Synthetic Biology Journal ›› 2021, Vol. 2 ›› Issue (5): 804-814.DOI: 10.12211/2096-8280.2021-081
Previous Articles Next Articles
Wentao SUN1, Xinzhe ZHANG2, Shengtong WAN2, Ruwen WANG2, Chun LI1,2
Received:
2021-08-06
Revised:
2021-09-24
Online:
2021-11-19
Published:
2021-11-19
Contact:
Chun LI
孙文涛1, 张昕哲2, 万盛通2, 王茹雯2, 李春1,2
通讯作者:
李春
作者简介:
基金资助:
CLC Number:
Wentao SUN, Xinzhe ZHANG, Shengtong WAN, Ruwen WANG, Chun LI. Regulation on oxidation selectivity for β-amyrin by Class Ⅱ cytochrome P450 enzymes[J]. Synthetic Biology Journal, 2021, 2(5): 804-814.
孙文涛, 张昕哲, 万盛通, 王茹雯, 李春. Ⅱ型细胞色素P450酶氧化β-香树脂醇的选择性调控研究[J]. 合成生物学, 2021, 2(5): 804-814.
Add to citation manager EndNote|Ris|BibTeX
URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2021-081
菌株 | 基因型 | 来源 |
---|---|---|
SynV | MATa his3Δ1;leu2;met15Δ;ura3-52 | 天津大学 |
SynV8 | SynV: ΔGal80 | 本研究构建 |
GA0-1 | SynV8: ΔHO:: FBA1p-bAS-CYC1-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-G418 | 本研究构建 |
GA0 | SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63-ADH1t-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3 | 本研究构建 |
GA160 | SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3 | 本研究构建 |
GA180-1 | SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-Gal7p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3 | 本研究构建 |
Tab. 1 Strains used in this study
菌株 | 基因型 | 来源 |
---|---|---|
SynV | MATa his3Δ1;leu2;met15Δ;ura3-52 | 天津大学 |
SynV8 | SynV: ΔGal80 | 本研究构建 |
GA0-1 | SynV8: ΔHO:: FBA1p-bAS-CYC1-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-G418 | 本研究构建 |
GA0 | SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63-ADH1t-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3 | 本研究构建 |
GA160 | SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-ENO2p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3 | 本研究构建 |
GA180-1 | SynV8: ΔHO:: FBA1p-bAS-CYC1t-Gal2p-CYP72A63(T338S)-ADH1t-Gal7p-Uni25647-TYS1t-PGK1p-GuCPR1-PGK1t-HIS3 | 本研究构建 |
成分 | 体积/μL |
---|---|
1 mol/L Tris-HCl(pH7.5) | 3000 |
2 mol/L MgCl2 | 150 |
dNTP mix | 240 |
1 mol/L DTT | 300 |
PEG-8000 | 1.5 g |
0.1 mol/L NAD | 300 |
ddH2O | 定容至6 mL,-80 ℃分装,保存 |
Tab. 2 Components of the 5×ISO buffer
成分 | 体积/μL |
---|---|
1 mol/L Tris-HCl(pH7.5) | 3000 |
2 mol/L MgCl2 | 150 |
dNTP mix | 240 |
1 mol/L DTT | 300 |
PEG-8000 | 1.5 g |
0.1 mol/L NAD | 300 |
ddH2O | 定容至6 mL,-80 ℃分装,保存 |
成分 | 体积/μL |
---|---|
5×ISO buffer | 320 |
10 U/μL T5 exonuclease | 0.64 |
2 U/μL Phusion polymerase | 20 |
40 U/μL | 160 |
ddH2O | 1200 |
Tab. 3 Components of Gibson reaction solution
成分 | 体积/μL |
---|---|
5×ISO buffer | 320 |
10 U/μL T5 exonuclease | 0.64 |
2 U/μL Phusion polymerase | 20 |
40 U/μL | 160 |
ddH2O | 1200 |
Fig. 2 Influence of the transmembrane structures on the substrate selectivity of CYP72A63 (T338S)[The same letters indicate no significant difference at the95% confidence level (P < 0.05)]
基因 | 注释 | FPKM Sgib | FPKM SynV |
---|---|---|---|
FAA1 | long-chain fatty acid-CoA ligase FAA1 | 1534.17 | 410.28 |
SUR2 | sphingosine C-4 hydroxylase | 480.16 | 162.61 |
Tab. 4 Transcription levels of genes associated to lipid metabolism in different strains
基因 | 注释 | FPKM Sgib | FPKM SynV |
---|---|---|---|
FAA1 | long-chain fatty acid-CoA ligase FAA1 | 1534.17 | 410.28 |
SUR2 | sphingosine C-4 hydroxylase | 480.16 | 162.61 |
Fig. 3 Product profile of the strains with FAA1or SUR2 knock out[The same letters indicate no significant difference at the 95% confidence level (P < 0.05)]
Fig. 4 Production of triterpenoids in the engineered strains overexpressing ELO1, ELO2 and ELO3[Asterisk (*) indicates significant difference at the 95% confidence level (P < 0.05)]
Fig. 5 Production of triterpenoids in the engineered strains overexpressing heterogenous GCSs[The same letters indicate no significant difference at the 95% confidence level (P < 0.05)]
Fig. 6 Product profile (a) and the relative transcription abundance of P450 enzymes after up-regulating Uni25647 expression (b)[Asterisk (*) indicates significant difference at 95% confidence level (P < 0.05)]
