Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (10): 304-310.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0387
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ZHAO Xin1,2(), DU Yu-yao1,2, YIN Zi-yang1,2, MAO Shu-hong1,2()
Received:
2023-04-22
Online:
2023-10-26
Published:
2023-11-28
Contact:
MAO Shu-hong
E-mail:18332862765@163.com;shuhongmao@tust.
ZHAO Xin, DU Yu-yao, YIN Zi-yang, MAO Shu-hong. Allogeneic Expression of Cholesterol 7α-hydroxylase in Pichia pastoris[J]. Biotechnology Bulletin, 2023, 39(10): 304-310.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
CYP7A1-F | CGCGGATCCATGATGACCACCTCTCTGATT |
CYP7A1-R | CCGGAATTCTTACAGATGTTTAAATTTATATTTAAATTCAAT |
H111A-F | AAAGCCTTTGGTGCTAGGTCTATTGATCCAATGGATGGTAATAC |
H111A-R | ACCAAAGGCTTTGGCAGAGGT |
I114A-F | GGTCATAGGTCTGCTGATCCAATGGATGGTAATACCACTGA |
I114A-R | AGACCTATGACCAAAGGCTTTGGC |
V281A-F | AAAACCCATCTGGCTGTTCTGTGGGCCTCTCAAGCC |
V281A-R | CAGATGGGTTTTGGCTTTTTCC |
W284A-F | CTGGTTGTTCTGGCYGCCTCTCAAGCCAATACCATTCC |
W284A-R | CAGAACAACCAGATGGGTTTTGG |
G485A-F | CAGTCTAGAGCTGCTCTGGGTATTCTGCCACCACTGA |
G485A-R | AGCTCTAGACTGATCCAGTGGTGGAC |
Table 1 Wild-type and mutant primers of CYP7A1 gene
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
CYP7A1-F | CGCGGATCCATGATGACCACCTCTCTGATT |
CYP7A1-R | CCGGAATTCTTACAGATGTTTAAATTTATATTTAAATTCAAT |
H111A-F | AAAGCCTTTGGTGCTAGGTCTATTGATCCAATGGATGGTAATAC |
H111A-R | ACCAAAGGCTTTGGCAGAGGT |
I114A-F | GGTCATAGGTCTGCTGATCCAATGGATGGTAATACCACTGA |
I114A-R | AGACCTATGACCAAAGGCTTTGGC |
V281A-F | AAAACCCATCTGGCTGTTCTGTGGGCCTCTCAAGCC |
V281A-R | CAGATGGGTTTTGGCTTTTTCC |
W284A-F | CTGGTTGTTCTGGCYGCCTCTCAAGCCAATACCATTCC |
W284A-R | CAGAACAACCAGATGGGTTTTGG |
G485A-F | CAGTCTAGAGCTGCTCTGGGTATTCTGCCACCACTGA |
G485A-R | AGCTCTAGACTGATCCAGTGGTGGAC |
Fig. 2 TLC analysis of biotransformation result of cholesterol catalyzed by recombinant P. pastoris Mixed labels: Mixed standards of cholesterol and 7α-hydroxycholesterol; CYP: CYP7A1(WT); GS115: P. pastoris GS115; 111: mutant H111A; 114: mutant I114A; 281: mutant V281A; 284: mutant W284A; 485: mutant G485A
Fig. 3 GC-MS analysis of the 7α-hydroxycholesterol catalyzed by recombinant P. pastoris A: Mass spectra of 7α-hydroxycholesterol. B: Mass spectra of recombinant P. pastoris products
Fig. 4 Production abilities of the mutants when compared with the wild type Product ratio of mutant H111A, I114A, V281A, W284A and G485A to CYP7A1(WT)recombinant P. pastoris
Fig. 5 Effects of P. pastoris co-expressed with CYP7A1 and CPR from different sources on cholesterol conversionThe growth rate of products of P. pastoris co-expressed with CYP7A1(WT)and CPR(ScCPR, hCPR, AnCPR, RatCPR and SgCPR)and P. pastoris co-expressed with G485A and SgCPR when compared with that of recombinant P. pastoris containing only CYP7A1(WT)
Fig. 6 Docking results of CYP7A1 and cholesterol molecules The yellow molecule is cholesterol, the others are CYP7A1, and the blue-labeled sites are mutation sites
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