Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (4): 114-123.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0572
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AI Lu(), CHEN Wen-hui, SHI Jing-hui, REN Zhi-yuan, SHEN Wen-qi, YANG Jia-ning, LUO Jian-mei(), WANG Min
Received:
2022-05-09
Online:
2023-04-26
Published:
2023-05-16
AI Lu, CHEN Wen-hui, SHI Jing-hui, REN Zhi-yuan, SHEN Wen-qi, YANG Jia-ning, LUO Jian-mei, WANG Min. Cloning and Expression of 11α Hydroxylase from Aspergillus ochraceus and Analysis of Key Amino Acid Sites[J]. Biotechnology Bulletin, 2023, 39(4): 114-123.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 酶切位点 Restriction site | 引物用途 Primer application |
---|---|---|---|
11αAO-F | ATGCCCTTCTTCACTGGGCTTCTGGCGAT | 无 | 从赭曲霉cDNA扩增11α羟化酶基因 |
11αAO-R | CTACACAGTTAAACTCGCCATATCGATCTCCGCC | 无 | Amplification of 11α hydroxylase gene from cDNA of Aspergillus ochraceus |
CYP68J5-F | GCCGCCAGTGTGCTGGAATTCATGCCATTTTTTA- CTGGTTTGTTG | EcoR I | 构建酿酒酵母表达CYP68J5重组菌株 Construction of the recombinant S. cerevisiae expressing CYP68J5 |
CYP68J5-R | TACATGATGCGGCCCTCTAGATTAAACAGTCAA- AGAAGCCATATCAA | Xba I | |
CYP68J5-F | CGGGATCCCATGAACATCAAGAAGTTCGCCAAG | BamH I | 构建毕赤酵母表达CYP68J5重组菌株 Construction of the recombinant P. pastorisexpressing CYP68J5 |
CYP68J5-R | CGGAATTCTCACTTGTTGACGGTGAGCTGCC | EcoR I | |
CYP68J5-F | CGGAATTCATGCCGTTCTTCACCGGCC | EcoR I | 构建紫红红球菌表达CYP68J5重组菌株 |
CYP68J5-R | GCTCTAGATCTAGATTAGACGGTGAGCGAGGCC | Xba I | Construction of the recombinant R. rhodochrous expressing CYP68J5 |
CYP68J5_W89A-F | GCGGGTTCTTTGATTGTTTTGCCAC | 无 | 构建CYP68J5突变体 |
CYP68J5_W89A-R | TTCAGACATCAATCTATATGGTTCATGAC | 无 | Construction of mutants of CYP68J5 |
CYP68J5_F111A-F | GCTGAAACTCCAACTACTGATGATTCTC | 无 | |
CYP68J5_F111 A-R | ATCCATTCTTGGATCATTTCTCAATTC | 无 | |
CYP68J5_T115A-F | GCTACTGATGATTCTCATGGTTATATTCC | 无 | |
CYP68J5_T115A-R | TGGAGTTTCAAAATCCATTCTTGGATC | 无 | |
CYP68J5_D118A-F | GCTTCTCATGGTTATATTCCTGGTTTTG | 无 | |
CYP68J5_D118A-R | ATCAGTAGTTGGAGTTTCAAAATCCAT | 无 | |
CYP68J5_S119A-F | GCTCATGGTTATATTCCTGGTTTTG | 无 | |
CYP68J5_S119A-R | ATCATCAGTAGTTGGAGTTTCAAAATC | 无 | |
CYP68J5_L129A-F | GCAAATGCTGATCCAAATTTGACTAAAG | 无 | |
CYP68J5_L129A-R | AGCATCAAAACCAGGAATATAACCATG | 无 | |
CYP68J5_F216A-F | GCTGGTGTTGGTGATAAATTGAGAAT | 无 | |
CYP68J5_F216A-R | AGCCAAAGCAGCATATTGAGAAGAAGTT | 无 | |
CYP68J5_R223A-F | GCAATTTATCCAAGAATGATTAGACC | 无 | |
CYP68J5_R223A-R | CAATTTATCACCAACACCAAAAGCC | 无 | |
CYP68J5_I303A-F | GCTGTTGCTATTCATACTACTTCTG | 无 | |
CYP68J5_I303A-R | AGACAAAGTAACTTGTTTCAAAACAG | 无 | |
CYP68J5_V304A-F | GCTGCTATTCATACTACTTCTGATTTGT | 无 | |
CYP68J5_V304A-R | AATAGACAAAGTAACTTGTTTCAAAAC | 无 | |
