Efficient secretory production of proline/alanine/serine (PAS) biopolymers in Corynebacterium glutamicum yielding a monodisperse biological alternative to polyethylene glycol (PEG)

Abstract

Background: PAS biopolymers are recombinant polypeptides comprising the small uncharged L-amino acids Pro, Ala and/or Ser which resemble the widely used poly-ethylene glycol (PEG) in terms of pronounced hydrophilicity. Likewise, their random chain behaviour in physiological solution results in a strongly expanded hydrodynamic volume. Thus, apart from their use as fusion partner for biopharmaceuticals to achieve prolonged half-life in vivo, PAS biopolymers appear attractive as substitute for PEG-or other poorly degradable chemical polymers-in many areas. As a prerequisite for the wide application of PAS biopolymers at affordable cost, we have established their highly efficient biotechnological production in Corynebacterium glutamicum serving as a well characterized bacterial host organism.

Results: Using the CspA signal sequence, we have secreted two representative PAS biopolymers as polypeptides with ~ 600 and ~ 1200 amino acid residues, respectively. Both PAS biopolymers were purified from the culture supernatant by means of a simple downstream process in a truly monodisperse state as evidenced by ESI-MS. Yields after purification were up to ≥ 4 g per liter culture, with potential for further increase by strain optimization as well as fermentation and bioprocess development. Beyond direct application as hydrocolloids or to exploit their rheological properties, such PAS biopolymers are suitable for site-specific chemical conjugation with pharmacologically active molecules via their unique terminal amino or carboxyl groups. To enable the specific activation of the carboxylate, without interference by the free amino group, we generated a blocked N-terminus for the PAS(1200) polypeptide simply by introducing an N-terminal Gln residue which, after processing of the signal peptide, was cyclised to a chemically inert pyroglutamyl group upon acid treatment. The fact that PAS biopolymers are genetically encoded offers further conjugation strategies via incorporation of amino acids with reactive side chains (e.g., Cys, Lys, Glu/Asp) at defined positions.

Conclusions: Our new PAS expression platform using Corynex^® technology opens the way to applications of PASylation^® technology in multiple areas such as the pharmaceutical industry, cosmetics and food technology.

Keywords: Disordered polypeptide; Hydrophilic polymer; PASylation; PEG; Pro/Ala/Ser.

Fig. 1

C. glutamicum expression vector for the secretion of PAS polypeptides (A) and schematic downstream process for the purification of the PAS biopolymers (B)

Fig. 2

Analysis of PAS polypeptide secretion in C. glutamicum and of the downstream purification by SDS-PAGE. PAS#1(600) (A) and PAS#1(1200) (B) were produced in MMTG-J medium containing 25 mg/L kanamycin either at pH 6.5 (lanes 1) or at pH 7.0 (lanes 2). Samples from the culture supernatant (SN), the ammonium sulfate precipitate ((NH₄)₂SO₄) and flow-through fractions from CEX and AEX were subjected to SDS-PAGE. The gels were either stained with Coomassie brilliant blue (left) or with BaI₂ (right). Free PAS polymer is only visible with BaI₂, not with Coomassie staining

Fig. 3

Biochemical analysis of the purified recombinant PAS biopolymers. A SEC profiles of PAS#1(1200) (dashed line) and PAS#1(600) (solid line). B Deconvoluted ESI–MS spectra (overlay) of PAS#1(600) (red, calculated mass: 49,644.3 Da) and PAS#1(1200) (blue, calculated mass with intact N-terminal Gln residue: 99,511.8 Da)

Fig. 4

N-terminal pyroglutamate formation of the recombinant PAS#1(1200) biopolymer. The deconvoluted ESI–MS spectrum indicates complete cyclisation of the N-terminal Gln residue after incubation in 99% acetic acid at 50 °C for 4 h (calculated mass: 99,494.0 Da)