Muehlenfeldstrasse 1, 37603 Holzminden, Germany
§ Justus Liebig University Giessen, Institute of Organic Chemistry, HeinrichꢀBuffꢀRing
17, 35392 Giessen, Germany
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*
Corresponding author (Tel: +49 641 99ꢀ34900; Fax: +49 641 99ꢀ34909; Eꢀmail:
holger.zorn@uniꢀgiessen.de)
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ABSTRACT: A number of methylꢀbranched aldehydes impart interesting flavor
impressions, and especially 12ꢀmethyltridecanal is a highly sought after flavoring
compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For
the biotechnological production of 12ꢀmethyltridecanal, the literature was screened
for fungi forming isoꢀfatty acids. Suitable organisms were identified and successfully
grown in submerged cultures. The culture medium was optimized to increase the
yields of branched fatty acids. A recombinant carboxylic acid reductase was used to
reduce 12ꢀmethyltridecanoic acid to 12ꢀmethyltridecanal. The efficiency of wholeꢀcell
catalysis was compared to that of the purified enzyme preparation. After lipase
catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were
converted to the corresponding aldehydes, including 12ꢀmethyltridecanal and 12ꢀ
methyltetradecanal.
KEYWORDS: biotransformation, carboxylic acid reductase, Conidiobolus
heterosporus, flavor, 12ꢀmethyltridecanal, 12ꢀmethyltridecanoic acid
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INTRODUCTION
Methylꢀbranched fatty acids (MBFAs) are suitable precursors for the biotechnological
production of their corresponding aldehydes. Naturally occurring MBFAs are typically
saturated and monoꢀmethylꢀbranched. They are common, but usually minor
constituents of lipids of bacteria and animals, and scarce in higher plants. Their
major physiological role may be to increase the fluidity of lipids as an alternative to
unsaturated fatty acids (FAs).1 MBFAs with a methyl branch at the penultimate
position (isopropyl group) are called isoꢀ and those with a methyl branch at the
antepenultimate carbon (secꢀbutyl group) anteisoꢀfatty acids. For meat and milk of
ruminants it is assumed that rumen bacteria are the major source of isoꢀ and
anteisoꢀFAs.2
While extensive research has been published on the occurrence and de novo
synthesis of branchedꢀchain fatty acids (BCFAs) in bacteria,1 only a few studies
report on fungal sources. Most of them date back to the late 1960s and early/midꢀ
1970s and cover Conidiobolus species.3ꢀ5 Up to 73% BCFAs of total FAs have been
reported for these Zygomycota.5 In general, the percentage of BCFAs in
Basidiomycota is much lower.6 For Armillaria mellea, ~10% of BCFAs have been
reported.7
Some methylꢀbranched aldehydes impart interesting flavor impressions.12ꢀ
Methyltridecanal (1) (Figure 1) especially is a highly sought after flavoring compound
for savory foods. Its smell is reminiscent of cooked beef and tallow.8 By aroma
extract dilution analysis (AEDA), stable isotope dilution analysis (SIDA),
determination of the corresponding odor thresholds, and calculation of odor activity
values (OAVs), 1 was found to be the most important contributor to the flavor of
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
other animal meat, but primarily in ruminants. For beef, concentrations of 55–150
ꢁg/g lipid were quantitated.8 While raw beef does not exhibit the typical smell of 1,
the methylꢀbranched aldehyde is released during cooking and roasting. It is assumed
that 1 is synthesized in the ruminants’ stomachs by bacteria, and small amounts are
incorporated into the plasmalogens after resorption and transport to the muscular
tissues.8 This hypothesis is supported by investigations on the occurrence of 1 in
microorganisms and physiological samples isolated from beef, in which the highest
amounts of 1 were detected in the bacteria isolated from the rumen of bovine
animals (475 ꢁg/g airꢀdry matter) followed by protozoa (229 ꢁg/g).10 The
concentration of 1 in beef is correlated to the animal’s phospholipid content and
increases with its age. Up to 810 ꢁg/g phospholipid were reported after SIDA.11
Additionally, the formation is effected by the feed. Pastureꢀfed bulls showed higher
concentrations compared to concentrateꢀfed animals.12 Nevertheless, the
concentrations of 1 in natural sources are generally very low. Even its chemical
synthesis is comparatively complex and therefore not economic or uses
environmental unfriendly chemicals.8,13 Albeit more convenient strategies have been
published recently, chemically synthesized 1 may not marketed as natural.14
Therefore, to the best of our knowledge, no natural 1 is available as a flavoring
substance on the world market, and no biotechnological route towards 1 has been
commercialized so far.
