天然产物的化学-酶法合成：方法与策略的演进

doi:10.12211/2096-8280.2024-028

摘要/Abstract

摘要：

天然产物是小分子药物和探针的重要来源，其合成研究一直以来是有机合成中一个备受关注而又极具挑战性的领域。随着色谱分离技术和结构分析技术的不断发展，微量活性天然产物的发现速度不断加快，其结构的多样性和复杂性也不断增加，而对其构效关系，靶标鉴定，体内活性等方面的研究则需要供应足够量的天然产物，因而对天然产物的合成在效率、经济性和规模等方面都提出了更高的要求。化学-酶法的方式为天然产物的合成研究提供了多维的视角，一方面提供了高效高选择性的酶催化合成方法，另一方面，酶催化反应的引入可以给原先合成策略的设计模式带来突破，并快速，高效地实现天然产物的多样化合成，从而成为近期研究的热点。其中酶催化反应如何有机地整合到天然产物的合成中便成为目前化学-酶法合成成功的关键，本文从当前天然产物化学-酶法的合成实践中总结了酶催化反应所发挥的三方面作用：①对合成起点的改变，即酶催化反应可以在合成原料中引入关键的手性中心或官能团，以体外酶促或体内发酵的方式提供复杂的合成前体，如多取代芳（杂）环，手性池等；②合成后期通过酶催化方式对多官能团底物或复杂骨架的惰性位置进行化学、区域和立体选择性的官能团化；③酶催化反应作为关键步骤在母核骨架构建中关键碳碳键形成方面的策略性应用。最后，本文从合成策略的设计，合成方法的开发，以及研究人员思维等三个方面讨论了化学-酶法策略在当下所面临的挑战和未来的发展趋势。在此背景下，化学合成与生物催化等多学科手段的深度交叉融合将为天然产物的合成科学带来新的活力。

关键词: 天然产物, 化学-酶法合成, 生物催化, 生物合成, 后期官能团化

Abstract:

Natural product is an important source of small-molecule drugs and probes, the synthesis of which is challenging, and has long attracted much more attention in the field of organic chemistry. With the continuous advancement of chromatographic techniques for separation and spectroscopic methods for structural analysis, the pace of discovering tiny bioactive natural products is accelerating, concomitantly leading to an increase in the diversity and complexity of the newly identified structures. However, to meet the demand of the quantity needed for the study of their structure-activity relationships, target identification, in vivo activity evaluation, etc., growing challenges in the requirement for the synthetic efficiency, economy, and scalability of natural products are emerging. Synthetic practices in a chemoenzymatic way have provided multi-dimensional visions for natural product research, which emerged as a hot research topic in recent years. On the one hand, enzymatic catalysis has provided highly efficient and selective synthetic methodologies that would complement traditional synthetic methods. On the other hand, the introduction of enzyme-catalyzed reactions would bring a new mode of strategic design for synthesis, enabling the rapid and diverse synthesis of natural products with high efficiency. In this context, how to integrate the enzyme-catalyzed reactions into the synthesis of natural products is the key to a successful chemoenzymatic synthesis. We herein summarized three roles played by the applications of enzyme-catalyzed reactions in the current practices of chemoenzymatic synthesis of natural products. ①The involvement of biocatalysis would introduce a chiral center or a key functional group into the starting material, or supply complex synthetic precursors (e.g., polysubstituted (hetero)aromatics, chiral pools, etc.) via in vitro enzyme-catalyzed reactions or fermentation, hence advancing the starting line of synthesis; ②Late-stage enzyme-catalyzed chemo-, regio-, and stereoselective modifications of substrates with heavily substituted functional groups or inert positions of complex skeletons; ③The strategic application of enzymatic catalysis as a key carbon-carbon bond-forming step in the construction of the skeleton of natural product. Finally, we have discussed the current challenges and future trends of the chemoenzymatic synthesis of natural products in three facets, including the design of synthetic strategy, the development of synthetic methods, as well as persons involved in the research. Thus, the integration of interdisciplinary methods and technologies, including chemical synthesis and biocatalysis, would invigorate the synthesis of natural products.

Key words: natural product, chemoenzymatic synthesis, biocatalysis, biosynthesis, late-stage functionalization

中图分类号:

Q816

张守祺, 王涛, 孔尧, 邹家胜, 刘元宁, 徐正仁. 天然产物的化学-酶法合成：方法与策略的演进[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-028.

Shouqi ZHANG, Tao WANG, Yao KONG, Jiasheng ZOU, Yuanning LIU, Zhengren XU. Chemoenzymatic Synthesis of Natural Products: Evolution of Synthetic Methodology and Strategy[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-028.

图/表 12

图1 酶催化反应在天然产物化学-酶法合成中的应用

Fig. 1 Applications of enzyme-catalyzed reactions in chemoenzymatic synthesis of natural products

图2 酶催化反应在底物中引入关键手性中心（A）酶催化的动态动力学拆分合成（R）-（-）-imperanene；（B）酶催化对二醇的去对称化反应合成（S）-（+）-imperanene

Fig. 2 Introduction of the key chiral center(s) to a simple substrate via enzymatic catalysis((A) Enzyme-catalyzed dynamic kinetic resolution for the synthesis of (R)-(-)-imperanene; (B) Enzyme-catalyzed assymmetrization of diol for the synthesis of (S)-(+)-imperanene.)