1 | CHEMLER J A, KOFFAS M A. Metabolic engineering for plant natural product biosynthesis in microbes[J]. Current Opinion in Biotechnology, 2008, 19(6): 597-605. |
2 | SCHLÄGER S, DRÄGER B. Exploiting plant alkaloids[J]. Current Opinion in Biotechnology, 2016, 37: 155-164. |
3 | CHANG M C, EACHUS R A, TRIEU W, et al. Engineering Escherichia coli for production of functionalized terpenoids using plant P450s[J]. Nature Chemical Biology, 2007, 3(5): 274-277. |
4 | GRANDNER J M, CACHO R A, TANG Y, et al. Mechanism of the P450-catalyzed oxidative cyclization in the biosynthesis of griseofulvin[J]. ACS Catalysis, 2016, 6(7): 4506-4511. |
5 | IGNEA C, ATHANASAKOGLOU A, IOANNOU E, et al. Carnosic acid biosynthesis elucidated by a synthetic biology platform[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(13): 3681-3686. |
6 | PARK H, PARK G, JEON W, et al. Whole-cell biocatalysis using cytochrome P450 monooxygenases for biotransformation of sustainable bioresources (fatty acids, fatty alkanes, and aromatic amino acids)[J]. Biotechnology Advances, 2020, 40: 107504. |
7 | NABAVI S M, ŠAMEC D, TOMCZYK M, et al. Flavonoid biosynthetic pathways in plants: Versatile targets for metabolic engineering[J]. Biotechnology Advances, 2020, 38: 107316. |
8 | SEKI H, SAWAI S, OHYAMA K, et al. Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin[J]. The Plant Cell, 2011, 23(11): 4112-4123. |
9 | SEKI H, OHYAMA K, SAWAI S, et al. Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(37): 14204-14209. |
10 | DENISOV I G, MAKRIS T M, SLIGAR S G, et al. Structure and chemistry of cytochrome P450[J]. Chemical Reviews, 2005, 105(6): 2253-2277. |
11 | CHEN J, FAN F, QU G, et al. Identification of Absidia orchidis steroid 11β-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone[J]. Metabolic Engineering, 2020, 57: 31-42. |
12 | SCHELER U, BRANDT W, PORZEL A, et al. Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast[J]. Nature Communications, 2016, 7: 12942. |
13 | CHEN W, FISHER M J, LEUNG A, et al. Oxidative diversification of steroids by nature-inspired scanning glycine mutagenesis of P450BM3 (CYP102A1)[J]. ACS Catalysis, 2020, 10(15): 8334-8343. |
14 | LI Y, WONG L L. Multi-functional oxidase activity of CYP102A1 (P450BM3) in the oxidation of quinolines and tetrahydroquinolines[J]. Angewandte Chemie International Edition, 2019, 58(28): 9551-9555. |
15 | LE-HUU P, HEIDT T, CLAASEN B, et al. Chemo-, regio-, and stereoselective oxidation of the monocyclic diterpenoid β-cembrenediol by P450 BM3[J]. ACS Catalysis, 2015, 5(3): 1772-1780. |
16 | URLACHER V B, GIRHARD M. Cytochrome P450 monooxygenases in biotechnology and synthetic biology[J]. Trends in Biotechnology, 2019, 37(8): 882-897. |
17 | BRANDENBERG O F, FASAN R, ARNOLD F H. Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions[J]. Current Opinion in Biotechnology, 2017, 47: 102-11. |
18 | ZHANG R K, HUANG X, ARNOLD F H. Selective CH bond functionalization with engineered heme proteins: new tools to generate complexity[J]. Current Opinion in Chemical Biology, 2019, 49: 67-75. |
19 | BULLER A R, ROYE P VAN, CAHN J K B, et al. Directed evolution mimics allosteric activation by stepwise tuning of the conformational ensemble[J]. Journal of the American Chemical Society, 2018, 140(23): 7256-7266. |
20 | SUN W, XUE H, LIU H, et al. Controlling chemo- and regioselectivity of a plant P450 in yeast cell toward rare licorice triterpenoid biosynthesis[J]. ACS Catalysis, 2020: 4253-4260. |
21 | 杨超凡,姜玉超,桑茉莉,等. 还原伴侣对细胞色素 P450 酶 MycG 的功能调控研究[J]. 合成生物学, 2021. doi: 10.12211/2096-8280.2021-053. |
YANG C F, JIANG Y C, SANG M L, et al. Studies on the functional modulating effects of redox partners on the cytochrome P450 enzyme MycG[J]. Synthetic Biology Journal, 2021. doi: 10.12211/2096-8280.2021-053. | |
22 | KROGH A, LARSSON B, HEIJNE G VON, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[J]. Journal of Molecular Biology, 2001, 305(3): 567-580. |
