Balancing carbon flux rebalancing around phosphoenolpyruvate node for enhancement of FK506 production in Streptomyces tsukubaensis

doi:10.16085/j.issn.1000-6613.2017-1482

Chemical Industry and Engineering Progress ›› 2018, Vol. 37 ›› Issue (06): 2347-2353.DOI: 10.16085/j.issn.1000-6613.2017-1482

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Balancing carbon flux rebalancing around phosphoenolpyruvate node for enhancement of FK506 production in Streptomyces tsukubaensis

LÜ Mengmeng^1,2, LIU Jiao^1,2, LIU Huanhuan^1,2, CHEN Hong^1,2, WANG Cheng^1,2, WEN Jianping^1,2

1 Key Laboratory of System Bioengineering(Tianjin University), Ministry of Education, Tianjin 300072, China;
2 SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering(Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Received:2017-07-18 Revised:2017-11-16 Online:2018-06-05 Published:2018-06-05

平衡磷酸烯醇式丙酮酸节点通量强化筑波链霉菌合成他克莫司

吕蒙蒙^1,2, 刘蛟^1,2, 刘欢欢^1,2, 陈红^1,2, 王成^1,2, 闻建平^1,2

1 天津大学化工学院系统生物工程教育部重点实验室, 天津 300072;
2 天津大学天津化学化工协同创新中心, 天津 300072

通讯作者: 闻建平,教授,研究方向为生物化工。
作者简介:吕蒙蒙(1991-),女,硕士研究生。
基金资助:
天津市重点项目（16YFZCSY00780）、国家973项目（2013CB733600）、国家自然科学基金重点项目（21236005）及国家自然科学基金项目（21376171）。

Abstract

Abstract: Tacrolimus(FK506),as one of the widely used immunosuppressants produced by Streptomyces species,has drawn much attention on clinic application. However,the low FK506 fermentation titer restricts its industrial production,which is mainly due to the insufficient precursor metabolism of the producing strain. In this work,balancing carbon flux rebalancing around phosphoenolpyruvate(PEP)node for enhancement of FK506 production were carried on. Firstly,the genes fkbO and fkbL were overexpressed in S. tsukubaensis D852,achieving S. tsukubaensis-OL1,of which FK506 production changed only slightly from 158.7mg/L to 163.9mg/L. Then,two precursor metabolic pathways,the anaplerotic and shikimate pathways emanating from PEP node,were fine-tuned for eliminating the inefficient supply of precursors of DHCHC and pipecolate. The genes encoding PPC and DAHPS were cloned from various species and expressed in S. tsukubaensis-OL1,respectively,and the FK506 production was separately increased by 40%(ppc,S. tsukubaensis)and 47%(dahP,S. roseosporus). Subsequently,the expression levels of the genes ppc and dahP were modulated under the control of four constitutive promoters(PermE*,Psco4503,Psco3410 and Psco5768)in nine engineering strains,resulting in the final increase of FK506 production from 163.9mg/L to 350.3mg/L. This work demonstrated that the optimization of the branches emanating from PEP node was a viable strategy to strengthen FK506 production in S. tsukubaensis.

Key words: tacrolimus(FK506), anaplerotic pathway, shikimate pathways, constitutive promoters, Streptomyces tsukubaensis

摘要： 他克莫司（FK506）是最重要的免疫抑制剂之一，然而前体代谢供应不足制约着工业化生产。通过优化平衡磷酸烯醇式丙酮酸（PEP）节点支路通量可提高FK506产量。本文首先在S.tsukubaensis D852中过表达基因fkbO（编码分支酸水合还原酶）和fkbL（编码赖氨酸环化酶）得到S.tsukubaensis-OL1，FK506的产量仅从158.7mg/L提高到163.9mg/L。随后调节PEP节点支路回补途径和莽草酸途径通量强化FK506的合成：先分别将不同菌株中编码磷酸烯醇式丙酮酸羧化酶（PPC）和3-脱氧-D-阿拉伯糖基-heptulosonate-7-磷酸合酶（DAHPS）的基因在S.tsukubaensis-OL1中过表达，FK506的产量分别提高40%（ppc，S.tsukubaensis）和47%（dahP，S.roseosporus）；然后采用4个不同强度的组成型启动子（PermE*，Psco4503，Psco3410 and Psco5768）平衡ppc和dahP的表达水平获得9株工程菌，最终使FK506的产量由163.9mg/L显著提高到350.3mg/L。这个结果说明优化平衡PEP节点竞争支路通量是提高FK506产量的有效策略。

