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Acta Microbiologica Sinica 2017, 57(3): 447-458 http://journals.im.ac.cn/actamicrocn DOI: 10.13343/j.cnki.wsxb.

1 Acta Microbiologica Sinica 2017, 57(3): DOI: /j.cnki.wsxb Research Article 研究报告操纵茶树类黄酮 3 - 羟基化酶生物合成 B 环 -3,4 - 二羟基黄酮类化合物周天山 *, 余有本, 肖斌, 鮑露, 高岳芳摘要 : 3 - B -3, (CsF3 H) P450 (ATR) SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA TOP10 DH5α BL21 12 S1 S CsF3 H pyes-dest52-csf3 H WAT11 S CsF3 H pes-ura-csf3 H 3- CsF3H AtFLS pes-his-csf3h::atfls 9AA WAT11 S14 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA TOP10 S6 25 C 1000 μmol/l μmol/l μmol/l μmol/l S μmol/l μmol/l μmol/l μmol/l S14 5 mmol/l h μmol/l μmol/l μmol/l μmol/l μmol/l 3 - B -3,4 - 关键词 : 3 - B -3,4 - [1] 2 (A B ) (C ) 15 [2] 基金项目 : (CARS-23) * 通信作者 Tel/Fax zhoutianshan@nwsuaf.edu.cn 收稿日期 : 修回日期 : 网络出版日期 :

2 448 Tianshan Zhou et al. Acta Microbiologica Sinica, 2017, 57(3) [3 5] (combinatorial biosynthesis) [6 7] [8] [9 10] B B 4 - B -3,4 - B -3, 4,5 - B 4 - A B 3 B 3,5 2 P (F3 H) 3,5 - (F3 5 H) [11 14] 3 - B % 24% ( ) [16] 3 - P B -3,4 - [15] 图 1. B 环 -3,4 - 二羟基黄酮类化合物生物合成途径 Figure 1. The biosynthetic pathway of B-3,4 -dihydroxylated flavones. F3 H: flavonoid 3 -hydroxylase; F3H: flavanone 3-hydroxylase; FLS: flavonol synthase [15]. actamicro@im.ac.cn

3 , 2017, 57(3) 材料和方法 ( ) Col-0 80 C Escherichia coli TOP10 E. coli DH5α E. coli BL21 Saccharomyces cerevisiae WAT11 pyes-dest52 pes-ura pes-his [(±)-naringenin] [(±)-eridodictyol] [(±)-dihydrokaempferol] (kaempferol) (dihydroquerctin) (quercetin) Sigma-Aldrich (USA) 1.2 TaKaRa RNAiso Plus Total RNA (Arabidopsis thaliana) (Camellia sinensis) RNA TaKaRa PrimeScript TM RT reagent kit cdna NCBI P450 (ATR) (FLS) 3 - (F3 H) 3- (F3H) PCR ( 1) 50 µl PCR 10 µl 5 HF PCR 4 µl dntps ( 10 mmol/l) 2.5 µl ( 10 µmol/l) 4 µl cdna ( 50 ng/µl) 0.5 µl Phusion High-Fidelity DNA 26.5 µl ddh 2 O PCR 98 C 3 min 98 C 20 s 63 C 30 s 68 C 60 s C 5 min 4 C 1% DNA PCR Invitrogen INC TOPO Cloning Kit PCR pentr E. coli TOP10 37 C h PCR 1.3 Expresso TM T7 sumo cloning kit (Lucigen) 1.2 pentr DNA CsF3 H ATR1 PCR CsF3 H ATR1 3 (Gly-Ser-Gly) 2 表 1. 用于基因克隆的引物 Table 1. Primers used for cloning process Access Nos. Primers Sequences (5 3 ) KT CsF3 H pentr F CACCATGACTTCCTTAGCTTTTGTTC CsF3 H pentr R TTAGGCCCGATACACATGGGGTG U AtFLS pentr F CACCATGGAGGTCGAAAGAGTCC AtFLS pentr R TCAATCCAGAGGAAGTTTATTGAG AY CsF3H pentr F CACCATGGCGCCAACAACAACGC CsF3H pentr R TCAAGCAAAAATCTCATCAGTGC NM ATR1 pentr F CACCATGACTTCTGCTTTGTATGCTTCCG ATR1 pentr R TCACCAGACATCTCTGAGGTATCTTCC NM ATR2 pentr F CACCATGTCCTCTTCTTCTTCTTCGTCAAC ATR2 pentr R TTACCATACATCTCTAAGATATCTTCCAC Underlined sequence indicates the 4 base pair sequences necessary for directional cloning on the 5 end of the forward primer.