For the reduction of MBFAs to their corresponding branched aldehydes, enzymes
with carboxylic acid reduction ability are required.15 Carboxylic acid reductases
(CAR) are oxidoreductases, which catalyze the reduction of aromatic and shortꢀchain
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
reduction of 3ꢀmethylbutyrate and 3ꢀmethylpentanoate has been reported for a CAR
from Clostridium thermoaceticum.18 No data on the reduction of longer MBFAs have
been published so far.
MATERIALS AND METHODS
Chemicals. Adenosineꢀ5'ꢀtriphosphate disodium salt (ATP), agarꢀagar (Kobe I),
glucose, nꢀhexane, ꢀleucine, 2ꢀmercaptoethanol, methanol, βꢀnicotinamide adenine
dinucleotide phosphate tetrasodium salt (NADPH), potassium hydrogen sulfate,
Dꢀ
L
sodium chloride, and sodium sulfate were purchased from Carl Roth GmbH + Co.
KG (Karlsruhe, Germany). tertꢀButyl methyl ether (TBME), dichloromethane, diethyl
ether, lipase from Candida rugosa (2.9 U/mg), magnesium chloride hexahydrate,
page ruler unstained protein ladder (10–200 kDa), and yeast extract were bought
from Fisher Scientific GmbH (Schwerte, Germany). Lithium aluminum hydride
(LiAlH4) (for synthesis) was purchased from Merck KGaA (Darmstadt, Germany).
Malt extract, petroleum ether (40–60 °C), and Supelco 37 component FAME mix
were purchased from SigmaꢀAldrich GmbH (Taufkirchen, Germany). 11ꢀ
Methyltridecanoic acid, 12ꢀmethyltridecanal (1), 12ꢀmethyltridecanoic acid (2) (Figure
1), 12ꢀmethyltetradecanal, 12ꢀmethyltetradecanoic acid, 13ꢀmethyltetradecanoic
acid, 13ꢀmethylpentadecanoic acid, and 14ꢀmethylpentadecanoic acid were provided
by Symrise AG (Holzminden, Germany). Boron trifluoride (20% in methanol) and
hydrochloric acid (32%) were obtained from VWR International GmbH (Darmstadt,
Germany).
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Microorganisms. The basidiomycetous fungus Armillaria mellea DSMZ 2941
(honey fungus) was obtained from the German Collection of Microorganisms and
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Cell Cultures (Brunswick, Germany). The fungi Conidiobolus denaeosporus CBS
137.57, Conidiobolus heterosporus CBS 543.63, C. heterosporus CBS 333.74, and
C. heterosporus CBS 138.57 (ATCC 12941) were obtained from CBSꢀKNAW Fungal
Biodiversity Centre (Utrecht, the Netherlands). Conidiobolus lobatus ATCC 18153
was purchased from the American Type Culture Collection (Manassas, VA).
Escherichia coli BL21(DE3) was purchased from Merck KGaA (Darmstadt,
Germany). The E. coli strain BL21(DE3)pETꢀPC2, which is able to coꢀexpress a
carboxylic acid reductase from Mycobacterium marinum and a phosphopantetheinyl
transferase (PPT) of Bacillus subtilis was provided by Pauli Kallio, University of
Turku, Finland and Patrick R. Jones, Imperial College London, England.
Cultivation. Stock cultures and precultures were maintained and grown as described
previously.19 Yeast extract (3 g/L) and malt extract (30 g/L) were used for stock
cultures and preculture media (YM medium). Growth periods on agar plates were: 5
days for C. heterosporus CBS 543.63 and CBS 333.74, 14 days for CBS 138.57, C.
denaeosporus, and C. lobatus, and 21 days for A. mellea. The standard cultivation
period of the precultures was 3 days, and 100 mL YM medium in 250 mL Erlenmeyer
flasks was used. Afterwards, the precultures were homogenized for 30 s at 9800 rpm
using an Ultra Turrax homogenizer (IKA, Staufen, Germany). For the main cultures,
20 mL of the homogenized suspension was used to inoculate 200 mL YM medium in
500 mL Erlenmeyer flasks. The cultivations were carried out in an Ecotron incubation
shaker (25 mm shaking diameter, 150 rpm, 24 °C) (Infors GmbH, Einsbach,
Germany) for 5 d (if not stated otherwise). Supplementation with Lꢀvaline was done
by adding 0.2 g/L or 2.0 g/L to the main culture medium prior to autoclaving. For
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
LabScience, Dassel, Germany).
