Biotransformation of oleic acid into

10-ketostearic acid by recombinant

Corynebacterium glutamicum-based
biocatalyst

Byeonghun Lee, Saebom Lee, Hyeonsoo

Kim, Kijun Jeong, Jinbyung Park,
Eunyeol Lee & Jinwon Lee

Biotechnology Letters

ISSN 0141-5492

Biotechnol Lett
DOI 10.1007/s10529-015-1775-7

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 Author's personal copy
Biotechnol Lett
DOI 10.1007/s10529-015-1775-7

ORIGINAL RESEARCH PAPER

Biotransformation of oleic acid into 10-ketostearic acid

by recombinant Corynebacterium glutamicum-based
biocatalyst
Byeonghun Lee • Saebom Lee • Hyeonsoo Kim •

Kijun Jeong • Jinbyung Park • Eunyeol Lee •

Jinwon Lee

Received: 17 December 2014 / Accepted: 20 January 2015

Ó Springer Science+Business Media Dordrecht 2015

Abstract Conclusion This is the first report of 10-ketostearic

Objective To produce 10-ketostearic acid from oleic acid production using a recombinant C. glutamicum.
acid.
Results Oleic acid was converted to 10-ketostearic Keywords Biocatalysts
Biotransformation

acid by a recombinant Corynebacterium glutamicum Corynebacterium glutamicum
10-Ketostearic acid

ATCC 13032 expressing oleate hydratase from Steno- Oleate hydratase
Oleic acid
Secondary alcohol
trophomonas maltophilia and a secondary alcohol dehydrogenase
dehydrogenase from Micrococcus luteus under the
control of a synthetic constitutive promoter. Optimal
conditions for 10-ketostearic acid production were pH
7.5 and 30 °C with 5 g cells l-1 and 2.5 g oleic acid Introduction
l-1. Under these conditions, the cells produced 1.96 g
10-ketostearic acid l-1 from oleic acid in 6 h, with a Corynebacterium glutamicum has traditionally been
conversion yield of 78 % (w) and a maximum used in the industrial production of various amino
volumetric productivity of 1.67 g l-1 h-1. acids (e.g., lysine and glutamic acid) (Hermann 2003)
and, furthermore, has been engineered to produce a
variety of bio-based chemicals, materials, and fuels
Byeonghun Lee and Saebom Lee have contributed equally to such as 2-ketoisovalerate, succinate, cadaverine,
this work. putrescine, 1,2-propanediol, ethanol, 1-butanol, and
polygalacturonic acid (Becker and Wittmann 2012).
Electronic supplementary material The online version of Many microorganisms, including Staphylococcus
this article (doi:10.1007/s10529-015-1775-7) contains supple-
mentary material, which is available to authorized users. spp. (Lanser 1993), Flavobacterium sp. strain DS5

B. Lee
S. Lee
H. Kim
J. Lee (&) J. Park

Department of Chemical and Biomolecular Engineering, Department of Food Science and Engineering, Ewha
Sogang University, Seoul 121-742, Republic of Korea Womans University, Seoul 120-750, Republic of Korea
e-mail: [email protected]
E. Lee
K. Jeong Department of Chemical Engineering, Kyung Hee
Department of Chemical and Biomolecular Engineering, University, Yongin-si, Gyeonggi-do 446-701, Republic of
Korea Advanced Institute of Science and Technology Korea
(KAIST), Yuseong-gu, Daejeon 305-701,
Republic of Korea

