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Increased Isoprene Production by the Recombinant Pantoea ananatis Strain due to the Balanced Amplification of Mevalonate Pathway Genes

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Abstract

The uncoordinated expression of a target biosynthetic pathway often results in low growth and productivity for a producing strain due to the accumulation of toxic metabolic intermediates. In this study, we report an increase in isoprene accumulation by an engineered strain of Pantoea ananatis that possesses foreign, artificial mevalonate-pathway genes. This was achieved due to the simultaneous, φ80-Int-dependent integration of a mixture of conditionally replicated plasmids carrying genes for the upper and lower mevalonate pathways and mevalonate kinase, followed by selection according to the isoprene accumulation of clones with optimal copy numbers of the integrative cassettes. A previously developed method, Dual In/Out, was modified to accelerate the construction of recipient strains containing several attB sites of the φ80 phage in their genomes.

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ACKNOWLEDGMENTS

The authors are extremely grateful to A.D. Kivero (AGRI) for valuable comments during the work with analytical equipment. The authors express their appreciation to Uehara (Nagai) Yuri for the fruitful cooperation and coordination of the joint work.

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Correspondence to E. D. Kazieva.

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Translated by I. Gordon

Abbreviations: BλattB site of λ phage; Bφ80attB site of φ80 phage; cat—chloramphenicol resistance gene; Cm—chloramphenicol; CmR—Cm-resistance; Int—phage integrase; IPTG—isopropyl-β-D-thiogalactopyranoside; kan—kanamycin resistance gene; Km—kanamycin; KmR—kanamycin resistance; MES—4-morpholineethanesulfonic acid; MOPS—4-morpholinepropanesulfonic acid; LB medium—lysogeny broth medium; LλattL site of λ phage; Lφ80attL site of φ80 phage; RλattR site of λ phage; Rφ80attR site of φ80 phage; Tc—tetracyclin; TcR—tetracyclin resistance; tetAR—tetracyclin resistance genes; Xis—excisionase.

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Katashkina, J.I., Kazieva, E.D., Tajima, Y. et al. Increased Isoprene Production by the Recombinant Pantoea ananatis Strain due to the Balanced Amplification of Mevalonate Pathway Genes. Appl Biochem Microbiol 55, 850–860 (2019). https://doi.org/10.1134/S0003683819090023

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