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Lipid Extraction. The harvested biomass was treated with 200 mL of 4 M
hydrochloric acid with boiling for 30 min. After addition of 100 mL hot water, filtration
of the hydrolysis solution and neutralization, the residue was dried in a drying cabinet
at 103 °C for 3 h. Subsequently, Soxhlet extraction was performed with 180 mL
petroleum ether for 4 h. The solvent was evaporated by means of a rotary
evaporator (40 °C, ≤150 mbar), and the lipid extract was dried in the drying cabinet
at 103 °C to constant weight. After addition of 2 mL nꢀhexane, the lipid extract was
stored at ꢀ20 °C until saponification and esterification.
Preparation of fatty acid methyl esters (FAMEs). 150 mg fungal lipid extract was
saponified in a derivatization vial with a Teflon septum by adding 4 mL 0.5 M
methanolic sodium hydroxide solution and heating to 80 °C for 10 min. After cooling
to room temperature, 3.5 mL of boron trifluoride (20% in methanol) was added. The
mixture was firmly shaken and incubated for 5 min (80 °C). After cooling, 5 mL nꢀ
hexane was added, the mixture was heated for 1 min (80 °C), and 5 mL saturated
sodium chloride solution was added. The organic phase was separated and dried
over anhydrous sodium sulfate. Reference standards were directly esterified by
addition of 1 mg fatty acid to 1 mL boron trifluoride solution. The generated FAMEs
were analyzed by means of gas chromatography (GC).
Protein expression. Overnight cultures were grown according to Akhtar et al. in Luriaꢀ
Bertani (LB) medium.17 100 ꢁL of these cultures was used for inoculation of 200 mL
Overnight Express Instant TB Medium (Merck KGaA). Cells were incubated for 20 h
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
pellets were employed for enzyme purification or biotransformation.
Enzyme purification. The Hisꢀtagged protein was purified according to the Protino Niꢀ
TED protocol (MachereyꢀNagel GmbH & Co. KG, Dueren, Germany) using 2 mg/mL
lysozyme.17 The enzyme containing fractions were combined and 2ꢀmercaptoethanol
(3%, v/v) was added. 10 mL aliquots were washed seven times with phosphate
buffer (50 mM, pH 7.5) using 30 K Macrosep Advance Centrifugal Devices (Pall Life
Sciences, Port Washington, NY) by means of centrifugation (4 °C, 4713 x g).
Determination of protein concentration. The protein concentration was measured by
means of a Roti NanoquantꢀKit K880 (Carl Roth GmbH + Co. KG) according to the
manufacturer’s protocol. The purified CAR enzyme solution was diluted with 50 mM
phosphate buffer (pH 7.5) to a final protein concentration of 100 ꢁg/mL and used for
in vitro bioconversion experiments.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSꢀPAGE) and
Coomassie staining. Discontinuous SDSꢀPAGE was performed according to
Laemmli.20 The gels were stained overnight at room temperature with Coomassie
brilliant blue G250 solution.21
Biotransformation – whole cell catalysis. Twentyꢀfive mg cells were suspended in 1
mL phosphate buffer (100 mM, pH 7.5) in a 10 mL reaction vial and 0.5 mM 2 (in nꢀ
hexane) and 100 mM Dꢀglucose were added. If not stated otherwise, the vials were
incubated for 24 h in an incubation shaker at 30 °C and 250 rpm. Prior to solvent
extraction with 1 mL dichloromethane, the pH was adjusted to pH 2 with hydrochloric
acid (18%, w/w). As controls, analogous transformations with BL21(DE3) E. coli
cultures and an empty plasmid were performed with 0.25 mM 1 and 0.5 mM 2 as
substrates. For additional negative controls, 25 mg E. coli BL21(DE3)pETꢀPC2 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 Dꢀglucose.
