In Vivo Production of Artificial Nonribosomal Peptide Products in the Heterologous Host Escherichia coli

Abstract

Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. By these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.

Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains

“…In vivo production of DPheLPro-diketopiperazine (DKP) in the heterologous host E. coli was performed by the expression of module pairs from two compatible plasmids as described elsewhere (16). Accordingly, genes of the TycA deletion mutants were subcloned from pTrcHis TOPO expression plasmids in the medium-copy vector pSU18 by using the restriction enzyme HincII.…”

“…Preparation and detection of DKP produced by two-plasmid systems were carried out as described (16).…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[190] One thing to note is that they can act together with modular type I PKSs, forming hybrid NRPS-PKS. Several successes in the nonribosomal peptides category include the production of echinomycin, [191] D-Phe-Pro-diketopiperazine, [192] yersiniabactin, [193] ikarugamycin, [194] and antimycins [195] in E. coli.…”

Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus

“…Most of the understanding of the genetic control of tyrocidine production has been garnered from expression systems in B. subtilis (Marahiel et al, 1987;Mootz & Marahiel, 1997), as the tyrocidine producer strain Bacillus aneurinolyticus, previously known as Bacillus brevis (Dubos, 1939), has been found to be genetically less accessible (Marahiel et al, 1987). Whilst a truncated dipeptide version of the tyrocidines has been produced successfully in Escherichia coli through the recombinant expression of the first two modules of the tyrocidine biosynthesis operon (Gruenewald et al, 2004), the synthesis of a complete tyrocidine peptide in a recombinant organism is yet to be achieved.…”

“…Most of the understanding of the genetic control of tyrocidine production has been garnered from expression systems in B. subtilis (Marahiel et al, 1987;Mootz & Marahiel, 1997), as the tyrocidine producer strain Bacillus aneurinolyticus, previously known as Bacillus brevis (Dubos, 1939), has been found to be genetically less accessible (Marahiel et al, 1987). Whilst a truncated dipeptide version of the tyrocidines has been produced successfully in Escherichia coli through the recombinant expression of the first two modules of the tyrocidine biosynthesis operon (Gruenewald et al, 2004), the synthesis of a complete tyrocidine peptide in a recombinant organism is yet to be achieved.Employing recombinant technology to improve tyrothricin production may not be necessary, as high natural production of tyrothricin has been achieved by several investigators (Appleby et al, 1947a, b;Baron, 1949;Lewis et al, 1945;Stokes & Woodward, 1943), including our group. The tyrocidines, as well as their structural analogues the tryptocidines and phenycidines (Table 1), are produced primarily during the late exponential growth phase by B.…”