Biosynthesis of Pinocembrin from Glucose Using Engineered Escherichia coli

Bong Gyu  Kim; Hyejin  Lee  and Joong-Hoon  Ahn

doi:10.4014/jmb.1406.06011

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Bioprocess and Metabolic Engineering

Biosynthesis of Pinocembrin from Glucose Using Engineered Escherichia coli

Bong Gyu Kim ¹, Hyejin Lee ² and Joong-Hoon Ahn ^2*

¹Department of Forest Resources, Gyeongnam National University of Science and Technology, Jinju-si 660-758, Republic of Korea, ²Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea

Received: June 5, 2014; Accepted: July 28, 2014

J. Microbiol. Biotechnol. 2014; 24(11): 1536-1541

Published November 28, 2014 https://doi.org/10.4014/jmb.1406.06011

Abstract

Pinocembrin is a flavonoid that exhibits diverse biological properties. Although the major source of pinocembrin is propolis, it can be synthesized biologically using microorganisms such as Escherichia coli, which has been used to synthesize diverse natural compounds. Pinocembrin is synthesized from phenylalanine by the action of three enzymes; phenylalanine ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS). In order to synthesize pinocembrin from glucose in Escherichia coli, the PAL, 4CL, and CHS genes from three different plants were introduced into an E. coli strain. Next, we tested the different constructs containing 4CL and CHS. In addition, the malonyl-CoA level was increased by overexpressing acetyl-CoA carboxylase. Through these strategies, a high production yield (97 mg/l) of pinocembrin was achieved.

Keywords

flavonoid biosynthesis, metabolic engineering, pinocembrin

References

Aboushoer MI, Fathy HM, Abdel-Kader MS, Goetz G, Omara AA. 2010. Terpenes and flavonoids from an Egyptian collection of Cleome droserifolia. Nat. Prod. Res. 24: 687-696.
Austin MB, Noel JP. 2003. The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 20: 79-110.
Cochrane FC, Davin LB, Lewis NG. 2004. The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoform. Phytochemistry 65: 1557-1564.
Dixon RA, Paiva NL. 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085-1097.
Hamberger B, Hahlbrock K. 2004. The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc. Natl. Acad. Sci. USA 101: 2209-2214.
Houghton PJ, Woldemariam TZ, Davey W, Basar A, Lau C. 1995. Quantitation of the pinocembrin content of propolis by densitomety and high performance liquid chromatography. Phytochem. Anal. 6: 207-210.
Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S. 2003. Production of plant-specific flavanoes by Escherichia coli containing an artificial gene cluster. Appl. Environ. Microbiol. 69: 2699-2707.
Jangaard NO. 1974. The characterization of phenylalanine ammonia-lyase from several plant species. Phytochemistry 13:1765-1768.
Jaganath IB, Crozier A. 2010. Dietary Flavonoids and Phenolic Compound in Plant Phenolics and Human Health. Fraga CG (ed.). John Wiley & Sons, Hoboken, New Jersey.
Kim B-G, Lee E-R, Ahn J-H. 2012. Analysis of flavonoid contents and expression of flavonoid biosynthetic genes in Populus euramericana Guinier in response to abiotic stress. J. Kor. Soc. Appl. Biol. Chem. 55: 141-145.
Kim BG, Kim HJ, Ahn J-H. 2012. Production of bioactive flavonol rhamnosides by expression of plant genes in Escherichia coli. J. Agric. Food Chem. 60: 11143-11148.
Kim MJ, Kim B-G, Ahn J-H. 2013. Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli. Appl. Microbiol. Biotechnol. 97: 7195-7204.
Lee Y-J, Jeon Y, Lee JS, Kim B-G, Lee CH, Ahn J-H. 2007. Enzymatic synthesis of phenolic CoAs using 4-coumarate:coenzyme A ligase (4CL) from rice. Bull. Kor. Chem. Soc. 28:365-366.
Leonard E, Lim H-K, Saw P-N, Koffas MAG. 2007. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl. Environ. Microbiol. 73: 3877-3886.
Leonard E, Yan Y, Fowler Z, Li Z, Kim C-C, Lim K-H, Koffas MAG. 2008. Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids. Mol. Pharm. 5: 257-265.
Lim CF, Fowler ZL, Hueller T, Schaffer S, Koffas MA. 2011. High-yield resveratrol production in engineered Escherichia coli. Appl. Environ. Microbiol. 77: 3451-3460.
Liu R, Wu C-X, Zhou D, Yang F, Tian S, Zhang L, et al. 2012. Pinocembrin protects against β-amyloid-induced toxicity in neurons through inhibiting receptor for advanced glycation end products (RAGE)-independent signaling pathways and regulating mitochondria-mediated apoptosis. BMC Med. 10: 105.
Miyahisa I, Funa N, Ohnishi Y, Martens S, Moriguchi T, Horinouchi S. 2006. Combinatorial biosynthesis of flavones and flavonols in Escherichia coli. Appl. Microbiol. Biotechnol. 71: 53-58.
Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S. 2005. Efficient production of (2S)flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl. Microbiol. Biotechnol. 68: 498-504.
Park SR, Ahn MS, Han AR, Park JW, Yoon YJ. 2011. Enhanced flavonoid production in Streptomyces venezuelae via metabolic engineering. J. Microbiol. Biotechnol. 21: 1143-1146.
Peng L, Yang S, Cheng YJ, Chen F, Pan S, Fan G. 2012. Antifungal activity and action mode of pinocembrin from propolis against Penicillium italicum. Food Sci. Biotechnol. 21:1533-1539.
Rasul A, Millimouno FM, Eltayb WA, Ali M, Li J, Li X. 2013. Pinocembrin: a novel natural compound with versatile pharmacological and biological activities. Biomed. Res. Int. 2013: 1.
Rösler J, Krekel F, Amrhein N, Schmid J. 1997. Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiol. 113: 175-179.
Santos CNS, Koffas M, Stephanopoulos G. 2011. Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab. Eng. 13: 392-400.
Vogt T. 2010. Phenylpropanoid biosynthesis. Mol. Plant 3: 2-20.
Weston RJ, Mitchella KR, Allen KL. 1999 Antibacterial phenolic components of New Zealand manuka honey. Food Chem. 64: 295-301.
Winkel-Shirley B. 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126: 485-493.
Wu J, Du G, Zhou J, Chen J. 2013. Metabolic engineering of Escherichia coli for (2S)-pincocembrin production from glucose by a modular metabolic strategy. Metab. Eng. 16: 48-55.
Yan Y, Kohli A, Koffas MAG. 2005. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 71: 5610-5613.
Yang N, Qin S, Wang M, Chen B, Yuan N, Fang Y, et al. 2013. Pinocembrin, a major flavonoid in propolis, improves the biological functions of EPCs derived from rat bone marrow through the PI3K-eNOS-NO signaling pathway. Cytotechnology 65: 541-551.
Yenjai C, Wanich S, Pitchuanchom S, Sripanidkulchai B. 2009. Structural modification of 5,7-dimethoxyflavone from Kaempferia parviflora and biological activities. Arch. Pharm. Res. 32: 1179-1184.