In vitro conversion with purified enzymes. For the conversion of 2, ATP (1 mM),
NADPH (1 mM), MgCl2 (10 mM), and 2 (0.5 mM) were added to 0.9 mL CARꢀ
solution (100 ꢁg/mL in 50 mM phosphate buffer, pH 7.5) in a total reaction volume of
1011 ꢁL. If not stated otherwise, the reaction mixture was incubated for 24 h at 30 °C
and subsequently extracted as described above. Alternatively, 150 mg lipid extract of
C. heterosporus CBS 333.74 was suspended in phosphate buffer containing ATP (1
mM), NADPH (1 mM), MgCl2 (10 mM), 2 (0.5 mM), 0.9 mL CARꢀsolution (100 ꢁg/mL
in 50 mM phosphate buffer, pH 7.5), and 200 ꢁL lipase solution (Candida rugosa; 12
mg/mL, 7 U in phosphate buffer) was added. The final volume of the reaction mixture
was 1210 ꢁL. The mixture was incubated for 2 h at 30 °C and subsequently
extracted as described above. For negative controls, the CAR solutions were heated
for 30 min at 95 °C prior to the reaction. In additional controls, the enzyme solution
was substituted by the same volume of 50 mM phosphate buffer.
Gas chromatography. GC analyses were performed according to Kleofas et al.22 For
compound identification, GCꢀmass spectrometry (MS) analyses on two columns of
different polarity and calculation of the corresponding retention indices (RIs) were
performed. For quantitation, response factors were determined by means of a GC
equipped with a flame ionization detector (FID). In general, 1 ꢁL sample was injected
with a split of 10:1 for GCꢀMS and 2:1 for GCꢀFID. The RIs of the methyl esters of
the MBFAs were as follows: methyl 11ꢀmethyltridecanoate 1977 (HPꢀInnowax) and
1761 (DBꢀ5), methyl 12ꢀmethyltridecanoate 1962 and 1754, methyl 12ꢀ
methyltetradecanoate 2080 and 1865, methyl 13ꢀmethyltetradecanoate 2065 and
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1857, methyl 13ꢀmethylpentadecanoate 2210 and 1969, and methyl 14ꢀ
methylpentadecanoate 2196 and 1961.
Synthesis of 12ꢀmethyltridecanol. For the synthesis of 12ꢀmethyltridecanol (3)
(Figure 1) 2.00 g 2 dissolved in 10 mL diethyl ether was added dropwise to 665 mg
of LiAlH4 in 20 mL diethyl ether, and the mixture was stirred overnight. The reaction
was stopped by addition of iceꢀwater and the crude product was extracted with 50
mL diethyl ether. After drying with sodium sulfate and removal of the solvent by
distillation, the crude product was purified by columnꢀchromatography (MTBE:nꢀ
hexane 1:3, v/v).
1.68 g (90%) of a clear oil was isolated. EIꢀMS: 69 (100), 55 (100), 43 (90), 41 (88),
83 (78), 97 (54), 111 (26), 140 (10), 168 (7), 125 (6); RI: 2118 (HPꢀInnowax), 1647
(DBꢀ5)
Statistics. Analyses were performed at least in duplicate, and results are reported as
means ± half range or standard deviations, respectively.
RESULTS AND DISCUSSION
Production of methylꢀbranched fatty acids. After screening of the literature for
MBFAsꢀproducing fungi, six different fungi were chosen for further investigations.
Five fungi were Conidiobolus species, three of them C. heterosporus strains from
different origins. All of them grew well in submerged cultures, and yeast extract / malt
extract medium was found to be an appropriate standard medium. After harvesting
and lipid extraction, the fatty acid profiles of the fungi were analyzed. BCFAs were
detected in all lipid samples, but in varying quantities (Table 1). The C. heterosporus
strains showed the highest share of isoꢀ and anteisoꢀFAs. More than 55% of MBFAs
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
Conidiobolus lobatus.4,5 In our studies, up to 40% 2 was observed with C.
heterosporus CBS 543.63 and CBS 333.74, while C. heterosporus CBS 138.57 and
C. lobatus only formed ~ 20% 2. The observed differences might be related to
different media used or culture parameters applied.
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According to Řezanka and Mareš the basidiomycetous fungus Armillaria mellea
(honey fungus) contains ~10% of BCFA, including small amounts of the oddꢀ
numbered isoꢀFA 13ꢀmethyltetradecanoic acid (1.8%).7 Oddꢀnumbered isoꢀFAs have
not been reported for other fungal species. Therefore, the fatty acid profile of A.
mellea was analyzed as well. However, all identified isoꢀFAs were evenꢀcarbonꢀ
numbered while the anteisoꢀFAs were oddꢀcarbonꢀnumbered (Table 1). For the
Conidiobolus species, this was in accordance with Tyrrell and Weatherston.5
C. heterosporus CBS 333.74 was chosen for all further experiments due to its overall
better ability to be cultivated in liquid media compared to other C. heterosporus
strains. Its growth in submerged cultures was investigated over a period of two
weeks. Both, the biomass and the lipid yield increased steadily over time (Figure 2).
