Identification of Caerulomycin A Gene Cluster Implicates a Tailoring Amidohydrolase

Abstract

The biosynthetic gene cluster for caerulomycin A (1) was cloned and characterized from the marine actinomycete Actinoalloteichus cyanogriseus WH1-2216-6, which revealed an unusual hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) system. The crmL disruption mutant accumulated caerulomycin L (2) with an extended L-leucine at C-7, implicating an amidohydrolase activity for CrmL. The leucine-removing activity was confirmed for crude CrmL enzymes. Heterologous expression of the 1 gene cluster led to 1 production in Streptomyces coelicolor.

Biochemical and Structural Insights into the Aminotransferase CrmG in Caerulomycin Biosynthesis

“…Bioinformatics analysis suggests that the crmG gene encodes a diaminobutyratepyruvate aminotransferase which is a candidate for incorporation of the amino group at C-7 in 1. 15 To verify this hypothesis, the crmG gene was inactivated by PCR-targeted insertional mutation using a previously established method ( Figure S1). 15,51 The resulting ΔcrmG mutant accumulated two products that are distinct from 1 ( Figure 2).…”

“…15 To verify this hypothesis, the crmG gene was inactivated by PCR-targeted insertional mutation using a previously established method ( Figure S1). 15,51 The resulting ΔcrmG mutant accumulated two products that are distinct from 1 ( Figure 2). One product was characterized to be CRM F 8 by comparing HRESIMS and NMR data ( Figure 2, Table S1 and Figure S2) with those of standard 8.…”

“…12−14 Recently, the biosynthetic pathways of 1 and its closely related analogue collismycin A have been demonstrated to involve a common origin of a hybrid polyketide synthase (PKS)/ nonribosomal peptide synthetase (NRPS) system. 15−19 Interestingly, the PKS/NRPS system in 1 was able to synthesize a bipyridyl product CRM L 2 ( Figure 1) with an L-leucine attached at C-7, 15 and a similar product was also identified in the collismycin A pathway. 16 However, the exact mechanism to form the bipyridine core in 1 and collismycin A remains unclear.…”

“…It has been speculated that the GirC/GriD-like dehydrogenase components, 22 CrmN/CrmO, function to reduce a carboxyl group to an aldehyde group, whereas the conversion of an aldehyde to an amino group may involve the aminotransferase CrmG. 15 Aminotransferases are PLP-dependent enzymes that transfer an amino group into a keto group and are ubiquitous in nature to play important roles in nitrogen metabolism in cells. 23 A number of aminotransferases involved in primary metabolism for the biosynthesis of amino acids have been biochemically and structurally well studied to provide detailed mechanistic insights.…”

Engineering the Biosynthesis of the Polyketide-Nonribosomal Peptide Collismycin A for Generation of Analogs with Neuroprotective Activity

“…This was confirmed through the isolation and characterization of the collismycin A gene cluster from Streptomyces sp. CS40 (Garcia et al, 2012) and, more recently, with the caerulomycin gene cluster from Actinoalloteichus cyanogriseus (Qu et al, 2012;Zhu et al, 2012). With the availability of the sequence of the collismycin A gene cluster, we were interested in understanding how collismycin A biosynthesis takes place and in generating collismycin analogs with potentially improved therapeutic properties.…”