Abstract
In silico metabolic network models are valuable tools for strain improvement with desired properties. In this work, based on the comparisons of each pathway flux under two different objective functions for the reconstructed metabolic network of Streptomyces roseosporus, three potential targets of zwf2 (code for glucose-6-phosphate hydrogenase), dptI (code for α-ketoglutarate methyltransferase), and dptJ (code for tryptophan oxygenase) were identified and selected for the genetic modifications. Overexpression of zwf2, dptI, and dptJ genes increased the daptomycin concentration up to 473.2, 452.5, and 489.1 mg/L, respectively. Furthermore, co-overexpression of three genes in series resulted in a 34.4% higher daptomycin concentration compared with the parental strain, which ascribed to the synergistic effect of the enzymes responsible for daptomycin biosynthesis. Finally, the engineered strain enhanced the yield of daptomycin up to 581.5 mg/L in the fed-batch culture, which was approximately 43.2% higher than that of the parental strain. These results demonstrated that the metabolic network based on in silico prediction would be accurate, reasonable, and practical for target gene identification and strain improvement.
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Alper H, Jin YS, Moxley JF, Stephanopoulos G (2005) Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metab Eng 7:155–164
Bai DM, Zhao XM, Li XG, Xu SM (2004) Strain improvement of Rhizopus oryzae for over-production of L(+)-lactic acid and metabolic flux analysis of mutants. Biochem Eng J 18:41–48
Baltz RH, Miao V, Wrigley SK (2005) Natural products to drugs: daptomycin and related lipopeptide antibiotics. Nat Prod Rep 22:717–741
Borodina I, Krabben P, Nielsen J (2005) Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism. Genome Res 15:820–829
Borodina I, Siebring J, Zhang J, Smith CP, van Keulen G, Dijkhuizen L, Nielsen J (2008) Antibiotic overproduction in Streptomyces coelicolor A3(2) mediated by phosphofructokinase deletion. J Biol Chem 283:25186–25199
Burgard AP, Pharkya P, Maranas CD (2003) OptKnock: a bilevel programming framework for identifying gene knockout strategies for microbial strain optimization. Biotechnol Bioeng 84:647–657
Butler MJ, Bruheim P, Jovetic S, Marinelli F, Postma PW, Bibb MJ (2002) Engineering of primary carbon metabolism for improved antibiotic production in Streptomyces lividans. Appl Environ Microbiol 68:4731–4739
Celik E, Calik P, Oliver SG (2010) Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: effects of methanol feeding rate. Biotechnol Bioeng 105:317–329
Christensen B, Nielsen J (2000) Metabolic network analysis of Penicillium chrysogenum using 13C-labeled glucose. Biotechnol Bioeng 68:652–659
Christensen B, Thykaer J, Nielsen J (2000) Metabolic characterization of high- and low-yielding strains of Penicillium chrysogenum. Appl Microbiol Biotechnol 54:212–217
Duan YX, Chen T, Chen X, Zhao XM (2010) Overexpression of glucose-6-phosphate dehydrogenase enhances riboflavin production in Bacillus subtilis. Appl Microbiol Biotechnol 85:1907–1914
Feist AM, Henry CS, Reed JL, Krummenacker M, Joyce AR, Karp PD, Broadbelt LJ, Hatzimanikatis V, Palsson BO (2007) A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1260 ORFs and thermodynamic information. Mol Syst Biol 3:121
Fowler ZL, Gikandi WW, Koffas MA (2009) Increased malonyl coenzyme A biosynthesis by tuning the Escherichia coli metabolic network and its application to flavanone production. Appl Environ Microbiol 75:5831–5839
Führer L, Kubicek CP, Röhr M (1980) Pyridine nucleotide levels and ratios in Aspergillus niger. Can J Microbiol 26:405–408
Hodgson DA (2000) Primary metabolism and its control in streptomycetes: a most unusual group of bacteria. Adv Microb Physiol 42:47–238
Huber FM, Pieper RL, Tietz AJ (1988) The formation of daptomycin by supplying decanoic acid to Streptomyces roseosporus cultures producing the antibiotic complex A21978C. J Biotechnol 7:283–292
Ishimura Y, Nozaki M, Hayaishi O (1970) The oxygenated form of L-tryptophan 2, 3-dioxygenase as reaction intermediate. J Biol Chem 245:3593–3602
Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Foundation, Norwich
Kim HB, Smith CP, Micklefield J, Mavituna F (2004) Metabolic flux analysis for calcium dependent antibiotic (CDA) production in Streptomyces coelicolor. Metab Eng 6:313–325
Kim SH, Lee HN, Kim HJ, Kim ES (2011) Transcriptome analysis of an antibiotic downregulator mutant and synergistic Actinorhodin stimulation via disruption of a precursor flux regulator in Streptomyces coelicolor. Appl Environ Microbiol 77:1872–1877
Lessie TG, Vander Wyk JC (1972) Multiple forms of Pseudomomas multivorans glucose-6-phosphate and 6-phosphogluconate dehydrogenases: differences in size, pyridine nucleotide specificity and susceptibility to inhibition by adenosine 5′-triphosphate. J Bacteriol 110:1107–1117
Li R, Townsend CA (2006) Rational strain improvement for enhanced clavulanic acid production by genetic engineering of the glycolytic pathway in Streptomyces clavuligerus. Metab Eng 8:240–252
