Polyhydroxyalkanoates (PHAs) are polyoxoesters produced by a wide range of bacteria when they find themselves in an environment with an available carbon source but limited in additional nutrient(s) required for growth (9
). The short-chain-length PHAs, where R is a methyl or ethyl, have properties of thermoplastics and are biodegradable (Fig. ). Much effort has focused on understanding the biology of PHA homeostasis for several reasons. First, this understanding could lead to expression of the appropriate gene set in heterologous systems to make PHA production economically competitive with oil-based polymers. Second, understanding PHA homeostasis serves as a paradigm for understanding the mechanism of homopolymerization reactions in which the product undergoes a phase transition during its formation, generating insoluble inclusions (granules). The intracellular PHAs can be degraded when the bacteria require carbon but are in otherwise nutrient-replete conditions, and the monomers and energy released can be reused to allow the bacteria to grow (17
). The insoluble PHA granules must, therefore, be biosynthesized in a controlled fashion to facilitate enzymatic degradation. A variety of proteins associated with PHA homeostasis have been identified and are being characterized (3
). As part of our research to understand the mechanisms that control polymer size and reuse, we have been interested in identifying the intracellular depolymerases that degrade poly[d
-(−)-3-hydroxybutyrate] (PHB) within the granules. The cloning, sequencing, and characterization in vivo of two new putative intracellular PHB depolymerases from Ralstonia eutropha
Thermoplastic short-chain-length PHAs are synthesized from monomers with short side chains.
Extracellular depolymerases, in contrast to the intracellular depolymerases, have been extensively characterized over the last decade (5
). These enzymes are secreted into the environment to degrade PHA released from dead bacteria. These proteins in general contain a N-terminal signal peptide of 25 to 38 amino acids. The peptide is cleaved during passage of the protein out of the cytosol. These depolymerases contain a large N-terminal catalytic domain with a lipase box (GXSXG), a C-terminal PHB binding domain, and a linker region that connects the N- and C-terminal domains (for an excellent review, see reference 6
). Several organisms contain extracellular depolymerase isozymes whose functions have not yet been clearly delineated.
The first sequence of an intracellular depolymerase was reported by Saegusa et al. (GenBank accession no. AB017612
) and was somewhat surprising, as the protein had no sequence similarity to the extracellular depolymerases. Recently this intracellular depolymerase, PhaZ (here designated PhaZ1), from R
was expressed, and its properties were examined in crude extracts. PhaZ1 is 47 kDa and has neither a lipase box nor an identifiable PHB binding domain. The depolymerase was shown to work best on amorphous PHB as found in the granules inside the cell. Furthermore, the enzyme was shown to convert the polymer into oligomers and monomers of hydroxybutyrate by the end of the degradation process (14
). The striking differences between the sequences of the extracellular and intracellular depolymerases make the intracellular depolymerases interesting subjects for mechanistic and biological studies.
The function of PhaZ1 in vivo has recently been addressed by two groups independently (4
). Each group generated a ΔphaZ1
H16 and then examined the fate of accumulated PHB under different growth conditions. The authors concluded from their studies that additional depolymerase(s) must be present to account for the observed phenotypes.
The availability of the R. eutropha phaZ1 sequence and the Ralstonia metallidurans genome sequence allowed us to successfully devise a strategy to find two additional candidate depolymerase genes in R. eutropha, designated phaZ2 and phaZ3. To understand the function of PhaZ2 and PhaZ3, a set of R. eutropha H16 phaZ gene deletion strains have been generated (ΔphaZ1, ΔphaZ2, ΔphaZ3, ΔphaZ1ΔphaZ2, ΔphaZ1ΔphaZ3, ΔphaZ2ΔphaZ3, and ΔphaZ1ΔphaZ2ΔphaZ3). The effect of these phaZ gene deletions on PHB accumulation and utilization when different R. eutropha strains were grown in rich medium and in PHB utilization medium are reported. These studies reveal that PhaZ2 is an intracellular depolymerase. The function of PhaZ3 remains to be established.