Document Type

Honors Project

Publication Date

Summer 2025

Abstract

Polyhydroxyalkanoates (PHAs), commonly known as biodegradable plastics, are polymers of hydroxyalkanoate monomers that are produced by over 75 genera of bacteria [1]. The most common PHA is polyhydroxybutyrate (PHB), which is composed of 3-hydroxybutyrate (3HB) monomers. PHB is degraded to 3HB by PHB depolymerases [2]. Streptomyces nymphaeiformis is a Gram positive, filamentous bacterium with a genomic G+C content of ~70% [3]. Production of PHB depolymerase in this organism is induced by the presence of PHB and 3HB, with maximal activity observed after 24 to 48 hrs, [4]. The PHB depolymerase protein can be purified from cell-free supernatants of PHB-grown cells of S. nymphaeiformis. Unfortunately, the amount of purified protein is very low (~10 μg/L of culture). We would like to overexpress the PHB depolymerase gene (phaZ) to obtain much larger amounts of purified PHB depolymerase for use in crystallization and/or preparation of antibodies. Streptomyces lividans TK24 is often used as a host for overexpression of genes from other Streptomyces species [4]. Foreign genes can be efficiently introduced into S. lividans TK24 by interspecific conjugation with Escherichia coli. The “shuttle” plasmid pIJ86 can replicate in both E. coli and Streptomyces (Fig. 2) [5]. It has an apramycin resistance marker and genes for proteins involved in bacterial conjugation, enabling transfer of the plasmid from E. coli to Streptomyces through a tube called the sex pilus. However, DNA replicated in E. coli is usually methylated at certain bases and unstable when introduced into Streptomyces. Therefore, a non-methylating strain of E. coli, 12567/pUZ8002 is used as a host for pIJ86. It also has a large plasmid, pUZ8002 which has genes for additional conjugation functions. The overall goal of the research was to overexpress the PHB depolymerase gene of S. nymphaeiformis in S. lividans TK24 and obtain large amounts of PHB depolymerase protein.

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