Effects of Increased NADPH Concentration by Metabolic Engineering of the Pentose Phosphate Pathway on Antibiotic Production and Sporulation in Streptomyces lividans TK24
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Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.
Abstract
Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.
참고문헌 (30)
- Horinouchi S., "A microbial hormone, A-factor, as a master switch for morphological differentiation and secondary metabolism in Streptomyces griseus", Front. Biosci, 7, pp.d2045~d2057 (2002)
- Craney A, "Towards a new science of secondary metabolism", J. Antibiot, 66, pp.387~400 (2013)
- Butler MJ, "Engineering of primary carbon metabolism for improved antibiotic production in Streptomyces lividans", Appl. Environ. Microbiol, 68, pp.4731~4739 (2002)
- Poulsen BR, "Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger", FEBS J, 272, pp.1313~1325 (2005)
- Corre C, "Evidence for the unusual condensation of a diketide with a pentulose in the methylenomycin biosynthetic pathway of Streptomyces coelicolor A3(2)", Chembiochem, 6, pp.2166~2170 (2005)
- Huppe HC, "Coordination of chloroplastic metabolism in N-limited Chlamydomonas reinhardtii by redox modulation (II. Redox modulation activates the oxidative pentose phosphate pathway during photosynthetic nitrate assimilation)", Plant Physiol, 105, pp.1043~1048 (1994)
- Hood DW, "Molecular genetic analysis of proline and tryptophan biosynthesis in Streptomyces coelicolor A3(2): interaction between primary and secondary metabolism — a review", Gene, 115, pp.5~12 (1992)
- Zhang CS, "Biosynthesis of the C(7)-cyclitol moiety of acarbose in Actinoplanes species SE50", 110. 7-OPhosphorylation of the initial cyclitol precursor leads to proposal of a new biosynthetic pathway, 2002, pp.277, 22853~22862
- Minagawa K, "ValC, a new type of C7-cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin A", Chembiochem, 8, pp.632~641 (2007)
- Choi WS, "Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-epi-5-epi-valiolone synthase gene in Streptomyces albus ATCC 21838", Appl. Microbiol. Biotechnol, 80, pp.637~645 (2008)
- Miclet E, "NMR spectroscopic analysis of the first two steps of the pentose-phosphate pathway elucidates the role of 6-phosphogluconolactonase", J. Biol. Chem, 276, pp.34840~34846 (2001)
- Bentley SD, "Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2)", Nature, 417, pp.141~147 (2002)
- Sundaram S, "Multiple oligomeric forms of glucose-6-phosphate dehydrogenase in cyanobacteria and the role of OpcA in the assembly process", Microbiology, 144, pp.1549~1556 (1998)
- Moritz B, "Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo", Eur. J. Biochem, 267, pp.3442~3452 (2000)
- Taguchi T, "Biosynthetic conclusions from the functional dissection of oxygenases for biosynthesis of actinorhodin and related Streptomyces antibiotics", Chem. Biol, 20, pp.510~520 (2013)
- Kieser T, "Practical Streptomyces Genetics" (2000), John Innes Foundation
- Vara J, "Cloning of genes governing the deoxysugar portion of the erythromycin biosynthesis pathway in Saccharopolyspora erythraea (Streptomyces erythreus)", J. Bacteriol, 171, pp.5872~5881 (1989)
- Green MR, "A Laboratory Manual" (2012), Cold Spring Harbor Laboratory Press
- Wamelink MM, "Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LCMS", MS: application to two new inherited defects of metabolism, 2005, pp.823, 18~25
- Bystrykh LV, "Production of actinorhodin-related 'blue pigments' by Streptomyces coelicolor A3(2)", J. Bacteriol, 178, pp.2238~2244 (1996)
- Takano E, "Transcriptional regulation of the redD transcriptional activator gene accounts for growth-phase-dependent production of the antibiotic undecylprodigiosin in Streptomyces coelicolor A3(2)", Mol. Microbiol, 6, pp.2797~2804 (1992)
- Avignone Rossa C, "Carbon flux distribution in antibiotic-producing chemostat cultures of Streptomyces lividans", Metab. Eng, 4, pp.138~150 (2002)
- Coze F, "Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-producing strain M145 and its non-producing derivative M1146", PLoS One, 8, pp.e84151~ (2013)
- Kim HB, "Metabolic flux analysis for calcium dependent antibiotic (CDA)production in Streptomyces coelicolor", Metab. Eng, 6, pp.313~325 (2004)
- Borodina I, "Antibiotic overproduction in Streptomyces coelicolor A3(2) mediated by phosphofructokinase deletion", J. Biol. Chem, 283, pp.25186~25199 (2008)
- Ryu YG, "Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor", Appl. Environ. Microbiol, 72, pp.7132~7139 (2006)
- Bartel PL, "Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in streptomycetes: clarification of actinorhodin gene functions", J. Bacteriol, 172, pp.4816~4826 (1990)
- Beltran- Alvarez P, "Dissecting the component reactions catalyzed by the actinorhodin minimal polyketide synthase", Biochemistry, 46, pp.14672~14681 (2007)
- Williamson NR, "The biosynthesis and regulation of bacterial prodiginines", Nat. Rev. Microbiol, 4, pp.887~899 (2006)
- Trutko S, "The role of prodigiosin biosynthesis in the regulation of oxidative metabolism of the producer Serratia marcescens", Mikrobiologiia, 58, pp.723~729 (1989)
