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Participation of putative glycoside hydrolases SlgC1 and SlgC2 in the biosynthesis of streptolydigin in Streptomyces lydicus.

Gómez C, Horna DH, Olano C, Méndez C, Salas JA - Microb Biotechnol (2012)

Bottom Line: Both genes are expressed when streptolydigin is produced.Inactivation of these genes affects streptolydigin production when the microorganism is grown in minimal medium containing either glycerol or d-glucans as carbon source.Streptolydigin yields in S. lydicus were increased by overexpression of either slgC1 or slgC2.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Funcional, Universidad de Oviedo, 33006, Oviedo, Spain.

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A. Production of streptolydigin by S. lydicus (WT), SLMC1 (C1‐), SLMC2 (C2‐), and S. lydicus carrying pEM4T (Ct), pEM4TslgC1 (C1) or pEM4slgC2 (C2) grown in MMG (MM containing glycerol at 10 g l−1).B. Production of streptolydigin in MML (MM containing laminarin at 10 g l−1).C. Production of streptolydigin in MMS (MM containing starch at 10 g l−1). In all cases streptolydigin production was determined by UPLC analysis. Experiments were run in triplicate. Minimal medium MM is composed of MOPS 21 g l−1, MgSO4 0.6 g l−1, CaCl2 5 mg l−1, MnCl2 1 mg l−1, ZnSO4 1 mg l−1, FeSO4 5 mg l−1, K2HPO4 1.75 g l−1, NH4Cl 1.6 g l−1 and fumarate 2%, pH 6.8, in deionized water. Laminarin and starch were purchased form Sigma.
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f3: A. Production of streptolydigin by S. lydicus (WT), SLMC1 (C1‐), SLMC2 (C2‐), and S. lydicus carrying pEM4T (Ct), pEM4TslgC1 (C1) or pEM4slgC2 (C2) grown in MMG (MM containing glycerol at 10 g l−1).B. Production of streptolydigin in MML (MM containing laminarin at 10 g l−1).C. Production of streptolydigin in MMS (MM containing starch at 10 g l−1). In all cases streptolydigin production was determined by UPLC analysis. Experiments were run in triplicate. Minimal medium MM is composed of MOPS 21 g l−1, MgSO4 0.6 g l−1, CaCl2 5 mg l−1, MnCl2 1 mg l−1, ZnSO4 1 mg l−1, FeSO4 5 mg l−1, K2HPO4 1.75 g l−1, NH4Cl 1.6 g l−1 and fumarate 2%, pH 6.8, in deionized water. Laminarin and starch were purchased form Sigma.

Mentions: Streptomycetes are usually soil living microorganisms. Therefore, we wonder if the presence of slgC1 and slgC2 genes in the streptolydigin cluster could be an evolutionary advantage for the producer microorganism by facilitating sugar supply for streptolydigin biosynthesis in its natural soil environment. To evaluate the potential participation of SlgC1 and SlgC2 enzymes in the degradation of β‐ and α‐glucans, cultures of S. lydicus, mutants SLMC1 and SLMC2 and S. lydicus carrying pEM4T, pEM4TslgC1 or pEM4TSlgC2 were performed on three minimal media using different carbon sources: glycerol (MMG), β‐glucan laminarin (MML) and α‐glucan starch (MMS). Differences in the production of streptolydigin were observed in MMG between S. lydicus wild type and mutants SLMC1 and SLMC2. Streptolydigin yields in these mutants decreased 1.6‐ and 12‐fold respectively. In contrast, a positive effect of the expression of slgC1 or slgC2 in S. lydicus was observed. In this medium, production of streptolydigin increased 5.5‐ and 5‐fold respectively, when compared with S. lydicus/pEM4T used as a control (Fig. 3A). Total production yields of streptolydigin by S. lydicus decreased when grown on MML or MMS (Fig. 3B and C). Inactivation of slgC1 or slgC2 also conducted to a clear decrease in streptolydigin yields when mutant strains SLMC1 and SLMC2 were grown in MML (2.8‐ and 5‐fold respectively) or in MMS (3.3‐ and 13‐fold respectively) (Fig. 3B and C). In addition, expression of slgC1 or slgC2 in S. lydicus also conducted to increase production yields in both MML (1.3‐ and 7‐fold respectively) and MMS (3‐ and 10‐fold respectively) (Fig. 3B and C). In both cases, yields of streptolydigin were higher when slgC2 was expressed in S. lydicus than when slgC1 was, being the streptolydigin titres higher than in the wild‐type strain when grown in MML, or similar to the wild‐type strain when grown in MMS. Curiously, the presence of the empty vector in S. lydicus used as control dramatically reduced the amount of streptolydigin produced in all media tested. This effect has been observed previously in S. lydicus (Gómez et al., 2011; Horna et al., 2011). In the present and previous works all strains were grown in the same conditions including the antibiotic for selection. However, the lower production observed in the control culture cannot be solely attributed to the presence of the antibiotic for selection (data not shown).


