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Lohße, Anna; Kolinko, Isabel; Raschdorf, Oliver; Uebe, René; Borg, Sarah; Brachmann, Andreas; Plitzko, Jürgen M.; Müller, Rolf; Zhang, Youming; Schüler, Dirk (2016): Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium. In: Applied and Environmental Microbiology, Vol. 82, No. 10: pp. 3032-3041
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Abstract

Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacterium Magnetospirillum gryphiswaldense by chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of the mms6 operon resulted in the formation of increasingly larger crystals (increase of similar to 35%), the duplication of all major magnetosome operons (mamGFDC, mamAB, mms6, and mamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of the mam and mms clusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches. IMPORTANCE Before our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.