Fig 1.
ACVS domain organization and product formation.
The ACVS consists of a total of 10 domains arranged in three modules with distinct specificities for the incorporation of L-α-aminoadipic acid (L-α-Aaa), L-cysteine and L-valine into the tripeptide δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine ((L,L,D)-ACV). The domain arrangement is conserved [30] and follows the order: AT-CAT-CATETe. The resulting (L,L,D)-ACV is converted into isopenicillin-N (IPN) by the isopenicillin-N synthetase (IPNS). Following different biosynthetic routes, IPN can further be converted into penicillins, cephalosporins, cephamycins and related compounds. In the circle, a schematic representation of the strategy [33] adopted to engineer the specificity of the first module of Nl ACVS is shown.
Fig 2.
Ni2+ affinity chromatography purification of the Nocardia lactamdurans ACVS.
Nl ACVS was isolated from E. coli HM0079 cells and harvested after overnight expression at 18 ºC. A cell-free lysate was obtained through sonication and subsequently separated into a clear supernatant (CFL) and the pellet was resuspended in 8 M Urea (CFL (i)). The clear lysate was further purified using gravity flow in combination with a His-tag affinity chromatography, using two washing steps (W1, W2) and elution with 50, 150 and 250 mM imidazole, respectively (E1, E2, E3). Marker lane shows reference proteins corresponding to 170 and 130 kDa.
Fig 3.
Enzymatic characterization of ACVS.
Three reaction series were conducted using various concentrations of L-α-Aaa (A), L-Cys (B) and L-Val (C) and analyzed by LC/MS to quantify the amounts of the ACV tripeptide produced for Michaelis-Menten kinetics (D).
Fig 4.
Substrate promiscuity of the N. lactamdurans ACVS: Structures of expected tripeptides.
The predicted structures of the novel tripeptides and their corresponding reactions are numbered 1–26 (see Table 1). Italic numbers indicate production of the respective tripeptide. * = ACC tripeptide derived from L-α-Aaa and L-Cys only (26).
Table 1.
ACVS substrate promiscuity.
Fig 5.
Subdomains multi-alignment and boundaries determination.
(A) Multi-alignment of the subdomains used in the engineering strategy proposed by Kries and coworkers [33]. The two residues highlighted in yellow in the consensus sequence represent the first highly conserved motif with a phenylalanine and the aspartic acid which forms a hydrogen bond with the α-amino group of the amino acid substrate [35]. The motifs highlighted with the red boxes represent the conserved motifs identified as boundaries of the subdomains in the consensus sequence. (B) Multi-alignment of the subdomains selected for this work; in absence of structural information the conserved motifs at both ends were used to determine the subdomain boundaries.
Table 2.
Set of subdomains selected for the engineering strategy.
Fig 6.
Overexpression and activity assays of the hybrid NRPSs.
(A) SDS-PAGE analysis of the purified fractions of the hybrids: only fraction E2 (150 mM imidazole) is shown. Marker lane shows reference protein corresponding to 180 kDa. (B) In vitro tripeptide production assays with hybrid enzymes. (C) The production of the tripeptide ACV was confirmed by accurate monoisotopic mass and equal retention time compared to the wild-type enzyme product and an ACV synthetic standard.