Comparative Genomics and Metabolomics Analyses of Clavulanic Acid-Producing Streptomyces Species Provides Insight Into Specialized Metabolism

Abstract

Clavulanic acid is a bacterial specialized metabolite, which inhibits certain serine β-lactamases, enzymes that inactivate β-lactam antibiotics to confer resistance. Due to this activity, clavulanic acid is widely used in combination with penicillin and cephalosporin (β-lactam) antibiotics to treat infections caused by β-lactamase-producing bacteria. Clavulanic acid is industrially produced by fermenting Streptomyces clavuligerus, as large-scale chemical synthesis is not commercially feasible. Other than S. clavuligerus, Streptomyces jumonjinensis and Streptomyces katsurahamanus also produce clavulanic acid along with cephamycin C, but information regarding their genome sequences is not available. In addition, the Streptomyces contain many biosynthetic gene clusters thought to be “cryptic,” as the specialized metabolites produced by them are not known. Therefore, we sequenced the genomes of S. jumonjinensis and S. katsurahamanus, and examined their metabolomes using untargeted mass spectrometry along with S. clavuligerus for comparison. We analyzed the biosynthetic gene cluster content of the three species to correlate their biosynthetic capacities, by matching them with the specialized metabolites detected in the current study. It was recently reported that S. clavuligerus can produce the plant-associated metabolite naringenin, and we describe more examples of such specialized metabolites in extracts from the three Streptomyces species. Detailed comparisons of the biosynthetic gene clusters involved in clavulanic acid (and cephamycin C) production were also performed, and based on our analyses, we propose the core set of genes responsible for producing this medicinally important metabolite.

Production of Plant-Associated Volatiles by Select Model and Industrially Important Streptomyces spp.

“…However, we did not observe a similar trend in all species analyzed as in some cases the number of predicted VOC BGCs did not correlate with those actually detected. For instance, S. clavuligerus , which is known to produce a wide range of specialized metabolites, including β-lactam antibiotics and molecules of plant origin [ 114 ], contains many predicted BGCs ( Supplemental Table S3 ) and produced five identifiable VOCs ( Figure 2 B, Supplemental Table S5 ). However, terpenes were not detected in S. clavuligerus headspace samples in our study ( Figure 2 B, Supplemental Table S5 ), even though the species possesses 12 predicted BGCs for such metabolites ( Figure 1 B, Supplemental Table S3 ).…”

“…However, terpenes were not detected in S. clavuligerus headspace samples in our study ( Figure 2 B, Supplemental Table S5 ), even though the species possesses 12 predicted BGCs for such metabolites ( Figure 1 B, Supplemental Table S3 ). Previous reports have shown that the plant-associated terpenoids carveol, cuminyl alcohol and hydroxyvalerenic acid were detected during liquid chromatography–MS/MS analysis of certain S. clavuligerus cultures extracted with methanol/ethyl acetate [ 114 ]. In a separate report, terpenes were not detected in n -hexane extracts of S. clavuligerus cultures grown on SFM or YMS media (also used in the current study) when subjected to GC-MS analysis [ 53 ].…”

“…In addition, inhibition of the feeding behavior of D. melanogaster by geosmin produced by the Streptomyces has already been discussed in detail [ 116 ]. Although the production of some plant-associated VOCs in the Streptomyces has been previously reported [ 114 ], we used three types of growth media and identified 27 such metabolites from cultures of just nine species ( Figure 3 and Figure 4 , Table 1 ). It should also be noted that our study detected such metabolites under laboratory conditions and that their production in the natural environment by some Streptomyces spp.…”

Specialized Metabolites from Ribosome Engineered Strains of Streptomyces clavuligerus

“…Streptomyces clavuligerus is the industrial producer of clavulanic acid, a β-lactamase inhibitor used in combination with β-lactam antibiotics to treat certain otherwise-resistant bacterial infections [ 3 ]. In addition, S. clavuligerus is also known to produce cephamycin C [ 4 ], 5 S clavams [ 5 ], naringenin [ 6 , 7 ], holomycin [ 8 ], and tunicamycins [ 8 ]. Different genome sequencing studies have reported the presence of 49–58 SM BGCs in S. clavuligerus [ 9 , 10 ], most of which are thought to be cryptic (or silent) as the identities of their SM products remain unknown [ 10 , 11 ].…”

“…Activation of production or improved yields of these cryptic SMs can lead to the identification of new biosynthetic capabilities. For example, S. clavuligerus was recently shown to produce certain plant-associated metabolites [ 6 , 7 ], previously not thought to be synthesized by bacteria [ 6 ]. In addition, genes for the biosynthesis of some of these metabolites do not reside in defined BGCs, providing additional challenges for their identification and heterologous expression.…”