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.

FIG. 1.

Tyrocidine biosynthetic system. The enzymatic assembly line of the cyclic decapeptide antibiotic tyrocidine consists of three peptide synthetases, TycABC, which are encoded by the polycistronic genes tycABC. The proteins are composed of one, three, and six modules, respectively, each one responsible for the integration of one amino acid into the nascent peptide chain (A). In this study, the first two modules (TycA and TycB1) were used as an artificial bimodular NRPS system for the production of d-Phe-Pro-DKP (B). Different catalytic domains are highlighted: condensation domain (light gray), adenylation domains (medium gray), peptidyl carrier protein domain (dark gray; wavy line, cofactor Ppant), epimerization domain (black), and thioesterase domain (striped).

FIG. 2.

Bimodular hybrid NRPS system TycA/TycB1. Shown are the different genetic setups investigated in this study (A) and their presumed consequences on the protein level (B). Catalytic domains are highlighted as in Fig. 1.

FIG. 3.

d-Phe-Pro-DKP formation in the heterologous host E. coli. Samples taken at certain time points (1 to 8 h after induction) and derived from cultures grown in LB rich medium (A) and M9 minimal medium (B) were analyzed by HPLC. Compound B coeluted with the chemical standard and corresponds to the expected d-Phe-Pro-DKP as verified by HPLC-MS (data not shown). The time-dependent production of d-Phe-Pro-DKP in LB (dots and solid line) and M9 (open circles and dashed line) media was normalized to OD₆₀₀ (C). Defined quantities of d-Phe-Pro-DKP (chemical standard) were analyzed by HPLC in order to establish a calibration curve (D). All samples were taken and analyzed in triplicate.

FIG. 4.

Determination of the optimal ratio of TycA to TycB1 for d-Phe-Pro-DKP formation. Both enzymes were incubated in the presence of l-phenylalanine, l-proline, ATP, and magnesium chloride, and the reactions were quenched after 1 h by immediate organic extraction. DKP was prepared as described in Materials and Methods and analyzed by HPLC. All samples were taken and analyzed in triplicate.

FIG. 5.

SDS-PAGE analysis for the quantification of TycA and TycB1 levels, as produced by the one-plasmid and two-plasmid systems. Amounts of total cell extracts applied to each lane were normalized to the OD₆₀₀. Lane 1, one-plasmid system without induction; lane 2, one-plasmid system with induction; lane 3, two-plasmid system without induction; lane 4, two-plasmid system with induction.

FIG. 6.

Effect of different genetic setups on in vivo d-Phe-Pro-DKP production. The one-plasmid system, fusion system, and two-plasmid system were individually studied at different growth temperatures, as well as with and without IPTG induction. All samples were taken and analyzed in triplicate.