图1 底物通道效应［7］(Different types of intermediate channeling in a two-step metabolic pathway, where a substrate is processed by enzyme E1 and turned into intermediate, which is then processed by enzyme E2 and turned into product)

Fig. 1 Substrate channel in a two-step metabolic pathway［7］

图2 多酶催化体系的组装技术［32］

Fig. 2 Assembly technology of multi-enzyme catalytic system［32］

图3 脱酰基-7-氨基头孢烷酸新旧合成路线比较［42］

Fig. 3 Comparison of two deacetyl-7-aminocephalosporanic acid synthetic routes［42］

图4 多酶催化合成D-2-氨基丁酸［50］［L-threonine deaminase catalyzes production of 2-oxobutyric acid, which is combined with cofactors to be reductively amination by D-amino acid dehydrogenase to obtain D-2-aminobutyric acid, and at same time combined with formate dehydrogenase (FDH) to achieve NADPH of recycling]

Fig. 4 Synthesis of D-2-aminobutyric acid catalyzed by multi-enzyme［50］

图5 肠球菌素的体外多酶催化合成途径［54］

Fig. 5 Multienzyme catalyzed synthesis pathway of enterococcin in vitro［54］

图6 一锅法多酶级联催化合成L-酪氨酸衍生物［66］[Regioselective and chemoselective hydroxylation of aromatics (1a-f) is catalyzed by highly selective P450 BM3 mutant to generate o-phenol intermediates (3a-f) and p-phenol intermediates (2a-f). In second step, under condition of consuming NH3, tyrosine phenol lyase (TPL) catalyzes C-C coupling of 3a-f to generate pyruvate to obtain L-4a-f]

Fig. 6 Synthesis of L-tyrosine derivatives by one-pot multi-enzyme cascade catalysis［66］

图7 一锅法多酶级联反应生产2'-脱氧鸟苷酸路线［70］［Original guanosine substrate is first cleaved by PNPase into guanine and 1-phosphate ribose. Subsequently， NDT-Ⅱ catalyzes reaction of guanine and thymidine to produce deoxyguanosine （dGR）. Finally， dGKase and ACKase utilize cytidine triphosphate （CTP） regeneration system of acetyl phosphate to phosphorylate intermediate dGR into dGMP. During reaction， original guanosine substrate is cleaved by PNPase into guanine and ribose-1-phosphate. Then， dGR is subsequently produced by reaction between guanine and thymidine， and NDT-Ⅱ catalyzes reaction to proceed. Finally， intermediate deoxyribonucleic acid is phosphorylated by deoxyglucosidase and CTP to generate deoxyribonucleic acid polysaccharide］

Fig. 7 One-pot multi-enzyme cascade production route of deoxyguanosine-2'-monophosphate［70］

图8 阿托伐他汀侧链前体生产路线［75］DERA—2-deoxyribose-5-phosphate aldolase； KRED—NADPH-dependent dehydrogenases； PGA—penicillin G-acylase

Fig. 8 Atorvastatin side chain precursor production route［75］

图9 合成熊去氧胆酸的关键中间体途径［82］

Fig. 9 Key intermediate synthesis pathway of ursodeoxycholic acid［82］

图10 体外多酶催化去消旋化体系生产（R）-1-苯基-1，2-乙二醇［87］［Racemic PED is reduced to (R)-enantiomer through selective oxidation and asymmetric reduction involving self-circulation of cofactor. Enzyme 1 oxidizes (S)-PED to 2-HAP and reduces NADP+ to NADPH. Subsequently, by enzyme 2, 2-HAP is reduced to (R)-PED, while NADPH is oxidized to NADP+]

Fig. 10 (R)-1-Phenyl-1,2-ethanediol were produced by in vitro multi-enzyme catalytic de-racemization system［87］

图11 多酶催化生产D-葡萄糖二酸［90］[Conversion of G1P to GlucA was achieved by six enzymes: i) a phosphoglucomutase (PGM) which catalyses intermolecular phosphate transfer of G1P to G6P, ii) a myo-inositol-3-phosphate synthase (IPS) which catalyses multi-step oxidation, cyclisation and reduction of G6P to I1P, iii) a inositol-1-monophosphatase (IMP) which catalyses dephosphorylation of I1P to myo-inositol, iv) a myo-inositol oxygenase (MIOX) which mediates oxygen-catalysed oxidation of myo-inositol to GA, v) uronate dehydrogenase (Udh) which catalyses oxidation of GA to glucaro-1,4-lactone (GlucA lactone) under reduction of NAD+ to NADH and vi) a NADH oxidase (Nox) for NAD+ regeneration to minimise supply of expensive coenzyme for continuous GlucA production. GlucA lactone ring is mostly stable in neutral solutions and is hydrolysed to GlucA with heating in alkaline conditions]

Fig. 11 Production of D-gluconic acid by multi-enzyme catalysis from glucose［90］

图12 多酶催化生产单萜［102］(Enzymes in each sub-module are marked with colored boxes, and steps of consuming and generating ATP are shown in purple and blue respectively. Purge valve that produces NADPH is shown in red, while top of mevalonate pathway that consumes NADPH is shown in orange. Solid arrow represents flow of cofactor, and dotted arrow emphasizes cycle of each step in pathway)

Fig. 12 Production of monoterpenes from glucose by multi-enzyme catalysis［102］

图13 无细胞多酶催化合成5-氨基乙酰丙酸［107］[(Succinyl-CoA synthase catalyzes succinate to produce succinyl-CoA, and CoA can be catalyzed by two reactions catalyzed by 5-aminolevulinic acid synthase and succinyl-CoA synthase recycling is carried out, and another required substrate, ATP, provides required ATP from polyphosphate through polyphosphate kinase regeneration system)]

Fig. 13 Synthesis of 5-aminolevulinic acid catalyzed by cell-free multi-enzyme［107］

图14 Cy5-LEVLVP-QD纳米探针多酶检测原理［109］

Fig. 14 Schematic representation of multi-enzyme detection using Cy5-LEVLVP-QD nanoprobe［109］

图15 SWCNT-（PDDA/ALP）n-PDDA-PSS-Ab2生物偶联物的制备［110］

Fig. 15 Illustration of preparation of SWCNT-(PDDA/ALP)n-PDDA-PSS-Ab2 bioconjugates［110］

图16 DNA支架组装CRISPR/Cas定向多酶复合物［111］

Fig. 16 General scheme of CRISPR/Cas-directed programmable assembly of multienzyme complexes on a DNA scaffold［111］