Structure, Biosynthetic Origin, and Engineered Biosynthesis of Calcium-Dependent Antibiotics from Streptomyces coelicolor

Abstract

The calcium-dependent antibiotic (CDA), from Streptomyces coelicolor, is an acidic lipopeptide comprising an N-terminal 2,3-epoxyhexanoyl fatty acid side chain and several nonproteinogenic amino acid residues. S. coelicolor grown on solid media was shown to produce several previously uncharacterized peptides with C-terminal Z-dehydrotryptophan residues. The CDA biosynthetic gene cluster contains open reading frames encoding nonribosomal peptide synthetases, fatty acid synthases, and enzymes involved in precursor supply and tailoring of the nascent peptide. On the basis of protein sequence similarity and chemical reasoning, the biosynthesis of CDA is rationalized. Deletion of SCO3229 (hmaS), a putative 4-hydroxymandelic acid synthase-encoding gene, abolishes CDA production. The exogenous supply of 4-hydroxymandelate, 4-hydroxyphenylglyoxylate, or 4-hydroxyphenylglycine re-establishes CDA production by the DeltahmaS mutant. Feeding analogs of these precursors to the mutant resulted in the directed biosynthesis of novel lipopeptides with modified arylglycine residues.

An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibiotics

“…for 5 days. The cells were removed by centrifugation and the CDA6a in the resulting supernatants was extracted onto Varian C-18 Bond Elute SPE cartridges (2 g), as described previously (Hojati et al, 2002). Purification of the crude extracts was achieved by preparative and semi-preparative reversed-phase HPLC: Phenomenex C-18 10 mm, 250621.2 mm and 5 mm, 250610 mm columns; solvent A H 2 O with 0.1 % HCO 2 H; solvent B acetonitrile with 0.1 % HCO 2 H. The flow rates were 20 and 5 ml min 21 , respectively, with a starting gradient of 0 % B and 100 % A, increasing to 100 % B over 30 min and then held for a further 5 min.…”

“…The calcium-dependent antibiotics (CDAs) from Streptomyces coelicolor (Hojati et al, 2002;Kempter et al, 1997) belong to the group of structurally related acidic lipopetide antibiotics, which include A54145 (Fukuda et al, 1990; Miao et al, 2006), daptomycin (Baltz et al, 2005;Miao et al, 2005;Raja et al, 2003), friulimicins and amphomycins (Vértesy et al, 2000). All of these nonribosomally biosynthesized lipopeptides contain N-terminal fatty acid side chains, which is a trans-2,3-epoxyhexanoyl moiety in the case of CDA (Fig.…”

“…As a result of this, there has been considerable interest in establishing the biosynthetic origins of the acidic lipopeptide group of antibiotics, with the specific aim of engineering new lipopeptide variants with improved therapeutic properties. To this end we have focused on engineering the biosynthesis of CDAs, using adenylation domain active site modifications to replace Asp at position 7 in CDAs with Asn (Uguru et al, 2004) and a mutasynthesis approach to replace the D-hydroxyphenylglycine (D-HPG) at position 6 with alternative arylglycines (Hojati et al, 2002).…”

“…In addition to D-HPG, CDA contains a number of other non-proteinogenic amino acids, including L-3-methylglutamic acid (3-MeGlu; at position 10), which is also present at the same relative position in the decapeptide cores of daptomycin and A54145 (Milne et al, 2006), and a Cterminal Z-dehydrotryptophan residue (Hojati et al, 2002). CDA variants which contain either (2R,3S)-3-hydroxyasparagine (3-OHAsn) or (2R,3S)-3-phosphohydroxyasparagine (3-OPAsn) at position 9 are also known.…”

“…As a result of this, there has been considerable interest in establishing the biosynthetic origins of the acidic lipopeptide group of antibiotics, with the specific aim of engineering new lipopeptide variants with improved therapeutic properties. To this end we have focused on engineering the biosynthesis of CDAs, using adenylation domain active site modifications to replace Asp In addition to D-HPG, CDA contains a number of other non-proteinogenic amino acids, including L-3-methylglutamic acid (3-MeGlu; at position 10), which is also present at the same relative position in the decapeptide cores of daptomycin and A54145 (Milne et al, 2006), and a Cterminal Z-dehydrotryptophan residue (Hojati et al, 2002). CDA variants which contain either (2R,3S)-3-hydroxyasparagine (3-OHAsn) or (2R,3S)-3-phosphohydroxyasparagine (3-OPAsn) at position 9 are also known.…”