关键词: 他克莫司, 回补途径, 莽草酸途径, 组成型启动子, 筑波链霉菌

CLC Number:

Q591

LÜ Mengmeng, LIU Jiao, LIU Huanhuan, CHEN Hong, WANG Cheng, WEN Jianping. Balancing carbon flux rebalancing around phosphoenolpyruvate node for enhancement of FK506 production in Streptomyces tsukubaensis[J]. Chemical Industry and Engineering Progress, 2018, 37(06): 2347-2353.

吕蒙蒙, 刘蛟, 刘欢欢, 陈红, 王成, 闻建平. 平衡磷酸烯醇式丙酮酸节点通量强化筑波链霉菌合成他克莫司[J]. 化工进展, 2018, 37(06): 2347-2353.

References

[1] PARSONS W H, SIGAL N H, WYVRATT M J. FK-506:a novel immunosuppressant[J]. Ann. N. Y. Acad. Sci., 1993, 685(1):22-36.
[2] SIERRA-PAREDES G, SIERRA-MARCUNO G. Ascomycin and FK506:pharmacology and therapeutic potential as anticonvulsants and neuroprotectants[J]. CNS Neurosci. Ther., 2008, 14:36-46.
[3] SHAPIRO R, YOUNG J B, MILFORD E L, et al. Immunosuppression:evolution in practice and trends[J]. Am. J. Transplant, 2005, 5:874-886.
[4] BARREIRO C, MARTINEZ-CASTRO M. Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506)[J]. Appl. Microbiol. Biotechnol., 2014, 98(2):497-507.
[5] MO S D, KIM H, LEE J H, et al. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues[J]. J. Am. Chem. Soc., 2011, 133(4):976-985.
[6] MOTAMEDI H, SHAFIEE A, CAI S J, et al. Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520[J]. J. Bacteriol., 1996, 178(17):5243-5248.
[7] ANDEXER J N, KENDREW S G, NUR-E-ALAM M, et al. Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate[J]. Proc. Natl. Acad. Sci. U S A, 2011, 108(12):4776-4781.
[8] GATTO G J, BOYNE M T, KELLEHER N L, et al. Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster[J]. J. Am. Chem. Soc., 2006, 128(11):3838-3847.
[9] COOMES M W, MITCHELL B K, BEEZLEY A, et al. Properties of an Escherichia coli mutant deficient in phosphoenolpyruvate carboxylase catalytic activity[J]. J. Bacteriol.,1985,164(2):646-652.
[10] MARTINEZ-CASTRO M, SALEHI-NAJAFABADI Z, ROMERO F, et al. Taxonomy and chemically semi-defined media for the analysis of the tacrolimus producer ‘Streptomyces tsukubaensis’[J]. Appl. Microbiol. Biotechnol., 2013, 97(5):2139-2152.
[11] TURLO J, GAJZLERSKA W, KLIMASZEWSKA M, et al. Enhancement of tacrolimus productivity in Streptomyces tsukubaensis by the use of novel precursors for biosynthesis[J]. Enzyme Microb. Technol., 2012, 51(6-7):388-395.
[12] MO S, BAN Y H, PARK J W, et al. Enhanced FK506 production in Streptomyces clavuligerus CKD1119 by engineering the supply of methylmalonyl-CoA precursor[J]. J. Ind. Microbiol. Biotechnol., 2009, 36(12):1473-8142.
[13] GORANOVIC D, KOSEC G, MRAK P, et al. Origin of the allyl group in FK506 biosynthesis[J]. J. Biol. Chem., 2010, 285(19):14292-142300.
[14] CHAN Y A, PODEVELS A M, KEVANY B M, et al. Biosynthesis of polyketide synthase extender units[J]. Nat. Prod. Rep., 2009, 26(1):90-114.
[15] BRAMWELL H, NIMMO H G, HUNTER I S, et al. Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2):purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete[J]. Biochem. J., 1993, 293:131-136.
[16] THOMAS L, HODGSON D A, WENTZEL A, et al. Metabolic switches and adaptations deduced from the proteomes of streptomyces coelicolor wild type and phoP mutant grown in batch culture[J]. Mol. Cell Proteomics, 2011, 11(2):M111.013797.