图3 甲苯双加氧酶催化的底物关键手性中心引入及其在多种天然产物合成中的应用

Fig. 3 Introduction of the key chiral center(s) to a simple substrate by the action of toluene dioxygenase and its application in the synthesis of various natural products.

图4 黄素依赖单加氧酶催化的酚类底物关键手性中心引入（A）SorbC对芳环C-5的羟化在bisorbicillinol和trichodimerol合成中的应用；（B）AzaH和AfoD对芳环C-7位的立体选择性羟化及其在trichoflextin合成中的应用

Fig. 4 Introduction of the key chiral center(s) to a phenolic substrate by the action of flavin-dependent monooxygenases(A) Hydroxylation of C-5 of aromatic ring by SorbC and its application in the synthesis of bisorbicillinol and trichodimerol; (B) Stereoselective hydroxylation of C-7 of aromatic ring by AzaH and AfoD and the application in the synthesis of trichoflextin.

图5 α-酮戊二酸依赖的非血红素铁双加氧酶催化的底物关键官能团引入（A）KDO1对L-赖氨酸的选择性羟化及其在tambromycin合成中的应用；（B）GetF对L-哌啶甲酸和GetI对L-瓜氨酸的选择性羟化及其在GE81112 B1合成中的应用；（C）ClaD对苄位甲基的羟化及其在（+）-xyloketal B合成中的应用。

Fig. 5 Introduction of the key functional group(s) to the substrate via α-ketoglutarate-dependent non-heme iron dioxygenase(A) Selective hydroxylation of L-lysine by KDO1 and its application in the synthesis of tambromycin; (B) Selective hydroxylation of L-pipecolic acid and L-citrulline by GetF and GetI, respectively, and their application in the synthesis of GE81112 B1; (C) Hydroxylation of the benzylic methyl group by ClaD and its application in the synthesis of (+)-xyloketal B.

图6 多取代芳香类骨架前体的酶法制备（A）六取代间苯二酚的异源合成及其在异色满衍生物中的合成应用；（B）酶催化的色氨酸二聚化制备四取代吡咯及其在spiroindimicin家族天然产物合成中的应用

Fig. 6 Enzymatic preparation of substrates with multiple-site substituted aromatic skeletons(A) Heterologous production of hexa-substituted resorcinol and its application in the synthesis of isochromene derivatives; (B) Enzyme-catalyzed dimerization of tryptophan for the preparation of tetra-substituted pyrrole and its application in the synthesis of spiroindimicin family of natural products.

图7 萜类前体的制备（A）萜类生物合成的MVA途径和MEP途径以及人工异戊烯醇利用途径；（B）异源表达制备萜类骨架前体及其在萜类天然产物合成中的应用；（C）体外酶催化制备萜类骨架及其在天然产物合成中的应用

Fig. 7 Preparation of terpenoid precursors(A) MVA, MEP and artificial isopentenol utilization pathways for the biosynthesis of terpenes; (B) Heterologous production of terpene skeletons and their applications in the synthesis of terpene natural products; (C) In vitro enzyme-catalyzed preparation of terpene skeleton and its application in the synthesis of terpene natural products.)

图8 基于细胞色素P450BM3突变体库筛选和改造的天然产物合成（A）P450BM3突变体在nigelladine A全合成中的后期选择性羟化；（B）P450BM3突变体的后期选择性羟化在萜类天然产物合成中的应用

Fig. 8 Natural product synthesis based on screening of cytochrome P450BM3 mutants and its engineering(A) Stereoselective late-stage hydroxylation in the synthesis of nigelladine A by P450BM3 mutant; (B) Selective late-stage hydroxylation by P450BM3 mutants and their applications in the synthesis of terpene natural products.

图9 基于生物合成途径修饰酶应用的天然产物合成（A）PtmO6和PtmO5催化羟基化在对映-贝壳杉烷类天然产物合成中的应用；（B）PtmO5催化对映-贝叶烷骨架羟基化及其在后续骨架重排中的应用

Fig. 9 Natural product synthesis based on enzymes from biosynthetic pathways(A) PtmO6- and PtmO5-catalyzed hydroxylation and its application in the synthesis of ent-kaurane natural products; (B) PtmO5-catalyzed hydroxylation of ent-beyerane skeleton and its application in the following skeleton rearrangement.

图10 基于生物合成/生物转化途径修饰酶应用的天然产物合成（A）基于生物合成途径中羟化酶的挖掘在甾体合成中的应用；（B）基于生物转化信息的羟化酶挖掘及其在甾体合成中的应用

Fig. 10 Natural product synthesis based on enzymes from biosynthetic or biotransformatic pathways(A) Mining hydroxylases from biosynthetic pathway and its application in the synthesis of steroids; (B) Mining hydroxylases based on the biotransformation information and its application in the synthesis of steroids.