Liu T, You D, Valenzano C, Sun Y, Li J, Yu Q, Zhou X, Cane DE, Deng Z (2006) Identification of NanE as the thioesterase for polyether chain release in nanchangmycin biosynthesis. Chem Biol 13:945–955
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408
MacNeil DJ, Gewain KM, Ruby CL, Dezeny G, Gibbons PH, MacNeil T (1992) Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111:61–68
Mahlert C, Kopp F, Thirlway J, Micklefield J, Marahiel MA (2007) Stereospecific enzymatic transformation of α-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesis. J Am Chem Soc 129:12011–12018
Mathai D, Biedenbach DJ, Jones RN, Bell JM, Turnidge J, Sader HS (2009) Activity of daptomycin against Gram-positive bacterial isolates from Indian medical centres. Int J Antimicrob Agents 34:497–499
Miao V, Coëffet-Legal MF, Brian P, Brost R, Penn J, Whiting A, Martin S, Ford R, Parr I, Bouchard M, Silva CJ, Wrigley SK, Baltz RH (2005) Daptomycin biosynthesis in Streptomyces roseosporus: cloning and analysis of the gene cluster and revision of peptide stereochemistry. Microbiology 151:1507–1523
Nailor MD, Sobel JD (2009) Antibiotics for gram-positive bacterial infections: vancomycin, teicoplanin, quinupristin/dalfopristin, oxazolidinones, daptomycin, dalbavancin, and telavancin. Infect Dis Clin North Am 23:965–982
Nguyen KT, Kau D, Gu JQ, Brian P, Wrigley SK, Baltz RH, Miao V (2006) A glutamic acid 3-methyltransferase encoded by an accessory gene locus important for daptomycin biosynthesis in Streptomyces roseosporus. Mol Microbiol 61:1294–1307
Obanye AIC, Hobbs G, Gardner DCJ, Oliver SG (1996) Correlation between carbon flux through the pentose phosphate pathway and production of the antibiotic methylenomycin in Streptomyces coelicolor A3(2). Microbiology 142:133–137
Okamoto S, Lezhava A, Hosaka T, Okamoto-Hosoya Y, Ochi K (2003) Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J Bacteriol 185:601–609
Paradkar AS, Mosher RH, Anders C, Griffin A, Griffin J, Hughes C, Greaves P, Barton B, Jensen SE (2001) Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production. Appl Environ Microbiol 67:2292–2297
Park JH, Lee KH, Kim TY, Lee SY (2007) Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc Natl Acad Sci USA 104:7797–7802
Peng L, Shimizu K (2003) Global metabolic regulation analysis for Escherichia coli K-12 based on protein expression by 2D electrophoresis and enzyme activity measurement. Appl Microbiol Biotechnol 61:163–178
Reeves AR, Cernota WH, Brikun IA, Wesley RK, Weber JM (2004) Engineering precursor flow for increased erythromycin production in Aeromicrobium erythreum. Metab Eng 6:300–312
Rhee KH, Davies J (2006) Transcription analysis of daptomycin biosynthetic genes in Streptomyces roseosporus. J Microbiol Biotechnol 16:1841–1848
Ryu YG, Butler MJ, Chater KF, Lee KJ (2006) Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor. Appl Environ Microbiol 72:7132–7139
Salas JA, Quiros LM, Hardisson C (1984) Pathways of glucose catabolism during germination of Streptomyces spores. FEMS Microbiol Lett 22:229–233
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Shi S, Chen T, Zhang Z, Chen X, Zhao X (2009) Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production. Metab Eng 11:243–252
Stephanopoulos G, Aristidou AA, Nielsen J (1998) Metabolic engineering: principles and methodologies, 1st edn. Academic, San Diego
Sun Y, He X, Liang J, Zhou X, Deng Z (2009) Analysis of functions in plasmid pHZ1358 influencing its genetic and structural stability in Streptomyces lividans 1326. Appl Microbiol Biotechnol 82:303–310
Tamehiro N, Hosaka T, Xu J, Hu H, Otake N, Ochi K (2003) Innovative approach for improvement of an antibiotic-overproducing industrial strain of Streptomyces albus. Appl Environ Microbiol 69:6412–6417
Taymaz-Nikerel H, Borujeni AE, Verheijen PJ, Heijnen JJ, van Gulik WM (2010) Genome-derived minimal metabolic models for Escherichia coil MG1655 with estimated in vivo respiratory ATP stoichiometry. Biotechnol Bioeng 107:369–381
Thykaer J, Nielsen J, Wohlleben W, Weber T, Gutknecht M, Lantz AE, Stegmann E (2010) Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster. Metab Eng 12:455–461
Tian WN, Braunstein LD, Pang J, Stuhlmeier KM, Xi QC, Tian X, Stanton RC (1998) Importance of glucose-6-phosphate dehydrogenase activity for cell growth. J Biol Chem 273:10609–10617
Wittmann M, Linne U, Pohlmann V, Marahiel MA (2008) Role of DptE and DptF in the lipidation reaction of daptomycin. FEBS J 275:5343–5354
Acknowledgments
This research was financially supported by the National 973 Project of China (No. 2011CB710800), the Key Program of National Natural Science Foundation of China (Grant No. 20936002), National Natural Science Foundation of China (No. 21076022), and the Programme of Introducing Talents of Discipline to Universities (No. B06006).
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Huang, D., Wen, J., Wang, G. et al. In silico aided metabolic engineering of Streptomyces roseosporus for daptomycin yield improvement. Appl Microbiol Biotechnol 94, 637–649 (2012). https://doi.org/10.1007/s00253-011-3773-6
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DOI: https://doi.org/10.1007/s00253-011-3773-6