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(Hou 1994), Sphingobacterium sp. strain 022 (Kuo Plasmid construction for expression of OhyA
et al. 1999), Staphylococcus warneri (Lanser and and ADH in C. glutamicum
Nakamura 1996), Mycobacterium, Nocardia, Asper-
gillus terreus, Sphingobacterium thalpophilum, and The primers used for PCR amplification of genes of
Bacillus sphaericus (Hou 2009), have been used for interest are shown in Supplementary Table 1. Restric-
the production of 10-ketostearic acid from oleic acid. tion enzymes were purchased from Takara (Kyoto,
In this study, the genes encoding oleate hydratase Japan) and oligonucleotides were synthesized by
(OhyA) in Stenotrophomonas maltophilia (Joo et al. Macrogen Inc. (Seoul, Korea). The OhyA gene of S.
2012) and secondary alcohol dehydrogenase (ADH) in maltophilia KCTC 1773 was ligated into pCES208
Micrococcus luteus (Huang et al. 2010) were cloned vectors containing synthetic promoters (H36, I16, or
into the pCES208-L10 plasmid and then introduced L10) using the ‘sequence and ligation independent
into C. glutamicum. Part of a previously-designed cloning (SLIC)’ method (Li and Elledge 2007). The
synthetic biotransformation pathway (Song et al. ADH gene of M. luteus NCTC 2665 was ligated into the
2013) in C. glutamicum was constructed using a vectors containing the OhyA gene using an In-fusion
constitutive expression vector for the production of cloning kit (Clontech, USA) (Clontechniques 2002). A
10-ketostearic acid from olive oil and oleic acid. To ribosomal-binding site (RBS) was encoded upstream
increase the conversion yield using whole cells, of the ADH gene. All PCR constructs were confirmed
reaction conditions (pH, temperature, substrate con- by Macrogen, Inc. (Seoul, Korea) and then transformed
centration, and cell concentration) were optimized, into C. glutamicum using electroporation with a Gene
after which a time-course assessment of the conver- Pulser Xcell electroporator (Bio-Rad, USA).
sion of oleic acid and olive oil to 10-ketostearic acid
was carried out under optimal conditions. This is the Optimization of reaction conditions
first report of microbial 10-ketostrearic acid produc-
tion using recombinant C. glutamicum cells. The OD600 values of the cultures were measured and
converted to dry cell weights (DCW). Unless other-
wise stated, reactions were performed at 30 °C in
50 mM Tris/HCl (pH 7.5) containing 2.5 g oleic acid
Materials and methods l-1, 5 g cells l-1, and 0.05 % Tween 80 (w/v) for 1 h
under aerobic conditions. To examine the effects of
Bacterial strains, cloning vectors, and culture pH and temperature on 10-ketostearic acid production
media using whole recombinant cells, pH was varied from
6.5–8.5 using 50 mM phosphate/citrate buffer (pH
The bacterial strains and plasmids used in this study are 6.5–7.5) or 50 mM Tris/HCl buffer (pH 7.5–8.5) at
listed in Supplementary Table 1. Escherichia coli 30 °C and temperature was varied from 20 to 40 °C at
DH5a was used as a host for gene cloning and plasmid pH 7.5.
maintenance, and C. glutamicum ATCC 13032 was Cell concentration was assessed at 0.5–10 g l-1
used as the main host for the production of 10-ketos- with 5 g oleic acid l-1 to determine the optimal cell
tearic acid. The OhyA and ADH genes were cloned into concentration for 10-ketostearic acid production and
E. coli/C. glutamicum shuttle vectors (pCES208, substrate concentration was assessed at 0.5–6.25 g l-1
KAIST, Daejeon, Korea) (Park et al. 2008) containing with a constant cell concentration of 5 g l-1 to
one of three synthetic promoters: H36, I16, or L10 determine the substrate concentration required for
(Yim et al. 2013). E. coli DH5a was cultivated in maximum production of 10-ketostearic acid. A fixed
lysogeny broth (10 g tryptone l-1, 5 g yeast reaction time (4 h) was used for the cell and substrate
extract l , and 5 g NaCl l-1) at 37 °C and for protein
-1
concentration experiments.
production C. glutamicum-pCES208-L10-OhyA-
ADH was cultivated in a 500 ml baffled flask contain- Biotransformation
ing 100 ml brain heart infusion (BHI) broth (Becton–
Dickinson, Sparks, MD) and 50 lg kanamycin ml-1 at Recombinant C. glutamicum expressing the OhyA and
30 °C for 12 h with shaking at 200 rpm. ADH genes was cultivated in BHI medium at 200 rpm

