Overcoming DAPA aminotransferase bottlenecks in biotin vitamers biosynthesis

Abstract

A method is disclosed for the increased production of biotin and the biotin precursor dethiobiotin using a bacterium that produces a lysine-utilizing DAPA aminotransferase. The method involves the use of a bacterium that is either grown in the presence of lysine or deregulated for lysine biosynthesis.

Description

BACKGROUND OF THE INVENTION [0001] The present invention is in the general field of the biosynthesis of biotin vitamers. [0002] Biotin biosynthesis in Escherichia coli and Bacillus sphaericus has been studied at both the biochemical and molecular biological levels (DeMoll, 1996. In F. C. Neidhardt et al., (eds.) E. coli and Salmonella typhimurium: Cellular and Molecular Biology, Second edition ed., vol 1., pp. 704-709, ASM Press, Washington, D.C.; Perkins et al., In A. L. Sonenshein et al. (eds.), In Bacillus subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, pp. 319-334, American Society for Microbiology, Washington, D.C.; Eisenberg, 1987. In F. Neidhardt et. al. (eds.), E. coli and Salmonella typhimurium, pp. 544-550. American Society for Microbiology, Washington, D.C.; Cronan, Cell 58:427-429, 1989, Izumi et al., Agric. Biol. Chem. 45:1983-1989, 1981; Gloeckler et al., Gene 87:63-70, 1990), although some steps and components in biotin syn...

AI Technical Summary

Benefits of technology

[0006] We have found that the conversion of KAPA to DAPA is a serious bottleneck in the biosynthesis of biotin using engineered cells that are fed pimelic acid. As other controls on biotin biosynthesis are removed, the KAPA to DAPA conversion is unable to keep pace with KAPA production, resulting in a build-up of KAPA, without a concomitant increase in the final product. We have also discovered that an important component of the bottleneck is the availability and identity of the amino donor used in the KAPA to DAPA conversion. In general, providing adequate quantities of the amino donor is an important strategy for overcoming the bottleneck. Moreover, a DAPA aminotransferase able to use lysine and related compounds as a source of the amino group to be transfered in the reaction which produces DAPA from KAPA, can significantly improve biosynthetic yields of the downstream biotin vitamers, especially dethiobiotin (DTB).

[0007] Although we do not wish to be limited to one specific explanation for our finding to the exclusion of other factors, it appears that providing higher levels of an amino donor which can be used by the available aminotransferase substantially ameliorates the bottleneck discussed above. For example, bacterial production of the biotin vitamers by bacteria whose DAPA aminotransferase uses lysine as an amino donor can be dramatically improved by making sufficient lysine available, either by including it in the fermentation medium or by deregulating the lysine biosynthetic pathway. Such a strategy can also be applied to the use of DAPA aminotransferases of B. subtilis and close relatives, including members of the cluster of Bacillus spp. represented by B. subtilis. The cluster includes, e.g., B. subtilis, B. pumilus, B. licheniformis, B. amyloliquefaciens, B. megaterium, B. cereus and B. thuringiensis. The members of the B. subtilis cluster are genetically and metabolically divergent from the more distantly related Bacillus spp. of clusters represented by B. sphaericus and B. stearothermophilus (Priest, In Bacillus subtilis and Other Gram-Positive Bacteria, supra pp. 3-16, hereby incorporated by reference; Stackebrant, et al. J. Gen. Micro. 133:2523-2529, 1987, hereby incorporated by reference).

[0018] Bacteria can be engineered by intentionally and specifically altering the wild-type genome to produce a desired biosynthetic phenotype—e.g., to synthesize more lysine than the corresponding wild-type, unengineered organism, or to remove a bottleneck in the biotin biosynthetic pathway.

Problems solved by technology

We have found that the conversion of KAPA to DAPA is a serious bottleneck in the biosynthesis of biotin using engineered cells that are fed pimelic acid.

As other controls on biotin biosynthesis are removed, the KAPA to DAPA conversion is unable to keep pace with KAPA production, resulting in a build-up of KAPA, without a concomitant increase in the final product.

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