树莓酮是一种在工业上有多种用途的重要香料物质。将来源于不同植物的4-香豆酰辅酶A连接酶(4-coumarate: CoA ligase,4CL1)、苄基丙酮合酶(benzalacetone synthase,BAS)和苄基丙酮还原酶(raspberry ketone/zingerone synthase,RZS1)基因分别克隆到载体pCDF-DUET-1和pET-DUET-1中,构建重组质粒并转入大肠杆菌W3110(DE3)中,成功获得了以对香豆酸为前体发酵生产覆盆子酮的重组菌株WCC-1。该菌摇瓶发酵培养2 h后,添加0.8 mmol/L的异丙基硫代半乳糖苷(isopropyl β-D-thiogalactoside,IPTG),30 ℃诱导,发酵72 h条件下,产覆盆子酮的质量浓度达到125.86 mg/L;在3 L发酵罐中进行补料分批发酵68 h,覆盆子酮的质量浓度提高到178.13 mg/L。该研究展现了微生物发酵生产树莓酮的前景。
Raspberry ketone is a multifunctional and important spice used in industries. In this study, the genes of 4-coumarate CoA ligase (4CL1), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from different plants were cloned into plasmids pCDF-DUET-1 and pET-DUET-1, respectively. The recombinant plasmids were transferred into Escherichia coli W3110 (DE3). A recombinant strain WCC-1 that produced raspberry ketone from p-coumaric acid was successfully obtained. After the strain was cultured for 2 h, 0.8 mmol/L isopropyl thiogalactoside was added and induced at 30 ℃. The concentration of raspberry ketone reached 125.86 mg/L after 72 h fermentation. When the fed-batch fermentation was carried out for 68 h in a 3 L fermentor, the concentration of raspberry ketone increased to 178.13 mg/L. This study suggests the prospect of microbial fermentation to produce raspberry ketone.
[1] 孙宝国.日用化工辞典[M].北京:化学工业出版社精细化工出版中心,2002:171-172.
[2] 唐健.复盆子酮的合成及应用[J].化工技术与开发,2006,35(9):21-23.
[3] 孙宝国,何坚.香料化学与工艺学[M].第二版.北京:化学工业出版社,2004:166-196.
[4] LEE J. Further research on the biological activities and the safety of raspberry ketone is needed[J].NFS Journal,2016,2:15-18.
[5] BEEKWILDER J,VAN DER MEER I M,SIBBESEN O,et al.Microbial production of natural raspberry ketone[J].Biotechnol Journol,2007,2(10):1 270.
[6] 李红武,张强,孙力,等.一种烟梗丝状成型加工工艺:中国, CN102631016A[P]. 2012-08-15.
[7] KIM M,BAEK H S,LEE M,et al.Rhododenol and raspberry ketone impair the normal proliferation of melanocytes through reactive oxygen species-dependent activation of GADD45[J].Toxicology in Vitro,2016,32:339-346.
[8] 丹尼尔·雷德尔斯多夫,彼得·韦伯,斯文·沃尔夫拉姆.含没食子酸表儿茶素和树莓酮的营养补充组合物:中国, CN1717268[P]. 2006-01-04.
[9] MORIMOTO C,SATOH Y,HARA M,et al.Anti-obese action of raspberry ketone[J].Life Sciences,2005,77(2):194-204.
[10] WANG Lili,MENG Xianjun,ZHANG Fengqing.Raspberry ketone protects rats fed high-fat diets against nonalcoholic steatohepatitis[J].Journal of Medicinal Food,2012,15(5):495-503.
[11] HAKKINEN S T,SEPPANEN-LAAKSO T,OKSMAN-CALDENTEY K M,et al.Bioconversion to raspberry ketone is achieved by several non-related plant cell cultures[J].Frontiers in Plant Sciences,2015,6:1 035.
[12] LARSEN M,POLL L,CALLESEN O,et al.Relations between the content of aroma compounds and the sensory evaluation of 10 raspberry varieties (Rubus idaeus L.)[J].Acta Agriculturae Scandinavica,1991,41(4):447-454.
[13] FISCHER M,BÖKER A,BERGER R G.Raspberry ketone from submerged cultured cells of the Basidiomycete Nidula niveo-tomentosa[J].Biotechnology Progress,2001,17(3):568-572.
[14] STABNIKOVA O,Wang Jing-yuan,IVANOV V. Value-Added Biotechnological Products from Organic Wastes[M].Humana Press,2010.
[15] 张奋强,刘欢,黄丽娜,等.树莓酮生物合成途径及关键酶功能研究进展[J].生物技术进展,2017,7(2):111-115.
[16] LEE D,LLOYD N D,PRETORIUS I S,et al.Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via pathway engineering and synthetic enzyme fusion[J].Microbial Cell Factories,2016,15(1):49.
[17] LIM C G,FOWLER Z L,HUELLER T,et al.High-yield resveratrol production in engineered Escherichia coli[J].Applied and Environmental Microbiology,2011,77(10):3 451-3 460.
[18] ABE I,TAKAHASHI Y,MORITA H,et al.Benzalacetone synthase. A novel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum[J].European Journal of Biochemistry,2001,268(11):3 354-3 359.
[19] KOEDUKA T,WATANABE B,SUZUKI S,et al.Characterization of raspberry ketone/zingerone synthase, catalyzing the alpha, beta-hydrogenation of phenylbutenones in raspberry fruits[J]. Biochemical and Biophysical Research Communications,2011,412(1):104-108.