4 450 Tianshan Zhou et al. Acta Microbiologica Sinica, 2017, 57(3) Primers CsF3 H[7-517] sumo F Table 2. CsF3 H [28-517] sumo F CsF3 H[7/28-517]::ATR1 mid R CsF3 H::ATR1[49-688] mid F ATR1 [49-688] sumo R CsF3 H [7/28-517]::ATR2 mid R CsF3 H::ATR2[75-711] mid F ATR2[75-711] sumo R CsF3H sumo F CsF3H::AtFLS mid 9 AA R CsF3H::AtFLS mid 9 AA F AtFLS sumo R CsF3 H EcoR I F CsF3 H Spe I R CsF3H::AtFLS EcoR I F CsF3H::AtFLS Spe I R 表 2. 用于构建表达质粒的引物 The primers used for constructing expression plasmids Sequences (5 3 ) CGCGAACAGATTGGAGGTGCAACTCTATTTCTCACA CGCGAACAGATTGGAGGTCGTCGCAACCGCAGCCAC ATCCGCCGTCGTTTTACCCGAACCACCCGACCCGAGTCC GGACTCGGGTCGGGTGGTTCGGGTAAAACGACGGCGGAT GTGGCGGCCGCTCTATTACCAGACATCTCTGAGGTATCTTCC ATTCCCAGAACCGGAACCCGAACCACCCGACCCGAGTCC GGACTCGGGTCGGGTGGTTCGGGTTCCGGTTCTGGGAAT GTGGCGGCCGCTCTATTACCATACATCTCTAAGATATCTTCC CGCGAACAGATTGGAGGTGCGCCAACAACAACGCTTACG GACTCTTTCGACCTCACCACCGGAGCCACCAGAGCCACCGCTAGCAAAAATCTCATC GATGAGATTTTTGCTAGCGGTGGCTCTGGTGGCTCCGGTGGTGAGGTCGAAAGAGTC GTGGCGGCCGCTCTATTATCAATCCAGAGGAAGTTTATT GAATTCATGACTTCCTTAGCTTTTGTTC ACTAGTTTAGGCCCGATACACATGGGGTG GAATTCGCGCCAACAACAACGCTTACG ACTAGTTTCAATCCAGAGGAAGTTTATT Underlined sequence indicates the sequences necessary for performing enzyme-free cloning. The linker sequences of the fusion cdna are indicated in italic. Boldface indicates restriction enzyme cleavage site. 1 PCR CsF3 H[7-517] sumo F CsF3 H[7/28-517]::ATR1 mid R CsF3 H[28-517] sumo F CsF3 H[7/28-517]::ATR1 mid R CsF3 H CsF3 H::ATR1[49-688] mid F ATR1[49-688] sumo R ATR1 2 PCR 1 PCR 2 DNA CsF3 H[7-517] sumo F ATR1[49-688] sumo R CsF3 H[28-517] sumo F ATR1[49-688] sumo R CsF3 H::ATR1 3 AA PCR SUMO SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA CsF3 H ATR2 SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA 9 (Ser-Gly-Gly-Ser- Gly-Gly-Ser-Gly-Gly) CsF3H::AtFLS cdna SUMO SUMO-CsF3H::AtFLS 9 AA Invitrogen LR Clonase II Plus Enzyme Mix LR pentr pyes-dest52 pyes-dest52-csf3 H CsF3 H CsF3H::AtFLS 9 AA pes-ura pes-his EcoRⅠ SpeⅠ pentr-csf3 H SUMO-F3H::FLS 9 AA pes-ura-csf3 H pes-his- CsF3H::AtFLS 9AA E. coli BL21 TOP10 DH5α S. cerevisiae WAT11 ZYMO RESEARCH Frozen-EZ Yeast Transformation Ⅱ TM ( 3) actamicro@im.ac.cn