图11 环化酶在天然产物spinosyn A（A）和equisetin（B）骨架形成中的应用。

Fig. 11 Application of cyclases to the skeleton-forming step in the synthesis of spinosyn A (A) and equisetin (B)

图12 酶催化反应形成骨架的关键碳碳键（A）酶催化氧化在podophyllotoxin骨架形成中的应用；（B）CylK-L411A催化芳环的烷基化反应在cylindrocyclophane类天然产物合成中的应用；（C）LolT和LolD在生物碱absouline合成中的应用

Fig. 12 Enzyme-catalyze reaction for the formation of key C-C bond of skeleton(A) Application of enzyme-catalyzed oxidative skeleton formation in the synthesis of podophyllotoxin; (B) CylK-L411A-catalyzed aromatic alkylation reaction and its application in the synthesis of the cylindrocyclophane family natural products; (C) Application of LolT and LolD in the synthesis of alkaloid absouline.

参考文献 129

1	NEWMAN D J, CRAGG G M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019[J]. Journal of Natural Products, 2020, 83(3): 770-803.
2	CRAGG G M, NEWMAN D J. Natural products: a continuing source of novel drug leads[J]. Biochimica et Biophysica Acta (BBA) - General Subjects, 2013, 1830(6): 3670-3695.
3	CARLSON E E. Natural products as chemical probes[J]. ACS Chemical Biology, 2010, 5(7): 639-653.
4	PYE C R, BERTIN M J, LOKEY R S, et al. Retrospective analysis of natural products provides insights for future discovery trends[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(22): 5601-5606.
5	BUTLER M S. The role of natural product chemistry in drug discovery[J]. Journal of Natural Products, 2004, 67(12): 2141-2153.
6	KOEHN F E, CARTER G T. The evolving role of natural products in drug discovery[J]. Nature Reviews Drug Discovery, 2005, 4(3): 206-220.
7	ATANASOV A G, ZOTCHEV S B, DIRSCH V M, et al. Natural products in drug discovery: advances and opportunities[J]. Nature Reviews Drug Discovery, 2021, 20(3): 200-216.
8	WILSON B A P, THORNBURG C C, HENRICH C J, et al. Creating and screening natural product libraries[J]. Natural Product Reports, 2020, 37(7): 893-918.
9	NEWMAN D J. Natural products as leads to potential drugs: an old process or the new hope for drug discovery?[J]. Journal of Medicinal Chemistry, 2008, 51(9): 2589-2599.
10	LI L, CHEN Z, ZHANG X W, et al. Divergent strategy in natural product total synthesis[J]. Chemical Reviews, 2018, 118(7): 3752-3832.
11	MULZER J. Trying to rationalize total synthesis[J]. Natural Product Reports, 2014, 31(4): 595-603.
12	WENDER P A. Toward the ideal synthesis and molecular function through synthesis-informed design[J]. Natural Product Reports, 2014, 31(4): 433-440.
13	DRAUZ K . GRÖGER H . MAY O. Enzyme catalysis in organic synthesis[M/OL]. Weinheim : Wiley‐VCH Verlag GmbH & Co. KGaA, 2012. (2012-02-22)[2024-03-01]. .
14	RODRÍGUEZ BENÍTEZ A, NARAYAN A R H. Frontiers in biocatalysis: profiling function across sequence space[J]. ACS Central Science, 2019, 5(11): 1747-1749.
15	LEWIS R D, FRANCE S P, MARTINEZ C A. Emerging technologies for biocatalysis in the pharmaceutical industry[J]. ACS Catalysis, 2023, 13(8): 5571-5577.
16	TRUPPO M D. Biocatalysis in the pharmaceutical industry: the need for speed[J]. ACS Medicinal Chemistry Letters, 2017, 8(5): 476-480.
17	REED J H, SEEBECK F P. Reagent engineering for group transfer biocatalysis[J]. Angewandte Chemie International Edition, 2024, 63(7): e202311159.
18	WU S K, SNAJDROVA R, MOORE J C, et al. Biocatalysis: enzymatic synthesis for industrial applications[J]. Angewandte Chemie International Edition, 2021, 60(1): 88-119.
19	TURNER N J, O'REILLY E. Biocatalytic retrosynthesis[J]. Nature Chemical Biology, 2013, 9(5): 285-288.
20	KIRSCHNING A, HAHN F. Merging chemical synthesis and biosynthesis: a new chapter in the total synthesis of natural products and natural product libraries[J]. Angewandte Chemie International Edition, 2012, 51(17): 4012-4022.
21	FRIEDRICH S, HAHN F. Opportunities for enzyme catalysis in natural product chemistry[J]. Tetrahedron, 2015, 71(10): 1473-1508.
22	MURRAY L A M, MCKINNIE S M K, MOORE B S, et al. Meroterpenoid natural products from Streptomyces bacteria - the evolution of chemoenzymatic syntheses[J]. Natural Product Reports, 2020, 37(10): 1334-1366.
23	CHAKRABARTY S, ROMERO E O, PYSER J B, et al. Chemoenzymatic total synthesis of natural products[J]. Accounts of Chemical Research, 2021, 54(6): 1374-1384.
24	ZHANG H L, TANG X Y. Combining microbial and chemical syntheses for the production of complex natural products[J]. Chinese Journal of Natural Medicines, 2022, 20(10): 729-736.
25	RODDAN R, CARTER E M, THAIR B, et al. Chemoenzymatic approaches to plant natural product inspired compounds[J]. Natural Product Reports, 2022, 39(7): 1375-1382.
26	STOUT C N, WASFY N M, CHEN F, et al. Charting the evolution of chemoenzymatic strategies in the syntheses of complex natural products[J]. Journal of the American Chemical Society, 2023, 145(33): 18161-18181.