Participation of putative glycoside hydrolases SlgC1 and SlgC2 in the biosynthesis of streptolydigin in Streptomyces lydicus.

Gómez C, Horna DH, Olano C, Méndez C, Salas JA - Microb Biotechnol (2012)

A. Production of streptolydigin by S. lydicus (WT), SLMC1 (C1‐), SLMC2 (C2‐), and S. lydicus carrying pEM4T (Ct), pEM4TslgC1 (C1) or pEM4slgC2 (C2) grown in MMG (MM containing glycerol at 10 g l−1).B. Production of streptolydigin in MML (MM containing laminarin at 10 g l−1).C. Production of streptolydigin in MMS (MM containing starch at 10 g l−1). In all cases streptolydigin production was determined by UPLC analysis. Experiments were run in triplicate. Minimal medium MM is composed of MOPS 21 g l−1, MgSO4 0.6 g l−1, CaCl2 5 mg l−1, MnCl2 1 mg l−1, ZnSO4 1 mg l−1, FeSO4 5 mg l−1, K2HPO4 1.75 g l−1, NH4Cl 1.6 g l−1 and fumarate 2%, pH 6.8, in deionized water. Laminarin and starch were purchased form Sigma.
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Related In: Results  -  Collection

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f3: A. Production of streptolydigin by S. lydicus (WT), SLMC1 (C1‐), SLMC2 (C2‐), and S. lydicus carrying pEM4T (Ct), pEM4TslgC1 (C1) or pEM4slgC2 (C2) grown in MMG (MM containing glycerol at 10 g l−1).B. Production of streptolydigin in MML (MM containing laminarin at 10 g l−1).C. Production of streptolydigin in MMS (MM containing starch at 10 g l−1). In all cases streptolydigin production was determined by UPLC analysis. Experiments were run in triplicate. Minimal medium MM is composed of MOPS 21 g l−1, MgSO4 0.6 g l−1, CaCl2 5 mg l−1, MnCl2 1 mg l−1, ZnSO4 1 mg l−1, FeSO4 5 mg l−1, K2HPO4 1.75 g l−1, NH4Cl 1.6 g l−1 and fumarate 2%, pH 6.8, in deionized water. Laminarin and starch were purchased form Sigma.
Mentions: Streptomycetes are usually soil living microorganisms. Therefore, we wonder if the presence of slgC1 and slgC2 genes in the streptolydigin cluster could be an evolutionary advantage for the producer microorganism by facilitating sugar supply for streptolydigin biosynthesis in its natural soil environment. To evaluate the potential participation of SlgC1 and SlgC2 enzymes in the degradation of β‐ and α‐glucans, cultures of S. lydicus, mutants SLMC1 and SLMC2 and S. lydicus carrying pEM4T, pEM4TslgC1 or pEM4TSlgC2 were performed on three minimal media using different carbon sources: glycerol (MMG), β‐glucan laminarin (MML) and α‐glucan starch (MMS). Differences in the production of streptolydigin were observed in MMG between S. lydicus wild type and mutants SLMC1 and SLMC2. Streptolydigin yields in these mutants decreased 1.6‐ and 12‐fold respectively. In contrast, a positive effect of the expression of slgC1 or slgC2 in S. lydicus was observed. In this medium, production of streptolydigin increased 5.5‐ and 5‐fold respectively, when compared with S. lydicus/pEM4T used as a control (Fig. 3A). Total production yields of streptolydigin by S. lydicus decreased when grown on MML or MMS (Fig. 3B and C). Inactivation of slgC1 or slgC2 also conducted to a clear decrease in streptolydigin yields when mutant strains SLMC1 and SLMC2 were grown in MML (2.8‐ and 5‐fold respectively) or in MMS (3.3‐ and 13‐fold respectively) (Fig. 3B and C). In addition, expression of slgC1 or slgC2 in S. lydicus also conducted to increase production yields in both MML (1.3‐ and 7‐fold respectively) and MMS (3‐ and 10‐fold respectively) (Fig. 3B and C). In both cases, yields of streptolydigin were higher when slgC2 was expressed in S. lydicus than when slgC1 was, being the streptolydigin titres higher than in the wild‐type strain when grown in MML, or similar to the wild‐type strain when grown in MMS. Curiously, the presence of the empty vector in S. lydicus used as control dramatically reduced the amount of streptolydigin produced in all media tested. This effect has been observed previously in S. lydicus (Gómez et al., 2011; Horna et al., 2011). In the present and previous works all strains were grown in the same conditions including the antibiotic for selection. However, the lower production observed in the control culture cannot be solely attributed to the presence of the antibiotic for selection (data not shown).

Bottom Line: Both genes are expressed when streptolydigin is produced.Inactivation of these genes affects streptolydigin production when the microorganism is grown in minimal medium containing either glycerol or d-glucans as carbon source.Streptolydigin yields in S. lydicus were increased by overexpression of either slgC1 or slgC2.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biología Funcional, Universidad de Oviedo, 33006, Oviedo, Spain.

Show MeSH