Carboxyethylarginine Synthase Genes Show Complex Cross-Regulation in Streptomyces clavuligerus

Abstract

Carboxyethylarginine synthase is the first dedicated enzyme of clavam biosynthesis in Streptomyces clavuligerus and is present in two isoforms encoded by two separate genes. When grown on a liquid soy medium, strains with ceaS1 deleted showed only a mild reduction of clavam biosynthesis, while disruption of ceaS2 abolished all clavam biosynthesis. Creation of an in-frame ceaS2 deletion mutant to avoid polarity did not restore clavam production, nor did creation of a site-directed mutant altered only in a single amino acid residue important for activity. Reverse transcriptase PCR analyses of these mutants indicated that the failure to produce clavam metabolites could be traced to reduced or abolished transcription of ceaS1 in the ceaS2 mutants, despite the location of ceaS1 on a replicon completely separate from that of ceaS2. Western analyses further showed that the CeaS1 protein (as well as the CeaS2 protein) was absent from the ceaS2 mutants. Complementation experiments were able to restore clavam production partially, but only by virtue of restoring CeaS2 production. CeaS1 was still absent from the complemented strains. While this dependence of CeaS1 production on the expression of ceaS2 from its native chromosomal location was seen in all of the ceaS2 mutants, the effect was limited to growth in liquid medium. When the same mutants were grown on solid soy medium, clavam production was restored and CeaS1 was produced, albeit at low levels compared to the wild type.

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

“…Therefore, our results provide additional support for the hypothesis that the clavam and paralog gene clusters are associated with 5 S clavam biosynthesis, and that some gene products from the two clusters augment CA production in S. clavuligerus by contributing to a common pool of precursors (Figure 1; Jensen, 2012; Hamed et al, 2013). Although, it should be noted that in S. clavuligerus , there is some cross regulation between the chromosomal CA and plasmid-borne paralog gene clusters (Kwong et al, 2013; Álvarez-Álvarez et al, 2017), which is again not expected to occur in the other two CA producers since they only contain the CA BGC. This also highlights the complexity of the regulatory pathways controlling CA and 5 S clavam production in S. clavuligerus (Liras et al, 2008).…”

The CagRS Two-Component System Regulates Clavulanic Acid Metabolism via Multiple Pathways in Streptomyces clavuligerus F613-1

“…The CcaR-encoding gene, ccaR , is located in the cephamycin C gene cluster, and CcaR is reported to positively regulate the expression of genes involved in the early stage of CA biosynthesis, such as ceaS2, bls2, pah2 , and cas2 (Santamarta et al, 2002, 2011; Alvarez-Alvarez et al, 2014). In addition, CcaR also binds the promoter of claR (Santamarta et al, 2011; Kurt et al, 2013), indicating that ClaR and CcaR may jointly form a regulatory system to regulate the biosynthesis of CA (Kurt et al, 2013; Kwong et al, 2013).…”