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J. Microbiol. Biotechnol. 2014; 24(11): 1536-1541

Published online November 28, 2014 https://doi.org/10.4014/jmb.1406.06011

Biosynthesis of Pinocembrin from Glucose Using Engineered Escherichia coli

Bong Gyu Kim ¹, Hyejin Lee ² and Joong-Hoon Ahn ^2*

¹Department of Forest Resources, Gyeongnam National University of Science and Technology, Jinju-si 660-758, Republic of Korea, ²Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul 143-701, Republic of Korea

Received: June 5, 2014; Accepted: July 28, 2014

Abstract

Pinocembrin is a flavonoid that exhibits diverse biological properties. Although the major
source of pinocembrin is propolis, it can be synthesized biologically using microorganisms
such as Escherichia coli, which has been used to synthesize diverse natural compounds.
Pinocembrin is synthesized from phenylalanine by the action of three enzymes; phenylalanine
ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS). In order
to synthesize pinocembrin from glucose in Escherichia coli, the PAL, 4CL, and CHS genes from
three different plants were introduced into an E. coli strain. Next, we tested the different
constructs containing 4CL and CHS. In addition, the malonyl-CoA level was increased by
overexpressing acetyl-CoA carboxylase. Through these strategies, a high production yield (97
mg/l) of pinocembrin was achieved.