The lipid content of the dry biomass was 25–30%. The amounts of saturated fatty
acids decreased until day 14 by about 10%, whereas the share of unsaturated fatty
acids increased slightly (data not shown). On day 14, the highest levels of 2 were
observed.
For certain bacteria and some Conidiobolus species it is known that supplementation
of the culture medium with the amino acid Lꢀvaline may increase the formation of
even numbered isoꢀFAs, i.e. 2 or 14ꢀmethylpentadecanoic acid.5,23,24 Based on these
observations, the culture media of C. heterosporus CBS 333.74 were supplemented
with 0.2 g/L and 2.0 g/L
Lꢀvaline, respectively. For 2, an increase of 10% (0.2 g/L)
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and 38% (2.0 g/L) was observed. For 14ꢀmethylpentadecanoic acid, the increase
was less pronounced with 4% and 10%.
Biotransformation to methyl branched aldehydes. Initially, several enzymes were
considered for the reduction of MBFAs to their corresponding aldehydes. Various
enzymes have been described to reduce aromatic carboxylic acids. The well
investigated CAR from Nocardia species are known especially to reduce acids with
aromatic residues, but successful reduction of aliphatic acids has not been reported
for them yet.25,26 However, for the CAR from M. marinum, the reduction of aliphatic
fatty acids, with a range from C8–C18, was published recently.17 Because of the
structural similarities, the CAR from M. marinum was chosen for the
biotransformation of MBFAs. The CAR was heterologously expressed and purified to
electrophoretic homogeneity. The molecular weight of the CAR was determined to
be 127 kDa (Figure 3) which was in agreement with the results of Akhtar et al.17
For whole cell biotransformation, 2 was incubated for 24 h with different amounts of
E. coli cell pellets expressing CAR. The formation of up to 28.0 ± 1.8 mol% 3 was
observed, but no 1 was detected. To further elucidate the formation of 1, shorter
biotransformation periods were checked. Here, 1 was identified, with the highest
amounts (3.1 ± 0.6 mol%) after 2 h (Figure 4A). The concentration of 3 steadily
increased over the first 6 h (20 ± 3.4 mol%). In contrast, in none of the negative
controls (without CAR, with heat inactivated CAR, with BL21(DE3) and empty
plasmid) a reduction of 2 to 1 or 3 was detected. On the other hand, the E. coli strain
BL21(DE3) with empty plasmid converted 1 to 3 (data not shown).
To prevent the accumulation of 3 and thus to increase the amounts of 1, the in vitro
conversion of 2 was further investigated with the purified CAR enzyme. Therefore,
the recombinant polyhistidineꢀtagged 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
presence of the required cofactors for 2–8 h. After 2 h, 22.1 ± 0.53 mol% 1 were
detected, and the highest conversion yield was found with 28 ± 7.8 mol% after 8 h
(Figure 4B). In all samples, only trace amounts of 3 were determined. This led us to
the conclusion, that the CAR is essential for the initial reduction of 2 to 1, whereas
native E. coli enzymes (most probably alcohol dehydrogenases) are responsible for
the reduction of the formed aldehyde to its corresponding alcohol.
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Next, the fatty acids produced by C. heterosporus CBS 333.74 were directly
employed. In addition to the CAR, a lipase from Candida rugosa was added to the
reaction mixture to release esterified fatty acids. Besides 12ꢀmethyltridecanal and
12ꢀmethyltetradecanal, several linear aldehydes, like dodecanal, tridecanal,
tetradecanal, pentadecanal, and hexadecanal were generated (Figure 5).
In conclusion, the enzymatic reduction of methylꢀbranched fatty acids to their
corresponding aldehydes has been shown in this study for the first time. 2 was
converted enzymatically to the highly sought after flavor compound 12ꢀ
methyltridecanal. Even though the initial concentrations obtained with the
investigated oneꢀpot reaction were rather low, the general feasibility was clearly
shown. Future studies will concentrate on aspects of increasing the yields of 1 by
direct enzymatic conversion of fungal lipids.
