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Identification and disruptional analysis of the Streptomyces cinnamonensis msdA gene, encoding methylmalonic acid semialdehyde dehydrogenase

  • Original Paper - JMBM
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Journal of Industrial Microbiology and Biotechnology

Abstract

The msdA gene encodes methylmalonic acid semialdehyde dehydrogenase (MSDH) and is known to be involved in valine catabolism in Streptomyces coelicolor. Using degenerative primers, a homolog of

msdA gene was cloned and sequenced from the monensin producer, Streptomyces cinnamonensis. RT-PCR results showed msdA was expressed in a vegetative culture, bump-seed culture and the early stages of oil-based monensin fermentation. However, isotopic labeling of monensin A by [2, 4-13C2]butyrate revealed that this MSDH does not play a role in providing precursors such as methylmalonyl-CoA for the monensin biosynthesis under these fermentation conditions. Using a PCR-targeting method, msdA was disrupted by insertion of an apramycin resistance gene in S. cinnamonensis C730.1. Fermentation results revealed that the resulting ΔmsdA mutant (CXL1.1) produced comparable levels of monensin to that observed for C730.1. This result is consistent with the hypothesis that butyrate metabolism in S. cinnamonensis in the oil-based fermentation is not mediated by msdA, and that methylmalonyl-CoA is probably produced through direct oxidation of the pro-S methyl group of isobutyryl-CoA. The CXL1.1 mutant and C730.1 were both able to grow in minimal medium with valine or butyrate as the sole carbon source, contrasting previous observations for S. coelicolor which demonstrated msdA is required for growth on valine. In conclusion, loss of the S. cinnamonensis msdA neither affects valine catabolism in a minimal medium, nor butyrate metabolism in an oil-based medium, and its role remains an enigma.

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Abbreviations

MSDH:

Methylmalonic acid semialdehyde dehydrogenase

PKS:

Polyketide synthase

ICM:

Isobutyryl-CoA mutase

CCR:

Crotonyl CoA reductase

PCC:

Propionyl CoA carboxylase

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health (GM 50542) and Eli Lilly and Company. We are grateful to Richard DeMaio and Vic Vinci at Lilly for providing S. cinnamonensis C730.1 and C730.7 as well as conditions for oil-based extended fermentations.

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  1. Institute for Structural Biology and Drug Discovery, Suite 212B, Biotechnology Park Virginia Commonwealth University, 800 East Leigh Street, Richmond, VA, 23219, USA

    Chaoxuan Li, Konstantin Akopiants & Kevin A. Reynolds

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  1. Chaoxuan Li
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  2. Konstantin Akopiants
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  3. Kevin A. Reynolds
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Correspondence to Kevin A. Reynolds.

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Li, C., Akopiants, K. & Reynolds, K.A. Identification and disruptional analysis of the Streptomyces cinnamonensis msdA gene, encoding methylmalonic acid semialdehyde dehydrogenase. J IND MICROBIOL BIOTECHNOL 33, 75–83 (2006). https://doi.org/10.1007/s10295-005-0053-4

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