Abstract
Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer, and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20°C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.
Highlights
Prodiginines are tripyrrolic red-colored secondary metabolites of microbial origin with highly valuable bioactivities, such as antibacterial, antitumor, immunosuppressive, and antimalarial activity (Han et al, 1998; Lapenda et al, 2014; Hassankhani et al, 2015)
Pseudomonas putida KT2440 was chosen as prodigiosin production host for several reasons
The strain offers specific advantages, rendering challenging biosyntheses of natural products like prodigiosin possible: (i) It harbors a phosphopantetheinyl transferase (PPTase) with broad substrate specificity (Gross et al, 2005). Enzymes of this family are required for functional PKS/NRPS expression, because they activate peptidyl carrier protein (PCP) as well as acyl carrier protein (ACP) domains, which occur in enzymes PigG and PigH of the prodigiosin pathway, respectively (Garneau-Tsodikova et al, 2006), by attaching the phosphopantetheinyl moiety to them
Summary
Prodiginines are tripyrrolic red-colored secondary metabolites of microbial origin with highly valuable bioactivities, such as antibacterial, antitumor, immunosuppressive, and antimalarial activity (Han et al, 1998; Lapenda et al, 2014; Hassankhani et al, 2015). Similar to that of the native producer S. marcescens (Figure 2A) The coloration of these strains was obviously more intense than in previously reported T7 RNA polymerase-dependent expression strains (P. putida pig-w1 + T7, Figure 2A), indicating a significantly higher prodigiosin production. Red pigmentation of the two prodigiosin producing P. putida strains was observed both in early and late stages of colony growth on plates, indicating the pig gene expression being under control of strong and constitutively active promoters. The foam cubes did adsorb the small fraction of prodigiosin accumulating in the supernatant, but seemed to facilitate prodigiosin secretion by continuously binding of the extracellular pigment, resulting in almost uncolored cells and culture medium (visualized in Supplementary Figure S5) These observations were corroborated by spectrophotometric quantification of the remaining cell-bound prodigiosin which revealed titers below 1 mg/L. The hereby yielded 56 ± 7 mg solid material (obtained from 1 L culture) contained 84% prodigiosin, as analyzed via qNMR
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