27	BRILL Z G, CONDAKES M L, TING C P, et al. Navigating the chiral pool in the total synthesis of complex terpene natural products[J]. Chemical Reviews, 2017, 117(18): 11753-11795.
28	BREUER M, DITRICH K, HABICHER T, et al. Industrial methods for the production of optically active intermediates[J]. Angewandte Chemie International Edition, 2004, 43(7): 788-824.
29	FACIN B R, MELCHIORS M S, VALÉRIO A, et al. Driving immobilized lipases as biocatalysts: 10 years state of the art and future prospects[J]. Industrial & Engineering Chemistry Research, 2019, 58(14): 5358-5378.
30	WANG H H, ZHANG Q, YU X, et al. Application of lipase B from Candida antarctica in the pharmaceutical industry[[J]. Industrial & Engineering Chemistry Research, 2023, 62(39): 15733-15751.
31	SLABU I, GALMAN J L, LLOYD R C, et al. Discovery, engineering, and synthetic application of transaminase biocatalysts[J]. ACS Catalysis, 2017, 7(12): 8263-8284.
32	TOOGOOD H S, SCRUTTON N S. Discovery, characterization, engineering and applications of ene-reductases for industrial biocatalysis[J]. ACS Catalysis, 2018, 8(4): 3532-3549.
33	YANG L C, DENG H P, RENATA H. Recent progress and developments in chemoenzymatic and biocatalytic dynamic kinetic resolution[J]. Organic Process Research & Development, 2022, 26(7): 1925-1943.
34	EGI M, SUGIYAMA K, SANETO M, et al. A mesoporous-silica-immobilized oxovanadium cocatalyst for the lipase-catalyzed dynamic kinetic resolution of racemic alcohols[J]. Angewandte Chemie International Edition, 2013, 52(13): 3654-3658.
35	MATSUNAGA K, SHIBUYA M, Imperanene OHIZUMI Y., a novel phenolic compound with platelet aggregation inhibitory activity from Imperata cylindrica [J]. Journal of Natural Products, 1995, 58(1): 138-139.
36	CARR J A, BISHT K S. Enantioselective synthesis of imperanene via enzymatic asymmetrization of an intermediary 1,3-diol[J]. Organic Letters, 2004, 6(19): 3297-3300.
37	GIBSON D T, KOCH J R, KALLIO R E. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene[J]. Biochemistry, 1968, 7(7): 2653-2662.
38	GIBSON D T, KOCH J R, SCHULD C L, et al. Oxidative degradation of aromatic hydrocarbons by microorganisms. II. Metabolism of halogenated aromatic hydrocarbons[J]. Biochemistry, 1968, 7(11): 3795-3802.
39	GIBSON D T, HENSLEY M, YOSHIOKA H, et al. Formation of (+)-cis-2, 3-dihydroxy-1-methylcyclohexa-4, 6-diene from toluene by Pseudomonas putida [J]. Biochemistry, 1970, 9(7): 1626-1630.
40	ZYLSTRA G J, GIBSON D T. Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli [J]. The Journal of Biological Chemistry, 1989, 264(25): 14940-14946.
41	HUDLICKY T, GONZALEZ D, GIBSON D T. Enzymatic dihydroxylation of aromatics in enantioselective synthesis: expanding asymmetric methodology [J]. Aldrichimica Acta, 1999, 32(2): 35-62.
42	HUDLICKY T, REED J. Celebrating 20 years of SYNLETT - special account onthe merits of biocatalysis and the impact of Arene cis-dihydrodiolson enantioselective synthesis[J]. Synlett, 2009(5): 685-703.
43	LEWIS S E. Applications of biocatalytic arene ipso, ortho cis-dihydroxylation in synthesis[J]. Chemical Communications, 2014, 50(22): 2821-2830.
44	TAHER E S, BANWELL M G, BUCKLER J N, et al. The exploitation of enzymatically-derived cis-1, 2-dihydrocatechols and related compounds in the synthesis of biologically active natural products[J]. Chemical Record, 2018, 18(2): 239-264.
45	LAN P, YE S, BANWELL M G. The application of dioxygenase-based chemoenzymatic processes to the total synthesis of natural products[J]. Chemistry, an Asian Journal, 2019, 14(22): 4001-4012.
46	HUDLICKY T, PRICE J D, FAN R L, et al. Efficient and enantiodivergent synthesis of (+)- and (-)-pinitol[J]. Journal of the American Chemical Society, 1990, 112(25): 9439-9440.
47	MATVEENKO M, WILLIS A C, BANWELL M G. A chemoenzymatic synthesis of the anti-influenza agent Tamiflu®[J]. Tetrahedron Letters, 2008, 49(49): 7018-7020.
48	MA X H, BANWELL M G, WILLIS A C. Chemoenzymatic total synthesis of the phytotoxic geranylcyclohexentriol (-)-phomentrioloxin[J]. Journal of Natural Products, 2013, 76(8): 1514-1518.
49	BANWELL M G, KOKAS O J, WILLIS A C. Chemoenzymatic approaches to the montanine alkaloids: a total synthesis of (+)-brunsvigine[J]. Organic Letters, 2007, 9(18): 3503-3506.
50	FINDLAY A D, BANWELL M G. A chemoenzymatic total synthesis of (+)-amabiline[J]. Organic Letters, 2009, 11(14): 3160-3162.
51	HUDLICKY T, OLIVO H F. A short synthesis of (+)-lycoricidine[J]. Journal of the American Chemical Society, 1992, 114(24): 9694-9696.
52	ENDOMA-ARIAS M A A, HUDLICKY T. Chemoenzymatic total synthesis of (+)-galanthamine and (+)-narwedine from phenethyl acetate[J]. Chemistry, 2016, 22(41): 14540-14543.