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and 30 °C until the stationary phase (for 12 h). Cells purposes, three pCES208 vectors each containing a
were harvested by centrifugation at 4 °C, washed, and synthetic promoter (H36, I16, or L10) allowing for the
resuspended in 50 mM Tris/HCl buffer (pH 7.5) to a constitutive expression of heterologous genes in C.
concentration of 5 g cells l-1. Reactions were initiated glutamicum were obtained. Of the three pCES208
by the addition of 2.5 g oleic acid l-1 and 0.05 % transformants, C. glutamicum-pCES208-L10-OhyA-
Tween 80 (w/v) to the buffer and were performed in a ADH (C. glutamicum expressing OhyA and ADH
shaking incubator at 200 rpm and 30 °C for 6 h. under control of the constitutive L10 promoter)
For the biotransformation of olive oil to 10-ketos- exhibited the highest 10-ketostearic acid yield (data
tearic acid, olive oil was hydrolyzed by the lipase from not shown) and was thus selected for 10-ketostearic
Candida rugosa (Sigma-Aldrich, USA) (Song et al. acid production by biotransformation.
2013) and the resulting hydrolysate was subjected to The biotransformation products of oleic acid and
biotransformation by recombinant OhyA- and ADH- olive oil (10-hydroxystearic acid and 10-ketostearic
expressing C. glutamicum. The biotransformation acid) were analyzed by GC/MS and the chemical
reaction was performed under optimal conditions. structures of these products were identified by com-
parison with the authentic reference compound
Analysis of products reported in a previous study (Song et al. 2013).

For product analysis, reactions were terminated by Optimization of reaction conditions

acidification to pH 2 with HCl and reaction products for the biotransformation of oleic acid
were then extracted with ethyl acetate containing to 10-ketostearic acid by whole recombinant
methyl palmitate as an internal standard. The solvent C. glutamicum cells
was removed from the extracts using a rotary evap-
orator and fatty acids present in the extract were The biotransformation of oleic acid to 10-ketostearic
silylated with a 3:1 mixture of pyridine:N-methyl-N- acid by whole recombinant C. glutamicum cells
(trimethylsilyl) trifluoroacetamide (TMS). TMS expressing the OhyA and ADH genes was optimized
derivatives were analyzed by GC/MS analysis which by assessing the biotransformation efficiency at var-
was performed on an Agilent 5975 series mass ious pH (6.5–8.5) (Fig. 1a) and temperatures (20–
spectrometer and a 7890A GC system equipped with 40 °C) (Fig. 1b) using a constant cell concentration of
non-polar capillary column (30 m length, 0.25 m film 5 g l-1. The optimal reaction conditions were selected
thickness, HP-5MS, Agilent Technologies, Palo Alto, for maximal conversion of oleic acid to 10-ketostearic
CA, USA). Helium was used as the carrier gas. The acid in terms of conversion yield (1 h reaction time). The
column temperature gradient was programmed as maximal conversion yield (%, w/w) of oleic acid to
follows: initially 80 °C, increased by 5 °C min-1 to 10-ketostearic acid was achieved at pH 7.5 and 30 °C.
125 °C, then by 15 °C min-1 to 200 °C, and finally by The optimal cell concentration for 10-ketostearic
5 °C min-1 to 255 °C. The injection port temperature acid production was determined by carrying out
was 230 °C and mass spectra were obtained using an biotransformation for 4 h (Fig. 2a). The optimal cell
electron impact ionization source at 70 eV. concentration was therefore determined to be 5 g l-1.
Moreover, to determine the optimal substrate concen-
tration for maximum conversion of oleic acid to
Results and discussion 10-ketostearic acid, oleic acid concentrations in reac-
tions were varied (0.5–6.25 g l-1) while the cell
Selection of recombinant Corynebacterium strains concentration was fixed (5 g l-1) (Fig. 2b). With
and product identification oleic acid concentrations of B2.5 g l-1, increasing
substrate concentrations resulted in proportional
In the expression of genes for protein production in increases in conversion, with a conversion yield of
microorganisms, it is important to choose an appro- *78 % (w/w) being measured for 2.5 g oleic acid/l;
priate promoter. Constitutive promoters are consid- however, conversion yields decreased with increasing
ered more cost-effective than inducible promoters concentrations of oleic acid[2.5 g l-1, and a conver-
(Yim et al. 2013; Lee 2014) and thus for cloning sion yield of 15 % (w/w) was measured for 6.25 g

