Secretion of polyhydroxybutyrate in Escherichia coli using a synthetic biological engineering approach

Abstract

BackgroundPolyhydroxyalkanoates (PHAs) are a group of biodegradable plastics that are produced by a wide variety of microorganisms, mainly as a storage intermediate for energy and carbon. Polyhydroxybutyrate (PHB) is a short-chain-length PHA with interesting chemical and physical properties. Large scale production of PHB is not wide-spread mainly due to the downstream processing of bacterial cultures to extract the PHB. Secretion of PHB from Escherichia coli could reduce downstream processing costs. PHB are non-proteinaceous polymers, hence cannot be directly targeted for secretion. Phasin, PhaP1, is a low molecular weight protein that binds to PHB, reducing PHB granule size. In this study PHB is indirectly secreted with PhaP1 from E. coli via type I secretion using HlyA signal peptides.ResultsThis study demonstrated the successful secretion of phasin and phasin bound PHB outside of the cell and into the culture medium. The secretion of PHB was initiated between 24 and 48 h after induction. After 48 h of culturing, 36% of the total PHB produced in the secreting strain was collected in the secreted fraction and 64% remained in the internal fraction. To further support the findings of this study, the PHB secretion phenomenon was observed using SEM.ConclusionsFrom this study, the ability to use type I secretion to: 1) secrete phasin and 2) successfully secrete PHB has been shown.

Microbubble assisted polyhydroxybutyrate production in Escherichia coli

“…The MBD bioreactor had an average PHB accumulation of 43.14 % 48 h post inoculation, which was similar to the 500 rpm agitation rate with 0.8 vvm air sparging and also outperformed the 350 rpm 0.8 vvm study. Accumulation of PHB to approximately 40–50 % after 48 h of culturing is similar to the results observed in shaker flask experiments in other studies with similar media composition and without pH or DO control [ 28 , 30 ].…”

“…The XL1-Blue strain of E. coli was chosen for its ability to out-perform other E. coli strains for PHB production [ 27 ]. The plasmid pBHR68 was selected as it contained the lactose inducible phaCAB operon and had demonstrated PHB accumulation up to approximately 50 % of the dry cell weight after 48 h of growth in a minimal media [ 28 ].…”

Beyond Intracellular Accumulation of Polyhydroxyalkanoates: Chiral Hydroxyalkanoic Acids and Polymer Secretion

“…The results obtained show that the PelB system was not effective in the translocation of GFP, while the other two T2SSs successfully exported GFP to the periplasm and with respect to the HlyA system, also secrete the GFP protein to the extracellular medium (Linton et al, 2012). Based on these results, the HlyA signal peptide fusion protein and a fascine that associates with the PHB granules were used to bind the signaling sequence to the PHB granule, leading to the secretion of PHB to the extracellular medium (Rahman et al, 2013). Results indicate that after 48 h of culture 36% of the total PHB produced by the secretory strain was collected in the secreted fraction while the remaining 64% corresponded to the internal fraction.…”

Recent strategies for efficient production of polyhydroxyalkanoates by micro-organisms

“…Also, developing a secretion method that eliminates the processes of separation and purification steps may reduce the production cost greatly. Rahman et al demonstrate the successful secretion of PHB in E. coli using recombinant approach so that reduce downstream processing costs (Rahman et al 2013). Furthermore, compared to digestion by enzymes or mechanical disruption and ultrasonication, a digestion condition by cheap chemicals can also be an attractive alternative in terms of PHAs recovery.…”