53	VARGHESE V, HUDLICKY T. Short chemoenzymatic total synthesis of ent-hydromorphone: an oxidative dearomatization/intramolecular[4+2]cycloaddition/amination sequence[J]. Angewandte Chemie International Edition, 2014, 53(17): 4355-4358.
54	LIN A, WILLIS A C, BANWELL M G. A chemoenzymatic and enantioselective total synthesis of the resorcylic acid lactone L-783, 290, the trans-isomer of L-783, 277[J]. Tetrahedron Letters, 2010, 51(7): 1044-1047.
55	BANWELL M G, HOCKLESS D C R, MCLEOD M D. Chemoenzymatic total syntheses of the sesquiterpene (-)-patchoulenone[J]. New Journal of Chemistry, 2003, 27(1): 50-59.
56	SCHWARTZ B D, MATOUŠOVÁ E, WHITE R, et al. A chemoenzymatic total synthesis of the protoilludane aryl ester (+)-armillarivin[J]. Organic Letters, 2013, 15(8): 1934-1937.
57	BAKER DOCKREY S A, NARAYAN A R H. Flavin-dependent biocatalysts in synthesis[J]. Tetrahedron, 2019, 75(9): 1115-1121.
58	FAHAD A A, ABOOD A, FISCH K M, et al. Oxidative dearomatisation: the key step of sorbicillinoid biosynthesis[J]. Chemical Science, 2014, 5(2): 523-527.
59	SIB A, GULDER T A M. Stereoselective total synthesis of bisorbicillinoid natural products by enzymatic oxidative dearomatization/dimerization[J]. Angewandte Chemie International Edition, 2017, 56(42): 12888-12891.
60	BAKER DOCKREY S A, LUKOWSKI A L, BECKER M R, et al. Biocatalytic site- and enantioselective oxidative dearomatization of phenols[J]. Nature Chemistry, 2018, 10(2): 119-125.
61	DAVISON J, FAHAD A A, CAI M H, et al. Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(20): 7642-7647.
62	ZABALA A O, XU W, CHOOI Y H, et al. Characterization of a silent azaphilone gene cluster from Aspergillus niger ATCC 1015 reveals a hydroxylation-mediated pyran-ring formation[J]. Chemistry & Biology, 2012, 19(8): 1049-1059.
63	SOMOZA A D, LEE K H, CHIANG Y M, et al. Reengineering an azaphilone biosynthesis pathway in Aspergillus nidulans to create lipoxygenase inhibitors[J]. Organic Letters, 2012, 14(4): 972-975.
64	PYSER J B, BAKER DOCKREY S A, BENÍTEZ A R, et al. Stereodivergent, chemoenzymatic synthesis of azaphilone natural products[J]. Journal of the American Chemical Society, 2019, 141(46): 18551-18559.
65	HERR C Q, HAUSINGER R P. Amazing diversity in biochemical roles of Fe(II)/2-oxoglutarate oxygenases[J]. Trends in Biochemical Sciences, 2018, 43(7): 517-532.
66	ITOH H, INOUE M. Comprehensive structure-activity relationship studies of macrocyclic natural products enabled by their total syntheses[J]. Chemical Reviews, 2019, 119(17): 10002-10031.
67	BAUD D, SAAIDI P L, MONFLEUR A, et al. Synthesis of mono- and dihydroxylated amino acids with new α-ketoglutarate-dependent dioxygenases: biocatalytic oxidation of C—H bonds[J]. ChemCatChem, 2014, 6(10): 3012-3017.
68	ZHANG X, KING-SMITH E, RENATA H. Total synthesis of tambromycin by combining chemocatalytic and biocatalytic C-H functionalization[J]. Angewandte Chemie International Edition, 2018, 57(18): 5037-5041.
69	MATTAY J, HÜTTEL W. Pipecolic acid hydroxylases: a monophyletic clade among cis-Selective bacterial proline hydroxylases that discriminates L-proline[J]. ChemBioChem, 2017, 18(15): 1523-1528.
70	ZWICK C R III, SOSA M B, RENATA H. Characterization of a citrulline 4-hydroxylase from nonribosomal peptide GE81112 biosynthesis and engineering of its substrate specificity for the chemoenzymatic synthesis of enduracididine[J]. Angewandte Chemie International Edition, 2019, 58(52): 18854-18858.
71	ZWICK C R III, SOSA M B, RENATA H. Modular chemoenzymatic synthesis of GE81112 B1 and related analogues enables elucidation of its key pharmacophores[J]. Journal of the American Chemical Society, 2021, 143(3): 1673-1679.
72	FAN J, LIAO G, KINDINGER F, et al. Peniphenone and penilactone formation in Penicillium crustosum via 1, 4-Michael additions of ortho-quinone methide from hydroxyclavatol to γ-butyrolactones from crustosic acid[J]. Journal of the American Chemical Society, 2019, 141(10): 4225-4229.
73	DOYON T J, PERKINS J C, BAKER DOCKREY S A, et al. Chemoenzymatic o-quinone methide formation[J]. Journal of the American Chemical Society, 2019, 141(51): 20269-20277.
74	ROMERO E O, PERKINS J C, BURCH J E, et al. Chemoenzymatic synthesis of (+)-xyloketal B[J]. Organic Letters, 2023, 25(9): 1547-1552.
75	ASAI T, YAMAMOTO T, SHIRATA N, et al. Structurally diverse chaetophenol productions induced by chemically mediated epigenetic manipulation of fungal gene expression[J]. Organic Letters, 2013, 15(13): 3346-3349.
76	ASAI T, TSUKADA K, ISE S, et al. Use of a biosynthetic intermediate to explore the chemical diversity of pseudo-natural fungal polyketides[J]. Nature Chemistry, 2015, 7(9): 737-743.
77	SÁNCHEZ C, MÉNDEZ C, SALAS J A. Indolocarbazole natural products: occurrence, biosynthesis, and biological activity[J]. Natural Product Reports, 2006, 23(6): 1007-1045.
