代谢工程改造益生菌E. coli Nissle 1917合成靛玉红

doi:10.13560/j.cnki.biotech.bull.1985.2025-0718

摘要/Abstract

摘要：

目的靛玉红源于传统中药青黛，是一种具有广谱抗菌、抗炎及抗肿瘤活性的双吲哚环类天然活性成分，在临床主要被用于联合给药治疗慢性粒细胞白血病、银屑病等。探索利用安全的益生菌大肠杆菌Nissle 1917（EcN）为底盘，构建靛玉红的生物合成体系，以开发此中药材活性成分的绿色供应体系。方法基于EcN天然内源质粒的工程化改造，系统评估利用噬甲基菌Methylophaga sp. SK1来源的黄素单加氧酶fmo及其突变体（fmo^K223R 、fmo^K223R/D317S ），构建靛玉红合成途径的产物输出效率。结合代谢工程改造，敲除色氨酸合成途径的竞争性支路关键基因，包括苯丙氨酸合成关键基因pheA及酪氨酸合成关键基因tyrA；以此为基础，再行失活三羧酸循环的上游途径基因，包括丙酮酸激酶基因pykA和磷酸烯醇式丙酮酸羧化酶基因ppc。最后，通过缺失色氨酸途径中的阻遏蛋白基因trpR，同时强化色氨酸途径中抗反馈抑制的trpE^S40F 等，进一步提升产物合成水平。结果经250 mL摇瓶（装液量50 mL）培养48 h显示，靛玉红产量达（176.9 ± 4.5）mg/L，相较于仅携带野生型fmo基因的亲本菌株提升约3.5倍，5 L发酵罐产量为（379.3 ± 12.3）mg/L。结论基于益生菌EcN内源质粒携带异源黄素单加氧酶突变体基因fmo^K223R，结合对色氨酸前体供应途径的代谢工程改造，获得的靛玉红产量达300 mg/L以上，展现出EcN作为细胞工厂生物合成传统中药活性成分的优良潜力。

关键词: 大肠杆菌Nissle 1917, 黄素单加氧酶bFMO, 中药青黛, 靛玉红, 生物合成

Abstract:

Objective Indirubin, a bi-indolic alkaloid derivative isolated from the traditional Chinese herbal medicine Indigo Naturalis, represents a potent therapeutic compound having broad-spectrum antibacterial, anti-inflammatory, and anti-tumor properties. Currently, it is clinically utilized primarily in combination therapy regimens for the treatment of chronic myeloid leukemia (CML), psoriasis, and related pathologies. This study investigates the potential of the safe Escherichia coli Nissle 1917 (EcN) as a chassis for constructing a green supply system for the active components of the traditional Chinese medicine indirubin. Method Based on the engineering modification of the natural endogenous plasmid of EcN, the product output efficiency of the indirubin biosynthesis pathway constructed using the flavin monooxygenase fmo and its mutants (fmo^K223R and fmo^K223R/D317S )) from Methylophaga sp. SK1 was systematically evaluated. Combined with metabolic engineering, key genes in the competitive branch of the tryptophan biosynthesis pathway were knocked out, including pheA, a key gene for phenylalanine biosynthesis, and tyrA, a key gene for tyrosine biosynthesis. Further optimization was achieved by inactivating the upstream genes of the tricarboxylic acid cycle, including pykA (pyruvate kinase) and ppc (phosphoenolpyruvate carboxylase). Finally, the product synthesis level was further improved by deleting the repressor protein gene trpR in the tryptophan pathway and strengthening the anti-feedback inhibition gene trpE^S40F in the tryptophan pathway. Result After 48 h of shaking culture in a 250 mL flask (50 mL culture), the indirubin titer reached (176.9 ± 4.5) mg/L, representing a 3.5-fold increase compared to the parent strain harboring only the wild-type fmo gene. The production in the 5 L bioreactor was (379.3 ± 12.3) mg/L. Conclusion Employing the endogenous cryptic plasmid of the probiotic EcN to express the heterologous flavin monooxygenase mutant gene fmo^K223R, and integrating this with systematic metabolic engineering of endogenous tryptophan biosynthesis pathways, the production of indirubin reached over 300 mg/L, demonstrating the excellent potential of EcN as a cell factory for biosynthesis of active ingredients in traditional Chinese medicines.

Key words: Escherichia coli Nissle 1917, flavin monooxygenase bFMO, traditional Chinese medicine Indigo Naturalis, indirubin, biosynthesis

毛李晶, 金晓萱, 施婉婷, 胡飞杨, 张苑蓉, 熊亮斌, 任璐. 代谢工程改造益生菌E. coli Nissle 1917合成靛玉红[J]. 生物技术通报, 2025, 41(12): 74-81.

MAO Li-jing, JIN Xiao-xuan, SHI Wan-ting, HU Fei-yang, ZHANG Yuan-rong, XIONG Liang-bin, REN Lu. Modifying the Probiotic Escherichia coli Nissle 1917 for the Biosynthesis of Indirubin via Metabolic Engineering[J]. Biotechnology Bulletin, 2025, 41(12): 74-81.

