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Biotechnol Lett
Biotechnol Lett
Biotechnology Letters
ISSN 0141-5492
Biotechnol Lett
DOI 10.1007/s10529-015-1775-7
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Biotechnol Lett
DOI 10.1007/s10529-015-1775-7
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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).
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Conclusion
References
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