78	BLAIR L M, SPERRY J. Total syntheses of (±)-spiroindimicins B and C enabled by a late-stage Schöllkopf-Magnus-Barton-Zard (SMBZ) reaction[J]. Chemical Communications, 2016, 52(4): 800-802.
79	ZHANG Z, RAY S, IMLAY L, et al. Total synthesis of (+)-spiroindimicin A and congeners unveils their antiparasitic activity[J]. Chemical Science, 2021, 12(30): 10388-10394.
80	ZHENG X K, LI Y, GUAN M T, et al. Biomimetic total synthesis of the spiroindimicin family of natural products[J]. Angewandte Chemie International Edition, 2022, 61(38): e202208802.
81	BUREAU J A, OLIVA M E, DONG Y M, et al. Engineering yeast for the production of plant terpenoids using synthetic biology approaches[J]. Natural Product Reports, 2023, 40(12): 1822-1848.
82	SIEMON T, WANG Z Q, BIAN G K, et al. Semisynthesis of plant-derived englerin A enabled by microbe engineering of guaia-6, 10(14)-diene as building block[J]. Journal of the American Chemical Society, 2020, 142(6): 2760-2765.
83	MOU S B, XIAO W, WANG H Q, et al. Syntheses of epoxyguaiane sesquiterpenes (-)-englerin A, (-)-oxyphyllol, (+)-orientalol E, and (+)-orientalol F: a synthetic biology approach[J]. Organic Letters, 2020, 22(5): 1976-1979.
84	MOU S B, XIAO W, WANG H Q, et al. Syntheses of the carotane-type terpenoids (+)-schisanwilsonene A and (+)-tormesol via a two-stage approach[J]. Organic Letters, 2021, 23(2): 400-404.
85	ZHOU Q H, CHEN X F, MA D W. Asymmetric, protecting-group-free total synthesis of (-)-englerin A[J]. Angewandte Chemie International Edition, 2010, 49(20): 3513-3516.
86	MOLAWI K, DELPONT N, ECHAVARREN A M. Enantioselective synthesis of (-)-englerins A and B[J]. Angewandte Chemie International Edition, 2010, 49(20): 3517-3519.
87	GAYDOU M, MILLER R E, DELPONT N, et al. Synthesis of (+)-schisanwilsonene A by tandem gold-catalyzed cyclization/1, 5-migration/cyclopropanation[J]. Angewandte Chemie International Edition, 2013, 52(25): 6396-6399.
88	LIU C G, CUI X Y, CHEN W, et al. Synthesis of oxygenated sesquiterpenoids enabled by combining metabolic engineering and visible-light photocatalysis[J]. Chemistry, 2022, 28(46): e202201230.
89	HSU S Y, PERUSSE D, HOUGARD T, et al. Semisynthesis of the neuroprotective metabolite, serofendic acid[J]. ACS Synthetic Biology, 2019, 8(10): 2397-2403.
90	BOTAS A, EITEL M, SCHWARZ P N, et al. Genetic engineering in combination with semi-synthesis leads to a new route for gram-scale production of the immunosuppressive natural product Brasilicardin A[J]. Angewandte Chemie International Edition, 2021, 60(24): 13536-13541.
91	NAKANO C, KUDO F, EGUCHI T, et al. Genome mining reveals two novel bacterial sesquiterpene cyclases: (-)-germacradien-4-ol and (-)-epi-α-bisabolol synthases from Streptomyces citricolor [J]. ChemBioChem, 2011, 12(15): 2271-2275.
92	GRANT P S, MEYRELLES R, GAJSEK O, et al. Biomimetic cationic cyclopropanation enables an efficient chemoenzymatic synthesis of 6,8-cycloeudesmanes[J]. Journal of the American Chemical Society, 2023, 145(10): 5855-5863.
93	HONG B K, LUO T P, LEI X G. Late-stage diversification of natural products[J]. ACS Central Science, 2020, 6(5): 622-635.
94	DECORTE B L. Underexplored opportunities for natural products in drug discovery[J]. Journal of Medicinal Chemistry, 2016, 59(20): 9295-9304.
95	FASAN R D. Tuning P450 enzymes as oxidation catalysts[J]. ACS Catalysis, 2012, 2(4): 647-666.
96	MÜNCH J, PÜLLMANN P, ZHANG W Y, et al. Enzymatic hydroxylations of sp³-carbons[J]. ACS Catalysis, 2021, 11(15): 9168-9203.
97	WHITEHOUSE C J C, BELL S G, WONG L L. P450_BM3(CYP102A1): connecting the dots[J]. Chemical Society Reviews, 2012, 41(3): 1218-1260.
98	LOSKOT S A, ROMNEY D K, ARNOLD F H, et al. Enantioselective total synthesis of nigelladine A via late-stage C-H oxidation enabled by an engineered P450 enzyme[J]. Journal of the American Chemical Society, 2017, 139(30): 10196-10199.
99	LI J, LI F Z, KING-SMITH E, et al. Merging chemoenzymatic and radical-based retrosynthetic logic for rapid and modular synthesis of oxidized meroterpenoids[J]. Nature Chemistry, 2020, 12(2): 173-179.
100	LI F Z, RENATA H. A Chiral-Pool-Based strategy to access trans-syn-fused drimane meroterpenoids: chemoenzymatic total syntheses of polysin, N-acetyl-polyveoline and the chrodrimanins[J]. Journal of the American Chemical Society, 2021, 143(43): 18280-18286.
101	LI J, CHEN F, RENATA H. Concise chemoenzymatic synthesis of gedunin[J]. Journal of the American Chemical Society, 2022, 144(42): 19238-19242.
102	LI F Z, DENG H P, RENATA H. Remote B-ring oxidation of sclareol with an engineered P450 facilitates divergent access to complex terpenoids[J]. Journal of the American Chemical Society, 2022, 144(17): 7616-7621.