Keywords: flavonoid biosynthesis, metabolic engineering, pinocembrin

Abstract

References

Aboushoer MI, Fathy HM, Abdel-Kader MS, Goetz G, Omara AA. 2010. Terpenes and flavonoids from an Egyptian collection of Cleome droserifolia. Nat. Prod. Res. 24: 687-696.
Austin MB, Noel JP. 2003. The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 20: 79-110.
Cochrane FC, Davin LB, Lewis NG. 2004. The Arabidopsis phenylalanine ammonia lyase gene family: kinetic characterization of the four PAL isoform. Phytochemistry 65: 1557-1564.
Dixon RA, Paiva NL. 1995. Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085-1097.
Hamberger B, Hahlbrock K. 2004. The 4-coumarate:CoA ligase gene family in Arabidopsis thaliana comprises one rare, sinapate-activating and three commonly occurring isoenzymes. Proc. Natl. Acad. Sci. USA 101: 2209-2214.
Houghton PJ, Woldemariam TZ, Davey W, Basar A, Lau C. 1995. Quantitation of the pinocembrin content of propolis by densitomety and high performance liquid chromatography. Phytochem. Anal. 6: 207-210.
Hwang EI, Kaneko M, Ohnishi Y, Horinouchi S. 2003. Production of plant-specific flavanoes by Escherichia coli containing an artificial gene cluster. Appl. Environ. Microbiol. 69: 2699-2707.
Jangaard NO. 1974. The characterization of phenylalanine ammonia-lyase from several plant species. Phytochemistry 13:1765-1768.
Jaganath IB, Crozier A. 2010. Dietary Flavonoids and Phenolic Compound in Plant Phenolics and Human Health. Fraga CG (ed.). John Wiley & Sons, Hoboken, New Jersey.
Kim B-G, Lee E-R, Ahn J-H. 2012. Analysis of flavonoid contents and expression of flavonoid biosynthetic genes in Populus euramericana Guinier in response to abiotic stress. J. Kor. Soc. Appl. Biol. Chem. 55: 141-145.
Kim BG, Kim HJ, Ahn J-H. 2012. Production of bioactive flavonol rhamnosides by expression of plant genes in Escherichia coli. J. Agric. Food Chem. 60: 11143-11148.
Kim MJ, Kim B-G, Ahn J-H. 2013. Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli. Appl. Microbiol. Biotechnol. 97: 7195-7204.
Lee Y-J, Jeon Y, Lee JS, Kim B-G, Lee CH, Ahn J-H. 2007. Enzymatic synthesis of phenolic CoAs using 4-coumarate:coenzyme A ligase (4CL) from rice. Bull. Kor. Chem. Soc. 28:365-366.
Leonard E, Lim H-K, Saw P-N, Koffas MAG. 2007. Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli. Appl. Environ. Microbiol. 73: 3877-3886.
Leonard E, Yan Y, Fowler Z, Li Z, Kim C-C, Lim K-H, Koffas MAG. 2008. Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids. Mol. Pharm. 5: 257-265.
Lim CF, Fowler ZL, Hueller T, Schaffer S, Koffas MA. 2011. High-yield resveratrol production in engineered Escherichia coli. Appl. Environ. Microbiol. 77: 3451-3460.
Liu R, Wu C-X, Zhou D, Yang F, Tian S, Zhang L, et al. 2012. Pinocembrin protects against β-amyloid-induced toxicity in neurons through inhibiting receptor for advanced glycation end products (RAGE)-independent signaling pathways and regulating mitochondria-mediated apoptosis. BMC Med. 10: 105.
Miyahisa I, Funa N, Ohnishi Y, Martens S, Moriguchi T, Horinouchi S. 2006. Combinatorial biosynthesis of flavones and flavonols in Escherichia coli. Appl. Microbiol. Biotechnol. 71: 53-58.
Miyahisa I, Kaneko M, Funa N, Kawasaki H, Kojima H, Ohnishi Y, Horinouchi S. 2005. Efficient production of (2S)flavanones by Escherichia coli containing an artificial biosynthetic gene cluster. Appl. Microbiol. Biotechnol. 68: 498-504.
Park SR, Ahn MS, Han AR, Park JW, Yoon YJ. 2011. Enhanced flavonoid production in Streptomyces venezuelae via metabolic engineering. J. Microbiol. Biotechnol. 21: 1143-1146.
Peng L, Yang S, Cheng YJ, Chen F, Pan S, Fan G. 2012. Antifungal activity and action mode of pinocembrin from propolis against Penicillium italicum. Food Sci. Biotechnol. 21:1533-1539.
Rasul A, Millimouno FM, Eltayb WA, Ali M, Li J, Li X. 2013. Pinocembrin: a novel natural compound with versatile pharmacological and biological activities. Biomed. Res. Int. 2013: 1.
Rösler J, Krekel F, Amrhein N, Schmid J. 1997. Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity. Plant Physiol. 113: 175-179.
Santos CNS, Koffas M, Stephanopoulos G. 2011. Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab. Eng. 13: 392-400.
Vogt T. 2010. Phenylpropanoid biosynthesis. Mol. Plant 3: 2-20.
Weston RJ, Mitchella KR, Allen KL. 1999 Antibacterial phenolic components of New Zealand manuka honey. Food Chem. 64: 295-301.
Winkel-Shirley B. 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126: 485-493.
Wu J, Du G, Zhou J, Chen J. 2013. Metabolic engineering of Escherichia coli for (2S)-pincocembrin production from glucose by a modular metabolic strategy. Metab. Eng. 16: 48-55.
Yan Y, Kohli A, Koffas MAG. 2005. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 71: 5610-5613.
Yang N, Qin S, Wang M, Chen B, Yuan N, Fang Y, et al. 2013. Pinocembrin, a major flavonoid in propolis, improves the biological functions of EPCs derived from rat bone marrow through the PI3K-eNOS-NO signaling pathway. Cytotechnology 65: 541-551.
Yenjai C, Wanich S, Pitchuanchom S, Sripanidkulchai B. 2009. Structural modification of 5,7-dimethoxyflavone from Kaempferia parviflora and biological activities. Arch. Pharm. Res. 32: 1179-1184.