Biosynthetic studies of lincomycin A, a thiosugar-containing natural product

The dissertation describes biosynthetic studies of lincomycin A, a thiosugarcontaining natural product. Lincomycin A was isolated from Streptomyces lincolnensis and has been used clinically as an effective antibiotic for more than 30 years. The structure of lincomycin A contains an N-methyl-4-propyl-L-proline moiety and a thiooctose. Although the clinical importance of lincomycin A has led to extensive efforts to optimize the fermentation conditions for its production, how it is biosynthesized, especially the construction of the thiooctose, remains poorly understood. The goal of my thesis research aims to identify the biosynthetic precursors of lincomycin and investigate the mechanism of the transformations involved in the biosynthetic pathway. Based on the results of early feeding experiments and bio-informatic analysis of the biosynthetic gene cluster, a biosynthetic pathway of lincomycin could be proposed. The enzymes predicted to be involved in the biosynthetic pathway were purified in order to examine their functions. Organic synthesis was utilized to construct the proposed sugar substrates and the anticipated products to support the activity and the structural confirmation of the enzymatic products. With this approach, ribose and fructose were found to be the precursors that are assembled in a transaldol condensation reaction catalyzed by LmbR to yield the eight-carbon sugar backbone of lincomycin. The stereochemistry of the enzymatic products was confirmed by the comparison with various synthetic stereoisomers. The later parts of this research focus on the subsequent modifications of the octose precursor. Our studies led to the identification of a key intermediate, guanosine diphosphate-activated octose, which was formed through a kinasephosphatase cascade reaction, catalyzed by LmbP (kinase), LmbK (phosphatase) and LmbO (guanylyltransferase). Further transformations of this GDP-sugar intermediate including epimerization, dehydration and transamination were also demonstrated via the catalysis of enzymes LmbM, LmbL/Z and LmbS, respectively. How the C-1 sulfur is incorporated into the highly decorated GDP-octose is the most intriguing question in the biosynthesis of lincomycin A. Sequence analysis of the lincomycin gene cluster showed the presence of a putative S-glycosyltransferase, which likely catalyzes the displacement of GDP with an appropriate sulfur donor. This proposal has been verified recently by others. In addition, the amide bond formation between the proline moiety and thiooctose was shown to be catalyzed by a protein complex consisted of LmbC, LmbN and LmbD. This process resembles the biosynthesis of nonribosomal peptides. In conclusion, this work unravels the biosynthetic origins of lincomycin A and also characterizes the full sequences of the enzymatic transformations of the octose intermediate. The significance of such biosynthetic studies of glycosylated natural products like lincosamide derivatives is due to the fact that these natural products are bioactive and pharmaceutically useful. Knowing their biosynthetic pathways gives us the opportunity to manipulate the biosynthetic machineries. Modifying the structure of the sugar components holds promise for varying their biological activities. Moreover, the information about the biosynthetic precursors and the intermediates is useful for the optimization of the fermentation condition