CYP68J5_T308A-F | GCTACTTCTGATTTGTTATTGCAAGCT | 无 | |
CYP68J5_T308A-R | ATGAATAGCAACAATAGACAAAGTAAC | 无 | |
CYP68J5_L368A-F | GCGTTGGGATCTTTCAGAAGACAAG | 无 | |
CYP68J5_L368A-R | AGTTGGTCTCAATCTTTGAGATTCTTTC | 无 | |
CYP68J5_R373A-F | GCAAGACAAGCTACTAATGATATTAAATT | 无 | |
CYP68J5_R373A-R | GAAAGATCCCAACAAAGTTGGTCTC | 无 | |
CYP68J5_M488A-F | GCGACTTATTTGGCTGATCCAAACAC | 无 | |
CYP68J5_M488A-R | ACCAATATTCAATGGTTGTGGTTTAAAAC | 无 | |
CYP68J5_T489A-F | GCTTATTTGGCTGATCCAAACACTAG | 无 | |
CYP68J5_T489A-R | CATACCAATATTCAATGGTTGTGGTTT | 无 |
Table 1 Primers used in this study
引物名称 Primer name | 引物序列 Primer sequence(5'-3') | 酶切位点 Restriction site | 引物用途 Primer application |
---|---|---|---|
11αAO-F | ATGCCCTTCTTCACTGGGCTTCTGGCGAT | 无 | 从赭曲霉cDNA扩增11α羟化酶基因 |
11αAO-R | CTACACAGTTAAACTCGCCATATCGATCTCCGCC | 无 | Amplification of 11α hydroxylase gene from cDNA of Aspergillus ochraceus |
CYP68J5-F | GCCGCCAGTGTGCTGGAATTCATGCCATTTTTTA- CTGGTTTGTTG | EcoR I | 构建酿酒酵母表达CYP68J5重组菌株 Construction of the recombinant S. cerevisiae expressing CYP68J5 |
CYP68J5-R | TACATGATGCGGCCCTCTAGATTAAACAGTCAA- AGAAGCCATATCAA | Xba I | |
CYP68J5-F | CGGGATCCCATGAACATCAAGAAGTTCGCCAAG | BamH I | 构建毕赤酵母表达CYP68J5重组菌株 Construction of the recombinant P. pastorisexpressing CYP68J5 |
CYP68J5-R | CGGAATTCTCACTTGTTGACGGTGAGCTGCC | EcoR I | |
CYP68J5-F | CGGAATTCATGCCGTTCTTCACCGGCC | EcoR I | 构建紫红红球菌表达CYP68J5重组菌株 |
CYP68J5-R | GCTCTAGATCTAGATTAGACGGTGAGCGAGGCC | Xba I | Construction of the recombinant R. rhodochrous expressing CYP68J5 |
CYP68J5_W89A-F | GCGGGTTCTTTGATTGTTTTGCCAC | 无 | 构建CYP68J5突变体 |
CYP68J5_W89A-R | TTCAGACATCAATCTATATGGTTCATGAC | 无 | Construction of mutants of CYP68J5 |
CYP68J5_F111A-F | GCTGAAACTCCAACTACTGATGATTCTC | 无 | |
CYP68J5_F111 A-R | ATCCATTCTTGGATCATTTCTCAATTC | 无 | |
CYP68J5_T115A-F | GCTACTGATGATTCTCATGGTTATATTCC | 无 | |
CYP68J5_T115A-R | TGGAGTTTCAAAATCCATTCTTGGATC | 无 | |
CYP68J5_D118A-F | GCTTCTCATGGTTATATTCCTGGTTTTG | 无 | |
CYP68J5_D118A-R | ATCAGTAGTTGGAGTTTCAAAATCCAT | 无 | |
CYP68J5_S119A-F | GCTCATGGTTATATTCCTGGTTTTG | 无 | |
CYP68J5_S119A-R | ATCATCAGTAGTTGGAGTTTCAAAATC | 无 | |
CYP68J5_L129A-F | GCAAATGCTGATCCAAATTTGACTAAAG | 无 | |
CYP68J5_L129A-R | AGCATCAAAACCAGGAATATAACCATG | 无 | |
CYP68J5_F216A-F | GCTGGTGTTGGTGATAAATTGAGAAT | 无 | |
CYP68J5_F216A-R | AGCCAAAGCAGCATATTGAGAAGAAGTT | 无 | |
CYP68J5_R223A-F | GCAATTTATCCAAGAATGATTAGACC | 无 | |
CYP68J5_R223A-R | CAATTTATCACCAACACCAAAAGCC | 无 | |
CYP68J5_I303A-F | GCTGTTGCTATTCATACTACTTCTG | 无 | |
CYP68J5_I303A-R | AGACAAAGTAACTTGTTTCAAAACAG | 无 | |
CYP68J5_V304A-F | GCTGCTATTCATACTACTTCTGATTTGT | 无 | |
CYP68J5_V304A-R | AATAGACAAAGTAACTTGTTTCAAAAC | 无 | |
CYP68J5_T308A-F | GCTACTTCTGATTTGTTATTGCAAGCT | 无 | |