5 , 2017, 57(3) 451 表 3. 所构建的生物发酵菌株 Table 3. Construction of strains used for fermentation No. Expressing plasmids Transformed strains S1 SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA E. coli BL21 S2 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA E. coli BL21 S3 SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA E. coli BL21 S4 SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA E. coli BL21 S5 SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA E. coli Top10 S6 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA E. coli Top10 S7 SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA E. coli Top10 S8 SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA E. coli Top10 S9 SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA E. coli DH5α S10 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA E. coli DH5α S11 SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA E. coli DH5α S12 SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA E. coli DH5α S13 pyes-dest52-csf3 H S. cerevisiae WAT11 S14 pes-ura-csf3 H pes-his-csf3h::atfls 9AA S. cerevisiae WAT S1-S12 LB 37 C 2 50 ml LB S1-S12 1 ml 37 C OD IPTG 0.5 mmol/l 25 C 30 C 2 h ( ) DMSO S13 ( S14) 15 ml SD-Urail ( SD-Urail-His) 30 C 24 h 50 ml 20 g/l SD-Urail 30 C 24 h 1600 g 5 min 20 ml 20 g/l SD-Urail ( SD-Urail-His) 1600 g 5 min 20 g/l SD-Urail ( SD-Urail-His) OD C 12 h ( ) h 1 ml 1 ml 100% 5 min g 10 min 0.2 μm SHIMADZU LC-20AT SPD-M20A Gl Sciences Inc Wondasil C 18 5 μm 4.6 mm 150 mm A B A 10 min 10% 40% 5 min 40% 60% 2 min 10% 1.0 ml/min 25 C 280 nm UV

6 452 Tianshan Zhou et al. Acta Microbiologica Sinica, 2017, 57(3) 2 结果和分析 CsF3 H P450 [15] N E. coli P450 (ATR) E. coli TMHMM [17] 4 CsF3 H::ATR E. coli TOP10 E. coli DH5α E. coli BL21 OD IPTG 2 h 25 C 30 C 24 h E. coli BL21 (S1 S4) SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA (S1 S3 S5 S7 S9 S11) SUMO-CsF3 H [28-517]::ATR1[49-688] 3 AA SUMO-CsF3 H [28-517]::ATR2[75-711] 3 AA TOP10 DH5α (S6 S8 S10 S12) C 30 C SUMO-CsF3 H[28-517]::ATR2[75-711] (S8 S12) SUMO-CsF3 H[28-517]:: ATR1[49-688] 3 AA (S6 S10) 1 DH5α (S10 S12) TOP10 (S6 S8) μmol/l μmol/l 1000 μmol/l μmol/l μmol/l μmol/l 图 2. CsF3 H 与 ATR 基因融合表达质粒构建 Figure 2. Constructing plasmid of fusion gene CsF3 H::ATR. A: SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA; B: SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA; C: SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA; D: SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA. SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA indicates construction of gene ensembles encoding CsF3 H[7-517] and ATR1[49-688]. Numbers correspond to the amino acids encoded by the first and last codons of the gene sequences. The other plasmids can be deduced by analogy. CsF3 H [15] S. cerevisiae WAT11 P450 [18] P450 [11 12,19] pyes-dest52-csf3'h WAT11(S13) h 1000 μmol/l h 4 actamicro@im.ac.cn

, 2017, 57(3) 453 图 3. 转化 CsF3 H 与 ATR 基因融合表达质粒菌株生成产物量 Figure 3. Content of product from different bacterial strains transformed fusion gene CsF3 H::ATR plasmids.