[17] COZE F, GILARD F, TCHERKEZ G, et al. Carbon-flux distribution within Streptomyces coelicolor Metabolism:a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146[J]. PLoS One, 2013, 8(12):e84151.
[18] DEKLEVA M L, STROHL W R. Activity of phosphoenolpyruvate carboxylase of an anthracycline-producing Streptomycete[J]. Can. J. Microbiol., 1988, 34:1241-1246.
[19] DEKLEVA M L, STROHL W R. Biosynthesis of epsilon-rhodomycinone from glucose by Streptomyces C5 and comparison with intermediary metabolism of other polyketide producing Streptomycetes[J]. Can. J. Microbiol., 1988, 34:1235-1240.
[20] NING Y, WU X, ZHANG C, et al. Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli[J]. Metab. Eng., 2016, 36:10-18.
[21] LEE K H, PARK J H, KIM T Y, et al. Systems metabolic engineering of Escherichia coli for L-threonine production[J]. Molecular Systems Biology, 2007, 3:149.
[22] HUANG D, LI S, XIA M, et al. Genome-scale metabolic network guided engineering of Streptomyces tsukubaensis for FK506 production improvement[J]. Microb. Cell Fact., 2013, 12:52.
[23] THYKAER J, NIELSEN J, WOHLLEBEN W, et al. Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster[J]. Metab. Eng., 2010, 12:455-461.
[24] GOSSET G, BONNER C A, JENSEN R A. Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate-and tryptophan-regulated enzyme from Xanthomonas campestris[J]. J. Bacteriol., 2001, 183(13):4061-4070.
[25] WU J, DU G, ZHOU J, CHEN J. Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy[J]. Metab. Eng., 2013, 16:48-55.
[26] DANG L, LIU J, WANG C, et al. Enhancement of rapamycin production by metabolic engineering in Streptomyces hygroscopicus based on genome-scale metabolic model[J]. J. Ind. Microbiol. Biotechnol., 2016, 44(2):259-270.
[27] WANG W, LI X, WANG J, et al. An engineered strong promoter for streptomycetes[J]. Appl. Environ. Microbiol., 2013, 79:4484-4492.
[28] GU P, YANG F, KANG J, et al. One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli[J]. Microb. Cell Fact., 2012, 11:30.
[29] XIA M, HUANG D, LI S, et al. Enhanced FK506 production in Streptomyces tsukubaensis by rational feeding strategies based on comparative metabolic profiling analysis[J]. Biotechnol. Bioeng., 2013, 110(10):2717-2730.
[30] WILKINSON C J, HUGHES-THOMAS Z A, MARTIN C J, et al. Increasing the effciency of heterologous promoters in actinomycetes[J]. J. Mol. Microbiol. Biotechnol., 2002, 4:417-426.
[31] KIESER T, BIBB M J, BUTTNER M J, et al. Practical streptomyces genetics[M]. Norwich:John Innes Foundation, 2000.
[32] LI S, WANG J, LI X, et al. Genome-wide dentifcation and evaluation of constitutive promoters in Streptomycetes[J]. Microb. Cell Fact., 2015, 14:172.
[33] SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative C(T) method[J]. Nat. Protoc., 2008, 3(6):1101-1108.
[34] PENG L, SHIMIZU K. Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement[J]. Appl. Microbiol. Biotechnol., 2003, 61:163-178.
[35] LIU Y J, LI P P, ZHAO KX, et al. Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features[J]. Appl. Environ. Microbiol., 2008, 74(17):5497-5503.

Balancing carbon flux rebalancing around phosphoenolpyruvate node for enhancement of FK506 production in Streptomyces tsukubaensis

平衡磷酸烯醇式丙酮酸节点通量强化筑波链霉菌合成他克莫司

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