103	DONG L B, ZHANG X, RUDOLF J D, et al. Cryptic and stereospecific hydroxylation, oxidation, and reduction in platensimycin and platencin biosynthesis[J]. Journal of the American Chemical Society, 2019, 141(9): 4043-4050.
104	RUDOLF J D, DONG L B, MANOOGIAN K, et al. Biosynthetic origin of the ether ring in platensimycin[J]. Journal of the American Chemical Society, 2016, 138(51): 16711-16721.
105	ZHANG X, KING-SMITH E, DONG L B, et al. Divergent synthesis of complex diterpenes through a hybrid oxidative approach[J]. Science, 2020, 369(6505): 799-806.
106	ZHAO Y, ZHANG B, SUN Z Q, et al. Biocatalytic C14-hydroxylation on androstenedione enabled modular synthesis of cardiotonic steroids[J]. ACS Catalysis, 2022, 12(16): 9839-9845.
107	SONG F Z, ZHENG M M, WANG J L, et al. Chemoenzymatic synthesis of C14-functionalized steroids[J]. Nature Synthesis, 2023, 2(8): 729-739.
108	JAMIESON C S, OHASHI M, LIU F, et al. The expanding world of biosynthetic pericyclases: cooperation of experiment and theory for discovery[J]. Natural Product Reports, 2019, 36(5): 698-713.
109	KIM H J, RUSZCZYCKY M W, CHOI S H, et al. Enzyme-catalysed[4+2]cycloaddition is a key step in the biosynthesis of spinosyn A[J]. Nature, 2011, 473(7345): 109-112.
110	KIM H J, CHOI S H, JEON B S, et al. Chemoenzymatic synthesis of spinosyn A[J]. Angewandte Chemie International Edition, 2014, 53(49): 13553-13557.
111	LI X J, ZHENG Q F, YIN J, et al. Chemo-enzymatic synthesis of equisetin[J]. Chemical Communications, 2017, 53(34): 4695-4697.
112	KATO N, NOGAWA T, HIROTA H, et al. A new enzyme involved in the control of the stereochemistry in the decalin formation during equisetin biosynthesis[J]. Biochemical and Biophysical Research Communications, 2015, 460(2): 210-215.
113	GAO L, SU C, DU X X, et al. FAD-dependent enzyme-catalysed intermolecular[4+2]cycloaddition in natural product biosynthesis[J]. Nature Chemistry, 2020, 12(7): 620-628.
114	LIU X J, YANG J, GAO L, et al. Chemoenzymatic total syntheses of artonin I with an intermolecular Diels-Alderase[J]. Biotechnology Journal, 2020, 15(11): e2000119.
115	GAO L, ZOU Y K, LIU X J, et al. Enzymatic control of endo- and exo-stereoselective Diels–Alder reactions with broad substrate scope[J]. Nature Catalysis, 2021, 4(12): 1059-1069.
116	LAU W, SATTELY E S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone[J]. Science, 2015, 349(6253): 1224-1228.
117	LI J, ZHANG X, RENATA H. Asymmetric chemoenzymatic synthesis of (-)-podophyllotoxin and related aryltetralin lignans[J]. Angewandte Chemie International Edition, 2019, 58(34): 11657-11660.
118	LAZZAROTTO M, HAMMERER L, HETMANN M, et al. Chemoenzymatic total synthesis of deoxy-, epi-, and podophyllotoxin and a biocatalytic kinetic resolution of dibenzylbutyrolactones[J]. Angewandte Chemie International Edition, 2019, 58(24): 8226-8230.
119	NAKAMURA H, SCHULTZ E E, BALSKUS E P. A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis[J]. Nature Chemical Biology, 2017, 13(8): 916-921.
120	WANG H Q, MOU S B, XIAO W, et al. Structural basis for the Friedel-Crafts alkylation in cyclindrocyclophane biosynthesis [J]. ACS Catalysis, 2022, 12(3): 2108-2117.
121	CHEN K Y, WANG H Q, YUAN Y, et al. Chemoenzymatic synthesis of cylindrocyclophanes A and F and merocyclophanes A and D[J]. Angewandte Chemie International Edition, 2023, 62(46): e202307602.
122	GAO J M, LIU S N, ZHOU C, et al. A pyridoxal 5'-phosphate-dependent Mannich cyclase[J]. Nature Catalysis, 2023, 6: 476-486.
123	LIU S N, HAI Y. Chemoenzymatic approaches to izidine alkaloids: an efficient total synthesis of (+)-absouline and laburnamine[J]. ACS Catalysis, 2023, 13(24): 15725-15729.
124	WU X M, GUAN Q Y, HAN Y B, et al. Regeneration of phytochemicals by structure-driven organization of microbial biosynthetic steps[J]. Angewandte Chemie International Edition, 2022, 61(8): e202114919.
125	KIEFER A F, LIU Y C, GUMMERER R, et al. An artificial in vitro metabolism to angiopterlactone B inspired by traditional retrosynthesis[J]. Angewandte Chemie International Edition, 2023, 62(23): e202301178.
126	YI D, BAYER T, BADENHORST C P S, et al. Recent trends in biocatalysis[J]. Chemical Society Reviews, 2021, 50(14): 8003-8049.
127	YANG Y, ARNOLD F H. Navigating the unnatural reaction space: directed evolution of heme proteins for selective carbene and nitrene transfer[J]. Accounts of Chemical Research, 2021, 54(5): 1209-1225.
128	PAN Y J, LI G B, LIU R X, et al. Unnatural activities and mechanistic insights of cytochrome P450 PikC gained from site-specific mutagenesis by non-canonical amino acids[J]. Nature Communications, 2023, 14(1): 1669.
129	ROMERO E O, SAUCEDO A T, HERNÁNDEZ-MELÉNDEZ J R, et al. Enabling broader adoption of biocatalysis in organic chemistry[J]. Journal of the American Chemical Society Au, 2023, 3(8): 2073-2085.