图/表 5

图1 利用大肠杆菌生物合成靛玉红的途径构建G6P：葡萄糖-6-磷酸；G3P：甘油醛-3-磷酸；PEP：磷酸烯醇式丙酮酸；E4P：赤藓糖-4-磷酸；DAHP：3-脱氧-D-阿拉伯庚酮糖-7-磷酸；CHA：分支酸；Prep：预苯酸；Phe：苯丙氨酸；Tyr：酪氨酸；PYR：丙酮酸；OAA：草酰乙酸；2-HID：2-羟基吲哚；3-HID：3-羟基吲哚

Fig. 1 Construction of the biosynthesis pathway of indirubin in Escherichia coliG6P: glucose-6-phosphate; G3P: glyceraldehyde-3-phosphate; PEP: phosphoenolpyruvate; E4P: erythrose-4-phosphate; DAHP: 3-deoxy-D-arabino-heptulosonate-7-phosphate; CHA: chorismate; Prep: prephenate; Phe: phenylalanine; Tyr: tyrosine; PYR: pyruvate; OAA: oxaloacetate; 2-HID: 2-hydroxyindole; 3-HID: 3-hydroxyindole

图2 向EcN引入fmo基因及其突变体对靛玉红产量的影响A：EIR01-03重组菌落PCR的凝胶电泳结果。依次为构建pI2e::fmo、pI2e::fmo K223R 及pI2e::fmoK223R/D317S重组菌的挑菌验证结果；B：过表达fmo及其突变体的工程菌（EIR01-03）靛玉红产量评估情况。采用t-检验判断数据差异性（***P<0.001）

Fig. 2 Effect of introducing the fmo gene and its mutants into E. coli Nissle 1917 on the production of indirubinA: Agarose gel electrophoresis analysis of colony PCR for recombinant strains EIR01-03. Lanes show verification results for colonies of constructs: pI2e::fmo, pI2e::fmoK223R and pI2e::fmoK223R/D317S. B: Indirubin production by engineered strains EIR01-03 overexpressing fmoand its mutants. Significant differences were determined using student’s t-test (***P<0.001)

图3 敲除前体合成相关途径基因对靛玉红产量的影响采用t-检验判断数据差异性（**P<0.01；***P<0.001，ns：无显著性差异）

Fig. 3 Effects of knocking-out of gene related to the precursor synthesis on indirubin productionSignificant differences were determined using student’s t-test (**P<0.01; ***P<0.001; ns: not significant)

图4 删除分支酸合成竞争路径基因对靛玉红合成的影响

Fig. 4 Effects of deleting genes in the competitive pathway of chorismate biosynthesis on the production of indirubin

图5 利用工程菌株EIR10分批补料发酵生产靛玉红

Fig. 5 Fed-batch fermentation of the engineered strain EIR10 for producing the indirubin

