Molecular characterization of phosphonate biosynthesis in nature

Phosphonates, compounds characterized by direct C-P bonds, comprise a structurally diverse class of natural products demonstrating an impressive range of biological activities. Although the first biologically produced phosphonate was described more than 50 years ago, the range and diversity of phosphonate production in nature is still not well understood. The biosynthetic pathways of almost all known phosphonates share the same initial step, in which phosphoenolpyruvate (PEP) is isomerized to phosphonopyruvate (PnPy) by PEP mutase (PepM). By using the pepM gene as a molecular marker for phosphonate biosynthetic capacity, I showed that phosphonate biosynthesis is both common and diverse across a wide range of environments, with pepM homologs detected in ~5% of sequenced microbial genomes and 7% of genome equivalents in metagenomic datasets. In addition, PEP mutase sequence conservation was found to be strongly correlated with conservation of other nearby genes, suggesting the diversity of phosphonate biosynthetic pathways could be inferred by examining PEP mutase diversity. By extrapolation, hundreds of unique phosphonate biosynthetic pathways were predicted to exist in nature. As part of a large screening program to uncover new phosphonate-containing natural products, two related phosphonate producers were identified by screening for the pepM gene: Glycomyces sp. NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338. These two actinomycetes produced high amounts of novel phosphonate-containing compounds, which were determined to be 2-hydroxyethylphosphonate (2-HEP) containing polysaccharides (also called phosphonoglycans). The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation and ultrafiltration. Sugar component analyses indicated the presence of various O-methylated galactoses in both phosphonoglycans; the O-methyl groups were shown to derive from S-adenosylmethionine. Partial acid hydrolysis of the purified phosphonoglycans from Glycomyces yielded 2-HEP in ester linkage to the O-5 or O-6 position of a hexose (presumably galactose) and a 2-HEP mono(2,3-dihydroxypropyl) ester. Partial acid hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl) ester. Examination of the genome sequences of the two strains revealed similar pepM-containing gene clusters that are likely to be required for phosphonoglycan synthesis. Like other classes of natural products, most of the phosphonate biosynthetic pathways remain silent under standard laboratory culture conditions. To elicit cryptic phosphonate gene clusters from actinomycetes, two strategies were employed: co-culturing with other microorganisms and selecting for antibiotic-resistant mutants. Although none of the methods successfully turned on phosphonate production, potential avenues for further exploration in terms of inducing the production of unknown phosphonates and increasing the yields of known phosphonates remain possible. To explore the possibility of using the industrial workhorse Corynebacterium glutamicum as a heterologous host to produce phosphonates, a synthetic biology approach was described. A synthetic pathway for the synthesis of 2-HEP was assembled and reconstituted in C. glutamicum, which afforded the production of 2-HEP. However, further modifications are required to improve the stability of the construct and optimize the product yield.