[1]	周强, 周大伟, 孙敬翔, 王靖楠, 姜万奎, 章文明, 蒋羽佳, 信丰学, 姜岷. 微生物发酵法合成虾青素的研究进展[J]. 合成生物学, 2024, 5(1): 126-143.
[2]	胡哲辉, 徐娟, 卞光凯. 自动化高通量技术在天然产物生物合成中的应用[J]. 合成生物学, 2023, 4(5): 932-946.
[3]	张凡忠, 相长君, 张骊駻. 进化与大数据导向生物信息学在天然产物研究中的发展及应用[J]. 合成生物学, 2023, 4(4): 629-650.
[4]	曾涛, 巫瑞波. 数据驱动的酶反应预测与设计[J]. 合成生物学, 2023, 4(3): 535-550.
[5]	赖奇龙, 姚帅, 查毓国, 白虹, 宁康. 微生物组生物合成基因簇发掘方法及应用前景[J]. 合成生物学, 2023, 4(3): 611-627.
[6]	董佳钰, 李敏, 肖宗华, 胡明, 松田侑大, 汪伟光. 米曲霉异源表达天然产物研究进展[J]. 合成生物学, 2022, 3(6): 1126-1149.
[7]	吕靖伟, 邓子新, 张琪, 丁伟. 基于深度学习识别RiPPs前体肽及裂解位点[J]. 合成生物学, 2022, 3(6): 1262-1276.
[8]	祁延萍, 朱晋, 张凯, 刘彤, 王雅婕. 定向进化在蛋白质工程中的应用研究进展[J]. 合成生物学, 2022, 3(6): 1081-1108.
[9]	唐士茗, 胡纪元, 郑穗平, 韩双艳, 林影. 基于无细胞体系的生物合成代谢模块设计、构建与快速途径原型[J]. 合成生物学, 2022, 3(6): 1250-1261.
[10]	杨健钊, 朱新广. 面向碳达峰与碳中和的植物合成生物学[J]. 合成生物学, 2022, 3(5): 847-869.
[11]	杨璐, 瞿旭东. 亚胺还原酶在手性胺合成中的应用[J]. 合成生物学, 2022, 3(3): 516-529.
[12]	楼玉姣, 徐鉴, 吴起. 生物催化惰性碳氢键的氘代反应研究进展[J]. 合成生物学, 2022, 3(3): 530-544.
[13]	王汇滨, 车昌丽, 游松. Fe/α-酮戊二酸依赖型卤化酶在绿色卤化反应中的研究进展[J]. 合成生物学, 2022, 3(3): 545-566.
[14]	金交羽, 周佳海. Z-基因组的生物合成奥秘被揭示[J]. 合成生物学, 2022, 3(1): 1-5.
[15]	严伟, 高豪, 蒋羽佳, 钱秀娟, 周杰, 董维亮, 章文明, 信丰学, 姜岷. 2-苯乙醇生物合成的研究进展[J]. 合成生物学, 2021, 2(6): 1030-1045.