参考文献 32

[1]	Meijer L, Skaltsounis AL, Magiatis P, et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins [J]. Chem Biol, 2003, 10(12): 1255-1266.
[2]	Hoessel R, Leclerc S, Endicott JA, et al. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases [J]. Nat Cell Biol, 1999, 1(1): 60-67.
[3]	Leclerc S, Garnier M, Hoessel R, et al. Indirubins inhibit glycogen synthase kinase-3β and CDK5/P25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease [J]. J Biol Chem, 2001, 276(1): 251-260.
[4]	Lee JY, Shin YS, Shin HJ, et al. Production of natural indirubin from indican using non-recombinant Escherichia coli [J]. Bioresour Technol, 2011, 102(19): 9193-9198.
[5]	Han GH, Gim GH, Kim W, et al. Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation [J]. J Biotechnol, 2013, 164(2): 179-187.
[6]	Shi YS, Wang D, Li RS, et al. Engineering yeast subcellular compartments for increased production of the lipophilic natural products ginsenosides [J]. Metab Eng, 2021, 67: 104-111.
[7]	Hu ZF, Gu AD, Liang L, et al. Construction and optimization of microbial cell factories for sustainable production of bioactive dammarenediol-II glucosides [J]. Green Chem, 2019, 21(12): 3286-3299.
[8]	Wang WJ, Wu Y, Xu HH, et al. Accumulation mechanism of indigo and indirubin in Polygonum tinctorium revealed by metabolite and transcriptome analysis [J]. Ind Crops Prod, 2019, 141: 111783.
[9]	熊亮斌, 唐红菊, 宋新巍, 等. 紫杉醇类抗肿瘤原料药生产的研究进展 [J]. 中草药, 2020, 51(15): 4042-4049.
	Xiong LB, Tang HJ, Song XW, et al. Recent advances in synthesis of paclitaxel antitumor pharmaceutical raw materials [J]. Chin Tradit Herb Drugs, 2020, 51(15): 4042-4049.
[10]	Liang FY, Xie YM, Zhang C, et al. Elucidation of the final steps in Taxol biosynthesis and its biotechnological production [J]. Nat Synth, 2025: 1-11.
[11]	Zhang J, Hansen LG, Gudich O, et al. A microbial supply chain for production of the anti-cancer drug vinblastine [J]. Nature, 2022, 609(7926): 341-347.
[12]	Hu TY, Zhou JW, Tong YR, et al. Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast [J]. Metab Eng, 2020, 60: 87-96.
[13]	Liu XN, Cheng J, Zhang GH, et al. Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches [J]. Nat Commun, 2018, 9: 448.
[14]	Wang YN, Liu XN, Chen BH, et al. Metabolic engineering of Yarrowia lipolytica for scutellarin production [J]. Synth Syst Biotechnol, 2022, 7(3): 958-964.
[15]	Mindt M, Ferrer L, Bosch D, et al. De novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum [J]. Appl Microbiol Biotechnol, 2023, 107(5): 1621-1634.
[16]	Yang WH, Zhou JL, Gu QY, et al. Combinatorial enzymatic catalysis for bioproduction of ginsenoside compound K [J]. J Agric Food Chem, 2023, 71(7): 3385-3397.
[17]	Wang D, Wang JH, Shi YS, et al. Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of Panax notoginseng using a synthetic biology platform [J]. Metab Eng, 2020, 61: 131-140.
[18]	Yu Z, Wei XJ, Liu LT, et al. Indirubin-3'-monoxime acts as proteasome inhibitor: Therapeutic application in multiple myeloma [J]. eBioMedicine, 2022, 78: 103950.
[19]	Zdioruk M, Jimenez-Macias JL, Nowicki MO, et al. PPRX-1701, a nanoparticle formulation of 6'-bromoindirubin acetoxime, improves delivery and shows efficacy in preclinical GBM models [J]. Cell Rep Med, 2023, 4(5): 101019.
[20]	Gowda SV, Kim NY, Harsha KB, et al. A new 1, 2, 3-triazole-indirubin hybrid suppresses tumor growth and pulmonary metastasis by mitigating the HGF/c-MET axis in hepatocellular carcinoma [J]. J Adv Res, 2025, 73: 341-356.
[21]	Liu XN, Zhu XX, Wang H, et al. Discovery and modification of cytochrome P450 for plant natural products biosynthesis [J]. Synth Syst Biotechnol, 2020, 5(3): 187-199.
[22]	Jiang Y, Chen B, Duan CL, et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system [J]. Appl Environ Microbiol, 2015, 81(7): 2506-2514.
[23]	Yin HF, Chen HP, Yan M, et al. Efficient bioproduction of indigo and indirubin by optimizing a novel terpenoid cyclase XiaI in Escherichia coli [J]. ACS Omega, 2021, 6(31): 20569-20576.
[24]	Sun BY, Sui HL, Liu ZW, et al. Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin [J]. Bioresour Bioprocess, 2022, 9(1): 70.
[25]	Du JK, Li YH, Chen ZZ, et al. Functional characterization of a novel flavin reductase from a deep-sea sediment metagenomic library and its application for indirubin production [J]. Appl Environ Microbiol, 2024, 90(6): e00429-24.
[26]	Dong MM, Song L, Xu JQ, et al. Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering [J]. Appl Microbiol Biotechnol, 2023, 107(16): 5257-5267.
[27]	Dong MM, Li YX, Xu M, et al. An Escherichia coli Nissle 1917-based live therapeutics platform with integrated phage resistance and programmable temperature sensitivity [J]. J Control Release, 2025, 387: 114188.
[28]	陈博, 宋凯, 何亚文. 微生物中邻氨基苯甲酸代谢与功能研究进展 [J]. 微生物前沿, 2024, 13(3): 175-186.
	Chen B, Song K, He YW. The metabolism and functions of anthranilic acid in microorganisms [J]. Adv Microbiol, 2024, 13(3): 175-186.
[29]	Rothman SC, Voorhies M, Kirsch JF. Directed evolution relieves product inhibition and confers in vivo function to a rationally designed tyrosine aminotransferase [J]. Protein Sci, 2004, 13(3): 763-772.
[30]	Du LH, Zhang Z, Xu QY, et al. Central metabolic pathway modification to improve L-tryptophan production in Escherichia coli [J]. Bioengineered, 2019, 10(1): 59-70.
[31]	Noda S, Shirai T, Oyama S, et al. Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives [J]. Metab Eng, 2016, 33: 119-129.
[32]	翁可欣, 张明亮, 李力. 微生物合成5-羟基色氨酸的研究进展 [J]. 热带生物学报, 2023, 14(1): 42-49.
	Weng KX, Zhang ML, Li L. Advances in microbial synthesis of 5-hydroxytryptophan [J]. J Trop Biol, 2023, 14(1): 42-49.