Enhanced production of the nonribosomal peptide antibiotic valinomycin in Escherichia coli through small-scale high cell density fed-batch cultivation

Abstract

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L⁻¹ by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L⁻¹. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD₆₀₀>50 and a valinomycin titer of appr. 10 mg L⁻¹. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.

Scale-up bioprocess development for production of the antibiotic valinomycin in Escherichia coli based on consistent fed-batch cultivations

“…For example, with more biocatalyst cell densities were always higher and oxygen contents were always lower in the first 12 h of cultivation. Finally, all cell densities reached high OD 600 values of ~20, although without addition of complex nutrient additives which were used in our previous studies [18]. With a biocatalyst concentration of 3 U L −1 , valinomycin reached the highest titer of nearly 800 μg L −1 and the corresponding yield was the second highest of 63 μg g −1 biomass (Figure 3d).…”

“…To circumvent this inconsistency, we used the fed-batch type of cultivation for valinomycin production over the whole process development procedure, which is also necessary to guarantee the long-term production of valinomycin. While in our earlier papers [18, 19], we applied media with complex additives for the production of valinomycin, here we aimed for simple glucose mineral salt based medium, for easier process development. As the building blocks for valinomycin all derive from the central carbon metabolism, a fermentation strategy based on glucose as the sole carbon and energy source should work out for the production of this NRP.…”

“…Valinomycin extraction and quantification were carried out as previously described [18]. Briefly, 1 mL of culture was centrifuged at 16,000× g for 3 min.…”

“…Autonomous formation of valinomycin could be achieved without substrate feeding, because the three substrates are available from the glycolysis as well as from the branched chain amino acid l -valine biosynthesis pathway in E. coli (Figure 1). The initial valinomycin titer was modest (0.3 mg L −1 ) in batch cultivations, while the titer was significantly enhanced up to 10 mg L −1 through flask scale high cell density fed-batch cultivations [18]. In addition, coexpression of the cognate type II thioesterase (TEII), which is encoded in the valinomycin gene cluster and responsible for regeneration of the NRPS activity, with Vlm1 and Vlm2 further improved valinomycin production to a final titer of 13 mg L −1 [19].…”

Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

“…The recent advances on synthetic biology have allowed to produce novel compounds (9), including high--performance enzymes with applications in the biotechnological sector such as the non ribosomal peptides synthetases (NRPs) and the polyketide synthetases (PKSs) (10) (11). In this context, aldolases constitute one of the most interesting groups of enzymes employed for organic synthesis of carbohydrates, aminoacids and hydroxyacids (12).…”