Streptomyces tsukubensis VKM Aс-2618D-an Effective Producer of Tacrolimus

Abstract

The Streptomyces sp. VKM Ac-2618D strain has been identified, and its morphological and physiological features have been studied in relation to the production of the immunosuppressant tacrolimus. The phenotypic variability of the strain was analyzed, and a dissociant with a high level of tacrolimus production was selected. Based on a comprehensive study of morphological, physiological, and chemotaxonomic properties and on phylogenetic analysis, the strain was named Streptomyces tsukubensis VKM Ac-2618D. The strain genome contains the full version of the tacrolimus biosynthetic gene cluster. The advantages of fed-batch cultivation mode for tacrolimus biosynthesis are shown. The results broaden the understanding of the characteristics of polyketide biosynthesis and can be used in the development of technology for tacrolimus production.

Keywords: FK-506; Streptomyces tsukubensis; biosynthesis; dissociant; genome; tacrolimus.

Fig. 1.

(а) Morphology (photographs) of the Streptomyces sp. VKM Ас-2618D strain colonies after 14 days of incubation at 30°C on ISP media: (а) ISP-2, (b) ISP-3, (c) ISP-4, and (d) ISP-5; (b) morphology (scanning electron micrographs) of spore chains after Streptomyces sp. VKM Ас-2618D growth on ISP 2 for 14 days at 30°C (scale bar is 5 μm).

Fig. 2.

Phylogenetic tree based on the sequencing of a 16S rRNA gene fragment of Streptomyces species with grouping via the neighbor-joining method. Bootstrap values based on 1000 replicates are shown at branch nodes. A bar corresponds to 0.005 substitutions per nucleotide position. The 16S rRNA gene nucleotide sequence of Micromonospora rifamycinica AM 105^T (AY561829) was used as an outgroup.

Fig. 3.

Gene cluster of tacrolimus biosynthesis in the S. tsukubensis VKM Ас-2618D genome. The gene numbers in the NCBI GenBank database are from EWI31_25195 to EWI31_25390. Genes that are absent in strains with the short version of the tacrolimus biosynthesis cluster are depicted in gray.

Fig. 4.

(a) Dissociants (D) of the S. tsukubensis VKM Ас-2618D strain on MDA medium. (b) Tacrolimus biosynthesis by the S. tsukubensis VKM Ас-2618D dissociants (the highest FK-506 yield in dissociant 2 (D2) is taken as 100%).

Fig. 5.

(a) Dynamics of biomass growth and tacrolimus biosynthesis by S. tsukubensis VKM Ac-2618D in the batch (1) and fed-batch (2) cultivation modes. (b) Dynamics of the content of bound glucose (1a and 1b) and the target product (2a and 2b) in the medium in batch (1a and 2a) and fed-batch (1b and 2b) cultivation modes during tacrolimus biosynthesis by S. tsukubensis VKM Ac-2618D.

Fig. 6.

Morphological changes in S. tsukubensis VKM Ac-2618D during tacrolimus biosynthesis: (a–c) batch cultivation mode, (d–f) fed-batch cultivation mode; (a) and (d) 1 day; (b) and (e) 6 days; (c) and (f) 10 days (scanning electron microscopy, scale bar is 5 μm). Arrows indicate destructive changes, such as hyphal profile curvature and loss of hyphal intracellular content.