7 , 2017, 57(3) 453 图 3. 转化 CsF3 H 与 ATR 基因融合表达质粒菌株生成产物量 Figure 3. Content of product from different bacterial strains transformed fusion gene CsF3 H::ATR plasmids. Products from the strains incubated at 30 C with naringenin (A), dihydrokaempferol (C) and kaempferol (E) as substrates; products from the strains incubated at 25 C with naringenin (B), dihydrokaempferol (D) and kaempferol (F) as substrates. Duncan s multiple range test (P<0.05, n=3). Bars superscripted by different letters are significantly different at the 0.05 probability level. 图 4. 菌株 S13 生物发酵过程中产物变化 Figure 4. Products accumulation in strain S13 bioconversion. A: eriodictyol; B: dihydroquercetin; C: quercetin. Duncan s multiple range test (P<0.05, n=3). Bars superscripted by different letters are significantly different at the 0.05 probability level.

8 454 Tianshan Zhou et al. Acta Microbiologica Sinica, 2017, 57(3) h 1000 μmol/l μmol/l 1000 μmol/l μmol/l 1000 μmol/l μmol/l ( 3) [20] CsF3 H CsF3H::AtFLS 9 AA pes-ura pes-his WAT11 5 mmol/l 5 图 5. 共表达酵母菌株 S14 生成产物液相色谱图 Figure 5. HPLC chromatograms of products from strain S14 co-transformed pes-ura-csf3 H and pes-his-csf3h::atfls 9 AA. A: standards; B: the strain WAT11 co-transformed with pes-ura-csf3 H and pes-his-csf3h::atfls 9 AA; C: the strain WAT11 co-transformed with pes-ura and pes-his. DHM: dihydromyricetin; DHQ: dihydroquercetin; MYC: myricetin; DHK: dihydrokaempferol; PEN: pentahydroxyflavonone; ERI: eridictyol; QUE: quercetin; NAR: naringenin; KAE: kaempferol. actamicro@im.ac.cn

, 2017, 57(3) 455 CsF3H::AtFLS 9 AA CsF3 H 6 48 h 1412.16 μmol/l 36 h 490.

DHQ: dihydroquercetin; DHK: dihydrokaempferol; ERI: eriodictyol; QUE: quercetin; KAE:

9 , 2017, 57(3) 455 CsF3H::AtFLS 9 AA CsF3 H 6 48 h μmol/l 36 h μmol/l 48 h μmol/l 48 h μmol/l μmol/l 图 6. 酵母共表达菌株 S14 生物发酵过程中各产物量变化 Figure 6. Products accumulation in coexpression strain S14 bioconversion. DHQ: dihydroquercetin; DHK: dihydrokaempferol; ERI: eriodictyol; QUE: quercetin; KAE: kaempferol. Duncan s multiple range test (P<0.05, n=3). Bars superscripted by different letters are significantly different at the 0.05 probability level.

10 456 Tianshan Zhou et al. Acta Microbiologica Sinica, 2017, 57(3) 3 讨论 [21 24] [25] P450 [17,26] P450 P450 P450 [27] P450 [28 29] CsF3 H P450 (ATR1 ATR2) 4 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA (S6 S8 S10 S12) SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA (S5 S7 S9 S11) P450 BL21 TOP10 DH5α 4 BL21 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[28-517]::ATR2 [75-711] 3 AA TOP10 DH5α (S6 S8 S10 S12) P450 [30] TOP10 DH5α JM109 BL21 ( 3) 30 C CsF3 H ( 3 4) 结论 (CsF3 H) P450 (ATR) SUMO-CsF3 H[7-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA SUMO-CsF3 H[7-517]::ATR2[75-711] 3 AA SUMO-CsF3 H[28-517]::ATR2[75-711] 3 AA TOP10 DH5α BL21 SUMO-CsF3 H[28-517]::ATR1[49-688] 3 AA TOP10 25 C 1000 μmol/l μmol/l μmol/l μmol/l pyes-dest52-csf3 H WAT μmol/l μmol/l μmol/l μmol/l 5 mmol/l pes-ura- actamicro@im.ac.cn

