Functional Analysis of a Gene Cluster from Chitinophaga pinensis Involved in Biosynthesis of the Pyrrolidine Azasugar DAB-1

Abstract

Azasugars, "nitrogen in the ring" analogues of monosaccharides, are known to be distributed in select plant, fungal. and bacterial species. We identify Chitinophaga pinensis DSM 2588 as the first bacterial source of the plant pyrrolidine azasugar 1,4-dideoxy-1,4-aminoarabinitol (DAB-1). Comparative sequence analyses identified C. pinensis as a putative azasugar producer, via observation of a three-gene cluster coding for putative aminotransferase, alcohol dehydrogenase, and sugar phosphatase enzymes, similar to the previously reported azasugar biosynthetic signature identified in Bacillus amyloliquefaciens FZB42. Multistep fractionation of C. pinensis culture media guided by a maltase inhibition assay yielded a component with a mass consistent with the structure of DAB-1. Heterologous expression of the three-gene cluster in E. coli, a non-azasugar producer, led to the isolation of nectrisine, a biosynthetic precursor to DAB-1, which displayed potent slow tight binding inhibition of maltase. Reduction of nectrisine with NaBH 4 removed the slow tight binding inhibition kinetics, and MS analysis provided evidence for the production of a compound matching that of the isolated DAB-1 from C. pinensis. 1 H NMR analysis of the nectrisine produced in E. coli after NaBD 4 reduction produced a spectrum consistent with DAB-1 deuterated at C-1, primarily at the pro-S position. These results support the idea that the azasugar three-gene cluster represents a general biosynthetic path leading to several different compounds, which may prove useful for the identification of other azasugarproducing organisms.

Genome Analysis of Streptomyces nojiriensis JCM 3382 and Distribution of Gene Clusters for Three Antibiotics and an Azasugar across the Genus Streptomyces

“…polymyxa may not be conserved in the genus Streptomyces . In Chitinophaga pinensis DSM 2588, orthologs of GabT1 and GutB1 are involved in the biosynthesis of another azasugar, 1,4-dideoxy-1,4-aminoarabinitol (DAB-1) [ 53 ].…”

Comparative genomic analysis of azasugar biosynthesis

“…For comparative purposes, the nodes representing either aminotransferase or dehydrogenase enzymes within a known azasugar producer were highlighted. The following symbol code was used: triangle, C. pinensis DSM7; parallelogram, P. polymyxa DSM365; square, B. velezensis FZB42; circle, B. amyloliquefaciens 140N; diamond, B. atrophaeus 1942; octagon, S. subrutilis ATCC 27467 (Schmidt et al 1979 ; Hardick et al 1992 ; Clark et al 2011 ; Gibbons et al 2011 ; Seo et al 2013 ; Nuñez and Horenstein 2019 ).

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“…Foundational work concerning the biosynthesis of azasugars in bacteria included feeding experiments using site specific isotope labeling in precursor sugars for Bacilli (Hardick et al 1992 ) and identification of genes coding for DNJ biosynthetic enzymes in Bacillus amyloliquefaciens FZB42 (Clark et al 2011 ), reclassified as Bacillus velezensis (Fan et al 2017 ), B. subtilis MORI 3K-85 (Kang et al 2011 ), and biosynthetic genes involved in production of 1,4-dideoxy-1,4-aminoarabinitol (DAB-1) in Chitinophaga pinensis (Nuñez and Horenstein 2019 ). The key biosynthetic machinery (Fig.…”

“…Spontaneous cyclization with the amino group attacking the newly formed carbonyl at O6, produces the azasugar. The three genes for the aminotransferase, phosphatase and dehydrogenase enzymes used in the biosynthesis are found clustered together in all known azasugar producers examined thus far (Clark et al 2011 ; Nuñez and Horenstein 2019 ) and we refer to them as the 3GC. In the case of DNJ biosynthesis, two more steps are needed to complete the work of the 3GC, namely the manno -configuration needs to be epimerized to the gluco -configuration at C2, and then the 1-OH group is reduced to the 1-deoxy form.…”

“…We originally identified 3GCs by using BLAST to search for aminotransferases and manually searching through their immediate genomic neighborhoods for phosphatase and dehydrogenase coding ORFs, a strategy used to successfully identify the 3GC in B. amyloliquefaciens FZB42 as well as the DAB-1 3GC in C. pinensis DSM 2588 (Clark et al 2011 ; Nuñez and Horenstein 2019 ). This method was also recently used to identify and compare potential DNJ producers in the Bacilli genus (Lee et al 2021 ).…”