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Fig. 2 Effects of cell concentration and substrate concentration

on 10-ketostearic acid production from oleic acid by
Fig. 1 Effects of reaction conditions on biotransformation.
recombinant C. glutamicum. a Effect of cell concentration.
a Effect of pH: reactions were performed in 50 mM phosphate/
b Effect of substrate concentration. Conversion yield (open
citrate buffer (pH 6.5–7.5, filled square) or 50 mM Tris/HCl
square) and 10-ketostearic acid concentration (filled triangle).
buffer (pH 7.5–8.5, filled triangle) with 2.5 g oleic acid l-1 and
Data represent the means of triplicate experiments and error
5 g cells l-1 for 1 h. b Effect of temperature: reactions were
bars represent standard deviation
performed in 50 mM Tris/HCl buffer (pH 7.5) containing 2.5 g
oleic acid l-1 and 5 g cells l-1 for 1 h. Data represent means of
triplicate experiments and error bars represent standard devia- pH 7.5, 30 °C, 5 g cells l-1, and 2.5 g oleic acid l-1.
tion. At the relative 100 % value, conversion of oleic acid to Under these optimal conditions, a time-course assess-
10-ketostearic acid was the highest in terms of conversion yield
ment was carried out and the whole recombinant C.
oleic acid l-1. With substrate concentrations of glutamicum cells were found to produce 1.96 g
[2.5 g l-1, the viscosity of the reaction solutions 10-ketostearic acid l-1 after 6 h, with a conversion
became so high that resistance to mass transfer in the yield of 78 % (w/w) and a maximum volumetric
aqueous phase became dominant and the conversion production rate of 1.67 g l-1 h-1 (Fig. 3). For bio-
rate decreased (Yu et al. 2008). transformation, a multistep approach by one biocata-
lyst unit enables simplification and intensification of
Production of 10-ketostearic acid by whole the conversion process and eliminates the need for
recombinant C. glutamicum cells under optimized expensive isolation of intermediates (Ladkau et al.
conditions 2014).
To extend the applicability of the biotransformation
The optimal reaction conditions for the conversion of described in this report, a plant oil (olive oil) with oleic
oleic acid to 10-hydroxystearic acid by whole acid as its major fatty acid component was used as
recombinant C. glutamicum cells were found to be a substrate instead of oleic acid. Olive oil was

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carried out using recombinant C. glutamicum express-

ing the OhyA and ADH genes, and 10-ketostearic acid
was produced at 1.3 g l-1 from oleic acid (Fig. 4).

Conclusion

The recombinant C. glutamicum expressing the OhyA

and ADH genes was first applied to the conversion of
oleic acid to 10-ketostearic acid. The conversion
reaction conditions (pH, temperature, cell concentra-
tion, and substrate concentration) for this system were
optimized and extended to produce 10-ketostearic acid
from olive oil, renewable oil. These findings thus
Fig. 3 Biotransformation of oleic acid into 10-ketostearic acid
by whole recombinant C. glutamicum cells. Time-course contribute to achieving the initial stage of the biolog-
assessment. The bioconversion of oleic acid (filled square) to ical process required for the industrial production of
10-hydroxystearic acid (filled circle) and 10-ketostearic acid 10-ketostearic acid.
(filled triangle) are shown. Reactions were performed in 50 mM
Tris/HCl buffer (pH 7.5) containing 2.5 g oleic acid l-1, 5 g Acknowledgments This work was supported by the MOTIE/
cells l-1, and 0.05 % Tween 80 for 6 h. Data represent the KEIT R&D Program (10044604, Bioproduction of long-chain
means of triplicate experiments and error bars represent dicarboxylic acids from fatty acids and lipids).
standard deviation
Supporting information Supplementary Table 1—Bacterial
strains, plasmids and oligonucleotides used in this study.

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