This is the first study to document alcoholysis activity in PHA synthases. == MATERIALS AND METHODS == == Bacterial strains, plasmids, and culture media. alcoholysis activity for PHA chains. These LTβR-IN-1 results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature. == INTRODUCTION == Polyhydroxyalkanoates (PHAs) are a category of aliphatic polyesters synthesized by a wide variety of bacteria as an intracellular carbon and energy storage material in response to various environmental conditions. Recently, PHAs have attracted industrial attention because of their potential use as biodegradable and biocompatible thermoplastics (1). The biosynthesis of poly[(R)-3-hydroxybutyrate] [P(3HB)], the most commonly found natural PHA, has been well studied inRalstonia eutrophaand requires only three enzymes from acetyl coenzyme A (acetyl-CoA): 3-ketothiolase (PhaA), encoded by thephaAgene; acetoacetyl-CoA reductase (PhaB), encoded by thephaBgene; and PHA synthase (PhaC), encoded by thephaCgene (2). The coexpression of these three enzymes enables P(3HB)-negative bacteria likeEscherichia colito accumulate P(3HB) (3). PHA synthases are grouped into four classes (classes I to IV) on the basis of subunit composition and substrate specificity (4). Class I synthases contain a single subunit, PhaC, and catalyze the polymerization of short-chain-length monomers (C3to C5); the synthase fromR. eutropha(PhaCRe) is an example of this class. Class II synthases, as represented by the synthase fromPseudomonas putida, also contain a single PhaC subunit but catalyze the polymerization of medium-chain-length monomers (C6to C14). Class III synthases, as represented by the synthase fromAllochromatium vinosum, contain two heterologous subunits, PhaE and PhaC, and catalyze the polymerization of short-chain-length monomers. Class IV synthases, a recently identified class of synthases, contain two heterologous subunits, PhaR and PhaC, and catalyze the RPTOR polymerization of short-chain-length monomers (C3to C5). On the basis of phylogenetic analysis (see Fig. S1 in the supplemental material), the PhaC subunits in class IV synthases can be further classified into two subgroups: theBacillus megateriumsubgroup and theBacillus cereussubgroup (57). These PhaC subunits recognize PhaR subunits from different subgroups (7). P(3HB) produced byE. coliexpressing class I to IV synthases exhibits a variety of molecular weights, depending on the characteristics of the synthase expressed (8). It is noteworthy that the synthase fromDelftia acidovorans(PhaCDa), a class I synthase, is capable of synthesizing high-molecular-weight P(3HB) inE. coli(9). In previous studies (7,10), it was shown thatB. cereusstrain YB-4 isolated from soil expresses a class IV PHA synthase consisting of two heterologous subunits, PhaRYB-4(18.5 kDa) and PhaCYB-4(41.7 kDa). RecombinantE. coliexpressing PhaRCYB-4showed an unusual decrease in the molecular weight of P(3HB) synthesized during cultivation, especially in the stationary phase of growth. In addition, kinetic analysis indicated that the decrease in molecular weight is the result of random scission of the polymer chain (7). The same phenomenon was observed withE. coliexpressing the class IV synthase fromBacillussp. strain INT005 (PhaRCBsp) (11). On the other hand,E. coliexpressing PHA synthase fromB. megaterium(PhaRCBm), another class IV synthase, did not produce P(3HB) with such a low molecular weight (7). A subunit recombination study of PhaR and PhaC fromB. cereusYB-4 andB. megateriumrevealed that the PhaCYB-4subunit is responsible for the scission activity. However, unlikeE. coli, the PHA-negative mutantR. eutrophaPHB4 produced high-molecular-weight P(3HB) even when PhaRCYB-4was expressed (12). From these observations, we hypothesized that there might be regulatory mechanisms governing the P(3HB) scission activity of PhaCYB-4. The aim of this study was to investigate the LTβR-IN-1 mechanism governing the P(3HB) scission activity of PhaRCYB-4. To this end, anin vivoscission assay was carried out, usingE. coliJM109 as the host strain, by LTβR-IN-1 expressing PhaRCYB-4from high- or low-copy-number plasmids. The results indicate that PhaRCYB-4has alcoholytic cleavage activity for P(3HB) chains in the presence of both endogenous and exogenous ethanol. Moreover, the results from coexpression of PhaRCYB-4with PhaCDa[PhaCDaproduces a high-molecular-weight P(3HB) used as the scissile substrate in this experiment] indicate that PhaRCYB-4is also able to cleave P(3HB) polymerized by another synthase. In addition, the alcoholytic cleavage activity of PhaRCYB-4was assayedin vitro. This is the first study to document alcoholysis activity in PHA synthases. == MATERIALS AND METHODS == == Bacterial strains, plasmids, and culture LTβR-IN-1 media. == The bacterial strains and plasmids used in this study are listed inTable 1.E. coliJM109 was used as a host strain LTβR-IN-1 for P(3HB) biosynthesis throughout the study.E. coliBL21(DE3) was used to produce His-tagged PhaRYB-4and PhaCYB-4from the expression plasmids pET15b-phaRYB-4and pET15b-phaCYB-4, respectively. For preculturing, the recombinant bacteria were grown in lysogeny.