11 , 2017, 57(3) 457 CsF3 H pes-his-csf3h::atfls 9AA WAT h μmol/l μmol/l μmol/l μmol/l μmol/l [1] Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 2001, 126(2): [2] Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. The Scientific World Journal, 2013, 2013: [3] Kumar S, Gupta A, Pandey AK. Calotropis procera root extract has the capability to combat free radical mediated damage. ISRN Pharmacology, 2013, 2013: [4] Ross JA, Kasum CM. Dietary flavonoids: bioavailability, metabolic effects, and safety. Annual Review of Nutrition, 2003, 22: [5] Peterson J, Dwyer J. Flavonoids: dietary occurrence and biochemical activity. Nutrition Research, 1998, 18(12): [6] Wang YC, Chen S, Yu O. Metabolic engineering of flavonoids in plants and microorganisms. Applied Microbiology and Biotechnology, 2011, 91(4): [7] Wang AM, Zhang FK, Huang LF, Yin XP, Li HF, Wang QY, Zeng ZW, Xie T. New progress in biocatalysis and biotransformation of flavonoids. Journal of Medicinal Plants Research, 2010, 4(10): [8] Trantas EA, Koffas MA, Xu P, Ververidis F. When plants produce not enough or at all: metabolic engineering of flavonoids in microbial hosts. Frontiers in Plant Science, 2015, 6: 7. [9] Kumar S, Mishra A, Pandey AK. Antioxidant mediated protective effect of Parthenium hysterophorus against oxidative damage using in vitro models. BMC Complementary and Alternative Medicine, 2013, 13: 120. [10] Kumar S, Pandey A K. Phenolic content, reducing power and membrane protective activities of Solanum xanthocarpum root extracts. International Journal of Plant Research, 2013, 26(1): [11] Wang YS, Xu YJ, Gao LP, Yu O, Wang XZ, He XJ, Jiang XL, Liu YJ, Xia T. Functional analysis of flavonoid 3, 5 -hydroxylase from tea plant (Camellia sinensis): critical role in the accumulation of catechins. BMC Plant Biology, 2014, 14: 347. [12] Olsen KM, Hehn A, Jugdé H, Slimestad R, Larbat R, Bourgaud F, Lillo C. Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum. BMC Plant Biology, 2010, 10: 21. [13] Brugliera F, Barri-Rewell G, Holton TA, Mason JG. Isolation and characterization of a flavonoid 3 -hydroxylase cdna clone corresponding to the Ht1 locus of Petunia hybrida. The Plant Journal, 1999, 19(4): [14] Schoenbohm C, Martens S, Eder C, Forkmann G, Weisshaar B. Identification of the Arabidopsis thaliana flavonoid 3 -hydroxylase gene and functional expression of the encoded P450 enzyme. Biological Chemistry, 2000, 381(8): [15] Zhou TS, Zhou R, Yu YB, Xiao Y, Li DH, Xiao B, Yu O, Yang YJ. Cloning and characterization of a flavonoid 3 -hydroxylase gene from tea plant (Camellia sinensis). International Journal of Molecular Sciences, 2016, 17(2): 261. [16].. 3. :, 2003: [17] Leonard E, Yan YJ, Koffas MAG. Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metabolic Engineering, 2006, 8(2): [18] Pompon D, Louerat B, Bronine A, Urban P. Yeast expression of animal and plant P450s in optimized redox environments. Methods in Enzymology, 1996, 272: [19] Teoh KH, Polichuk DR, Reed DW, Nowak G, Covello PS. Artemisia annua L. (Asteraceae) trichome-specific cdnas reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin. FEBS Letters, 2006, 580(5): [20] Kim DH, Kim BG, Jung NR, Ahn JH. Production of genistein from naringenin using Escherichia coli containing isoflavone synthase-cytochrome P450 reductase fusion protein. Journal of Microbiology and Biotechnology, 2009, 19(12): [21] Draths KM, Knop DR, Frost JW. Shikimic acid and quinic acid: replacing isolation from plant sources with recombinant microbial biocatalysis. Journal of the American Chemical Society, 1999, 121(7): [22] Huang QL, Roessner CA, Croteau R, Scott AI. Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol. Bioorganic & Medicinal Chemistry, 2001, 9(9): [23] Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology, 2003, 21(7): [24] Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE, Khosla C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science, 2001, 291(5509): [25] Alper H, Miyaoku K, Stephanopoulos G. Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nature Biotechnology, 2005, 23(5): [26] Chang MCY, Keasling JD. Production of isoprenoid pharmaceuticals by engineered microbes. Nature Chemical Biology, 2006, 2(12): [27] Sevrioukova IF, Li HY, Zhang H, Peterson JA, Poulos TL.