CYP68J5_T308A-R | ATGAATAGCAACAATAGACAAAGTAAC | 无 | |
CYP68J5_L368A-F | GCGTTGGGATCTTTCAGAAGACAAG | 无 | |
CYP68J5_L368A-R | AGTTGGTCTCAATCTTTGAGATTCTTTC | 无 | |
CYP68J5_R373A-F | GCAAGACAAGCTACTAATGATATTAAATT | 无 | |
CYP68J5_R373A-R | GAAAGATCCCAACAAAGTTGGTCTC | 无 | |
CYP68J5_M488A-F | GCGACTTATTTGGCTGATCCAAACAC | 无 | |
CYP68J5_M488A-R | ACCAATATTCAATGGTTGTGGTTTAAAAC | 无 | |
CYP68J5_T489A-F | GCTTATTTGGCTGATCCAAACACTAG | 无 | |
CYP68J5_T489A-R | CATACCAATATTCAATGGTTGTGGTTT | 无 |
Fig. 2 TLC results of 17α-hydroxyprogesterone at 48 h by 10 molds 1:Standard of 17α-hydroxyprogesterone. 2:Standard of 11α,17α-dihydroxy progesterone. 3:Aspergillus ochraceus. 4:Cunningpamycetes elegans. 5:Absidia coerulea. 6:Beauveria bassiana. 7:Metarhizium anisopliae. 8:Aspergillus niger. 9:Penicillium citreoviridin. 10:Aspergillus flavus. 11:Penicillium citrinum. 12:Penicillium raistrickii
Fig. 3 Molar production rate(a)and production intensity(b)of 11α,17α-dihydroxy progesterone from 17α-hydroxyprogesterone by Aspergillus ochraceus and Cunningpamycetes elegans,respectively
Fig. 4 Verification of the recombinant P. pastoris(a),S. cerevisiae(b)and R. rhodochrous(c)expressing CYP68J5 M:DL 2000 DNA marker;1:PCR product of pYES2-11αAo;2:PCR product of pNV18-11αAo
Fig. 5 TLC biotransformation results of 17α-hydroxypro-gesterone at 48 h by the recombinant S. cerevisiae,P. pastoris and R. rhodochrous expressing CYP68J5,respectively 1:Standard of 17α-hydroxyprogesterone. 2. Standard of 11α,17α-dihydroxy progesterone. 3:Saccharomyces cerevisiae. 4:Pichia pastoris. 5:Rhodococcus rhodochrous
Fig. 6 Molar production rate(a)and production inten-sity(b)of 11α, 17α-dihydroxy progesterone from 17α-hydroxyprogesterone by the recombinant S. cer-evisiae and P. pastoris expressing CYP68J5, respec-tively
Fig. 8 Predicted overall structure of CYP68J5 and the molecular docking with 17α-hydroxyprogesterone α helix,β folding and random curl are colored in bright blue,bright purple and pink,respectively. The active site residues and the substrate are colored in bright yellow and dark red,respectively
Fig. 9 Relative molar production rate of 11α, 17α-dihydr-oxy progesterone from 17α-hydroxyprogesterone by the mutants compared to that of the wild-type CYP68J5 The data represent the mean value of three independent experiments. The relative molar production rate is calculated by using the molar production rate of wild-type CYP68J5 as 100%
Fig. 10 Local model of the key amino acid site residues D118(a),F216(b)and M488(c)of CYP68J5 and the alanine mutation site residues A118(d),A216(e)and A488(f)
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