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

Asif Rahman,Alex D Hatch,Elisabeth Linton,Ronald C Sims,Charles D Miller

doi:10.1186/1754-1611-7-24

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.

Highlights

Polyhydroxyalkanoates (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
Analysis of phasin translocation For E. coli cells expressing pCMEL1 and pLG575, a phasin band is observed at 22–26 kDa in the cytoplasmic fraction, the periplasmic fraction, the membrane fraction, and the concentrated extracellular media (Figure 1)
This polyacrylamide gel and corresponding immunoblot demonstrated: 1) the ability for E. coli to produce non-codon optimized Phasin, a protein not naturally expressed in E. coli and 2) translocation of phasin into different fractions of the cell

Summary

Introduction

Polyhydroxyalkanoates (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. Secretion of PHB from Escherichia coli could reduce downstream processing costs. Based on an United States Environmental Protection Agency study in 2011, there was an increase in non-biodegradable plastic accumulation in municipal solid waste systems from 0.5% to 12.4% during 1960 to 2010 [1]. PHAs are produced by a variety of microorganisms as an intercellular storage medium for energy and carbon and can accumulate up to 90% of the cell dry weight [4]. PHAs have a variety of possible applications, that could replace traditional plastics derived from petroleum [11]. Some of the possible applications are highlighted in previous studies [12,13] and include: packaging, medical uses [14], agricultural uses, and in carbon nanotubes [15]

Objectives

Methods

Results

Conclusion