12 458 Tianshan Zhou et al. Acta Microbiologica Sinica, 2017, 57(3) Structure of a cytochrome P450-redox partner electron-transfer complex. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(5): [28] Barnes HJ, Arlotto MP, Waterman MR. Expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylase in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(13): [29] Williams PA, Cosme J, Sridhar V, Johnson EF, McRee DE. Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features. Journal of Inorganic Biochemistry, 2000, 81(3): [30] Leonard E, Koffas MAG. Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli. Applied and Environmental Microbiology, 2007, 73(22): Engineering of a flavonoid 3 -hydroxylase from tea plant (Camellia sinensis) for biosynthesis of B-3,4 -dihydroxylated flavones Tianshan Zhou *, Youben Yu, Bin Xiao, Lu Bao, Yuefang Gao College of Horticulture, Northwest A & F University, Yangling , Shaanxi Province, China Abstract: [Objective] A flavonoid 3 -hydroxylase from tea plant was engineered to synthesize B-3,4 -dihydroxylated flavones such as eriodictyol, dihydroquercetin and quercetin. [Methods] Four articifical P450 constructs harboring both flavonoid 3 -hydroxylase gene from Camellia sinensis (CsF3 H) and P450 reductase gene from Arabidopsis thaliana (ATR1 or ATR2) were introduced into Escherichia coli strains TOP10, DH5α and BL21, resultantly engineering strains S1 to S12. The plasmid pyes-dest52-csf3 H harboring CsF3 H gene was introduced into yeast Saccharomyces cerevisiae WAT11 designated as strain S13. The plasmid pes-his-csf3h::atfls 9 AA was constructed through fusing flavanone 3-hydroxylase gene from Camellia sinensis (CsF3H) and flavonol synthase gene from Arabidopsis thaliana (AtFLS). Plasmid pes-ura-csf3 H and pes-his-csf3h::atfls 9 AA were then co-introduced into yeast S. cerevisiae WAT11 designated as strain S14. [Results] Strain S6 generated highest bioconversion efficiency at 25 C among all E. coli strains during 24 h fernentation. Supplemented with 1000 μmol/l naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by strain S13 were μmol/l, μmol/l and μmol/l respectively. Supplemented with 5 mmol/l naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by strain S14 were μmol/l, μmol/l, μmol/l, μmol/l and μmol/l during h fermentaion respectively. [Conclusion] CsF3 H was engineered for biosynthesis of B-3,4 -dihydroxylated flavone. Keywords: flavonoid 3 -hydroxylase from Camellia sinensis, B-3,4 -dihydroxylated flavones, biosynthesis ( 本文责编 : 张晓丽 ) Supported by the Earmarked Fund for Modern Agro-industry Technology System (CARS-23) * Corresponding author. Tel/Fax: ; zhoutianshan@nwsuaf.edu.cn Received: 19 August 2016; Revised: 1 December 2016; Published online: 12 December 2016 actamicro@im.ac.cn

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