ABBREVIATIONS
AEDA, aroma extract dilution analysis; BCFA, branchedꢀchain fatty acid; MBFA,
methylꢀbranched fatty acid; OAV, odor activity value; PPT, phosphopantetheinyl
transferase; SIDA, stable isotope dilution analysis; YM, yeast extractꢀmalt extract.
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ACKNOWLEDGMENT
We are very grateful to Pauli Kallio, University of Turku, and Patrik R. Jones,
Imperial College London, who generously provided the BL21(DE3)pETꢀPC2 E. coli
strain.
ASSOCIATED CONTENT
Supporting Information
GCꢀMS chromatogram of a C. denaeosporus lipid extract after transesterification to
the corresponding fatty acid methyl esters and mass spectrum of methyl 12ꢀ
methyltridecanoate identified in the C. denaeosporus lipid extract. This material is
available free of charge via the Internet at http://pubs.acs.org.
Funding
The authors MAF and HZ are grateful for financial support by the excellence initiative
of the Hessian Ministry of Science and Art which encompasses a generous grant for
the LOEWE Center for Insect Biotechnology & Bioresources.
Notes
The authors declare no competing financial interest. Torsten Geissler, Egon Gross,
JensꢀMichael Hilmer, and Jakob Ley are members of Symrise AG holding intellectual
property on 12ꢀmethyltridecanal.
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(24) Kaneda, T. Biosynthesis of branchedꢀchain fatty acids: IV. Factors affecting
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FIGURE CAPTIONS
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Figure 1. Structures of 12ꢀmethyltridecanal (1), 12ꢀmethyltridecanoic acid (2), and
12ꢀmethyltridecanol (3).
Figure 2. Dry biomass, lipids, and relative lipid content of C. heterosporus CBS
333.74 grown in submerged cultures over a cultivation period of 14 d.
Figure 3. SDSꢀPAGE of the CAR enzyme preparation after heterologous expression
and purification according to the procedure described by Akhtar et al.;17 left lane
shows a molecular mass marker (kDa), right lane shows the isolated CAR.
Figure 4. Kinetics of the whole cell catalyzed reduction of 2 with CAR from M.
marinum coꢀexpressed with PPT in E. coli for 1–6 h (A) and by the recombinant
CAR, purified by means of immobilized metal ion affinity chromatography, for 2–8 h
(B).
Figure 5. Section of a GCꢀMS chromatogram (total icon current, scan mode, m/z 33–
300) recorded after injection of an organic solvent extract of a reaction mixture of the
enzymatic hydrolysis of a fungal lipid and reduction of the thereby generated free
fatty acids by CAR.
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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.
C.
C. heterosporus C. heterosporus C. heterosporus
fatty acid
A. mellea
C. lobatus
denaeosporus
CBS 543.63
n.d.
CBS 333.74
n.d.
CBS 138.57
n.d.
a14:0
n.d.
5.0
n.d.
8.6
n.d.
18.8
7.8
i14:0
40.1
13.6
n.d.
34.5
12.0
n.d.
20.1
12.7
n.d.
a15:0
1.4
11.1
n.d.
n.d.
0.4
i15:0
n.d.
n.d.
0.7
n.d.
n.d.
2.1
a16:0
n.d.
n.d.
n.d.
i16:0
4.2
7.0
2.1
∑ unassigned FAs
∑ saturated FAs*
∑ unsaturated
28.3
31.2
33.4
28.6
48.3
3.0
19.6
21.1
1.4
21.4
20.7
4.5
7.7
7.8
47.6
9.8
44.0
19.5
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FAs
a14:0 11ꢀmethyltridanoic acid, i14:0 12ꢀmethyltridanoic acid, a:15:0 12ꢀmethyltetradecanoic acid, i15:0 13ꢀmethyltetradecanoic
acid, a16:0 13ꢀmethylpentadecanoic acid, i16:0 14ꢀmethylpentadecanoic acid, n.d. not detected, * excluding listed methylꢀbranched
fatty acids
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FIGURES
Figure 1
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Figure 2
12
10
8
30
25
20
15
10
5
6
4
2
0
0
2
4
6
8
10
12
14
cultivation time [d]
dry biomass
lipids
lipids / dry biomass
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Figure 3
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Figure 4
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Figure 5
300000
250000
200000
150000
100000
50000
0
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46
[min]
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