Enhanced Production of (R)-3-Hydroxybutyrate Oligomers by Coexpression of Molecular Chaperones in Recombinant Escherichia coli Harboring a Polyhydroxyalkanoate Synthase Derived from Bacillus cereus YB-4.

Saki Goto,Takeharu Tsuge,Seiichi Taguchi,Ayaka Hiroe,Yuki Miyahara

doi:10.3390/microorganisms10020458

Abstract

The biodegradable polyester poly-(R)-3-hydroxybutyrate [P(3HB)] is synthesized by a polymerizing enzyme called polyhydroxyalkanoate (PHA) synthase and accumulates in a wide variety of bacterial cells. Recently, we demonstrated the secretory production of a (R)-3HB oligomer (3HBO), a low-molecular-weight P(3HB), by using recombinant Escherichia coli expressing PHA synthases. The 3HBO has potential value as an antibacterial substance and as a building block for various polymers. In this study, to construct an efficient 3HBO production system, the coexpression of molecular chaperones and a PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4) was examined. First, genes encoding enzymes related to 3HBO biosynthesis (phaRCYB4, phaA and phaB derived from Ralstonia eutropha H16) and two types of molecular chaperones (groEL, groES, and tig) were introduced into the E. coli strains BW25113 and BW25113ΔadhE. As a result, coexpression of the chaperones promoted the enzyme activity of PHA synthase (approximately 2–3-fold) and 3HBO production (approximately 2-fold). The expression assay of each chaperone and PHA synthase subunit (PhaRYB4 and PhaCYB4) indicated that the combination of the two chaperone systems (GroEL-GroES and TF) supported the folding of PhaRYB4 and PhaCYB4. These results suggest that the utilization of chaperone proteins is a valuable approach to enhance the formation of active PHA synthase and the productivity of 3HBO.

Highlights

Polyhydroxyalkanoates (PHAs) are biodegradable plastics that are synthesized by various microorganisms [1–3]
We focused on the expression level of PHA synthase to establish an efficient 3HB oligomer (3HBO) production system
To examine the effect of chaperones on 3HBO production, E. coli BW25113 harboring the plasmid pGEM-phaRCYB4 AB alone or the plasmid pair pGEM-phaRCYB4 AB+pG-Tf2 was grown in LB medium containing 20 g/L glucose (Table 2)

Summary

Introduction

Polyhydroxyalkanoates (PHAs) are biodegradable plastics that are synthesized by various microorganisms [1–3]. PHAs can be categorized into three groups: short-chain-length PHAs (SCL-PHAs), containing 3–5 carbon atoms in the monomer; medium-chain-length PHAs (MCL-PHAs), containing 6–14 carbon atoms in the monomer; and SCL-MCL-PHAs, containing both SCL and MCL monomer units. PHA synthase is a key enzyme in the polymerization of PHA. PHA synthases can be divided into four classes according to their substrate specificities and subunit compositions [4]. Whereas class I and II PHA synthases are composed of single subunits of PhaC, class III and IV PHA synthases are composed of two heterosubunits, with PhaE and PhaC forming PhaEC and. Class I, III, and IV PHA synthases prefer SCL monomers as substrates, whereas class II PHA synthases are specific to MCL monomers. Attempts have been made to synthesize PHAs with favorable properties

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Conclusion