23 | ZHANG L, REN S, LIU X, et al. Mining of UDP-glucosyltrfansferases in licorice for controllable glycosylation of pentacyclic triterpenoids[J]. Biotechnology and Bioengineering, 2020, 117(12): 3651-3663. |
24 | ZHAO Y J, LI C. Biosynthesis of plant triterpenoid saponins in microbial cell factories[J]. Journal of Agricultural and Food Chemistry, 2018, 66(46): 12155-12165. |
25 | LIU H, FAN J, WANG C, et al. Enhanced β-amyrin synthesis in Saccharomyces cerevisiae by coupling an optimal acetyl-CoA supply pathway[J]. Journal of Agricultural and Food Chemistry, 2019, 67(13): 3723-3732. |
26 | ESPINOZA R V, SHERMAN D H. Exploring the molecular basis for selective C-H functionalization in plant P450s[J]. Synthetic and Systems Biotechnology, 2020, 5(2): 97-98. |
27 | ZHAO F, BAI P, LIU T, et al. Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae[J]. Biotechnology and Bioengineering, 2016, 113(8): 1787-1795. |
28 | MAMODE CASSIM A, GOUGUET P, GRONNIER J, et al. Plant lipids: Key players of plasma membrane organization and function[J]. Progress in Lipid Research, 2019, 73: 1-27. |
29 | BRIGNAC-HUBER L M, PARK J W, REED J R, et al. Cytochrome P450 organization and function are modulated by endoplasmic reticulum phospholipid heterogeneity[J]. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 2016, 44(12): 1859-1866. |
30 | SEZGIN E, LEVENTAL I, MAYOR S, et al. The mystery of membrane organization: composition, regulation and roles of lipid rafts[J]. Nature Reviews Molecular Cell Biology, 2017, 18(6): 361-374. |
31 | HENRY S A, KOHLWEIN S D, CARMAN G M. Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae[J]. Genetics, 2012, 190(2): 317-349. |
32 | ZHANG G, CAO Q, LIU J, et al. Refactoring β-amyrin synthesis in Saccharomyces cerevisiae[J]. AIChE Journal, 2015, 61(10): 3172-3179. |
33 | XIE Z X, LI B Z, MITCHELL L A, et al. "Perfect" designer chromosome V and behavior of a ring derivative[J]. Science, 2017, 355(6329): eaaf4704. |
34 | PARISI L R, SOWLATI-HASHJIN S, BERHANE I A, et al. Membrane disruption by very long chain fatty acids during necroptosis[J]. ACS Chemical Biology, 2019, 14(10): 2286-2294. |
35 | DICKSON R C. Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast[J]. Journal of Lipid Research, 2008, 49(5): 909-921. |
36 | MASHIMA R, OKUYAMA T, OHIRA M. Biosynthesis of long chain base in sphingolipids in animals, plants and fungi[J]. Future Science OA, 2019, 6(1): FSO434. |
37 | RAMIREZ-GAONA M, MARCU A, PON A, et al. YMDB 2.0: a significantly expanded version of the yeast metabolome database[J]. Nucleic Acids Research, 2017, 45(D1): D440-D445. |
[1] | Yingjia PAN, Siyang XIA, Chang DONG, Jin CAI, Jiazhang LIAN. Mutator-driven continuous genome evolution of Saccharomyces cerevisiae [J]. Synthetic Biology Journal, 2023, 4(1): 225-240. |
[2] | Chaofan YANG, Yuchao JIANG, Moli SANG, Shengying LI, Wei ZHANG. Studies on the functional modulating effect of redox partners on the cytochrome P450 enzyme MycG [J]. Synthetic Biology Journal, 2022, 3(3): 587-601. |
[3] | Jianming LYU, Huan ZHAO, Dan HU, Hao GAO. Biosynthesis of alkyne moiety in natural products and application of alkyne biosynthetic machineries [J]. Synthetic Biology Journal, 2021, 2(5): 734-750. |
[4] | Xiaodong LI, Chengshuai YANG, Pingping WANG, Xing YAN, Zhihua ZHOU. Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae [J]. Synthetic Biology Journal, 2021, 2(5): 792-803. |
[5] | Yi LI, Zhenquan LIN, Zihe LIU. Advances in yeast based adaptive laboratory evolution [J]. Synthetic Biology Journal, 2021, 2(2): 287-301. |
[6] | Yue SHENG, Genlin ZHANG. Yeast terminator engineering: from mechanism exploration to artificial design [J]. Synthetic Biology Journal, 2020, 1(6): 709-721. |
[7] | Siyang XIA, Lihong JIANG, Jin CAI, Lei HUANG, Zhinan XU, Jiazhang LIAN. Advances in genome evolution of Saccharomyces cerevisiae [J]. Synthetic Biology Journal, 2020, 1(5): 556-569. |
Viewed | ||||||
Full text |
|
|||||
Abstract |
|
|||||