SUMMARY Poly(3-hydroxyalkanoates) (PHAs) are a class of microbially produced polyesters that have potential applications as conventional plastics, specifically thermoplastic elastomers. A wealth of biological diversity in PHA formation exists, with at least 100 different PHA constituents and at least five different dedicated PHA biosynthetic pathways. This diversity, in combination with classical microbial physiology and modern molecular biology, has now opened up this area for genetic and metabolic engineering to develop optimal PHA-producing organisms. Commercial processes for PHA production were initially developed by W. R. Grace in the 1960s and later developed by Imperial Chemical Industries, Ltd., in the United Kingdom in the 1970s and 1980s. Since the early 1990s, Metabolix Inc. and Monsanto have been the driving forces behind the commercial exploitation of PHA polymers in the United States. The gram-negative bacterium Ralstonia eutropha, formerly known as Alcaligenes eutrophus, has generally been used as the production organism of choice, and intracellular accumulation of PHA of over 90% of the cell dry weight have been reported. The advent of molecular biological techniques and a developing environmental awareness initiated a renewed scientific interest in PHAs, and the biosynthetic machinery for PHA metabolism has been studied in great detail over the last two decades. Because the structure and monomeric composition of PHAs determine the applications for each type of polymer, a variety of polymers have been synthesized by cofeeding of various substrates or by metabolic engineering of the production organism. Classical microbiology and modern molecular bacterial physiology have been brought together to decipher the intricacies of PHA metabolism both for production purposes and for the unraveling of the natural role of PHAs. This review provides an overview of the different PHA biosynthetic systems and their genetic background, followed by a detailed summation of how this natural diversity is being used to develop commercially attractive, recombinant processes for the large-scale production of PHAs.

Esherichia coli microcin B17 is a posttranslationally modified peptide that inhibits bacterial DNA gyrase. It contains four oxazole and four thiazole rings and is representative of a broad class of pharmaceutically important natural products with five-membered heterocycles derived from peptide precursors. An in vitro assay was developed to detect heterocycle formation, and an enzyme complex, microcin B17 synthase, was purified and found to contain three proteins, McbB, McbC, and McbD, that convert 14 residues into the eight mono- and bisheterocyclic moieties in vitro that confer antibiotic activity on mature microcin B17. These enzymatic reactions alter the peptide backbone connectivity. The propeptide region of premicrocin is the major recognition determinant for binding and downstream heterocycle formation by microcin B17 synthase. A general pathway for the enzymatic biosynthesis of these heterocycles is formulated.

SummaryMicrocin B17 (MccB17) is a ribosomally encoded DNAgyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N-terminal leader, which is removed during maturation, and a C-terminal structural peptide. PreMccB17, the translational product of mcbA, is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification-specific antibody rather than antimicrobial activity, we show that the 26-amino-acid N-terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17-␤-galactosidase fusion lacking the leader are post-translationally modified.

Expression ofEscherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHA MCL ) formation from fatty acids in an E. coli fadB mutant. The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional ␤-oxidation enzymes. E. coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHA MCL s when grown in the presence of fatty acids. Plasmid-based expression of yfcX in the FadB ؊ YfcX ؊ PhaC ؉ strain restores polymer formation. YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities. The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity. Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E. coli fadB mutant strains. Further studies are required to determine the natural role of YfcX in the metabolism of E. coli.Polyhydroxyalkanoates (PHAs) are a class of biopolyesters that are receiving considerable attention for use as renewable plastics in packaging and personal hygiene items (40). A broad spectrum of properties can be obtained from PHAs by varying the monomer unit composition (38,40). PHAs containing short-chainlength monomer units are thermoplastics, whereas PHAs containing medium-chain-length monomer units are elastomeric. While considerable progress has been made toward understanding and manipulating pathways for the formation of short-chainlength PHAs (23,34,38), such as poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate, our understanding of the biosynthesis of medium-chain-length PHAs (PHA MCL ) is not as extensive. Escherichia coli engineered with a PHA synthase possessing substrate specificity for medium-chain-length monomer units will only produce significant levels of PHA MCL when grown in the presence of fatty acids if the activity of the fatty acid ␤-oxidation complex has been disrupted by mutation or chemical inhibition (37). Since ␤-oxidation activities are required to process longer-chain fatty acids to PHA MCL monomer units (Fig. 1), the requirement of a disrupted fatty acid ␤-oxidation complex for PHA MCL formation is counterintuitive.In the study described here, we examined the ability of fadB mutants of E. coli to produce PHA MCL from longer-chain fatty acids. FadB encodes the ␣ subunit of a multienzyme complex that is involved in the degradation of fatty acids in E. coli (4). It has been previously suggested (37) that FadB activities are involved in PHA MCL formation in E. coli strain LS1298, a fadB mutant of E. coli, despite reports (9) that the strain is devoid of ␤-oxidation activities. These unexplained results encouraged us to evaluate the ability of fadB strains to produce PHA MCL from longer-chain fatty acids and to search for alternative activities that may play a role in PHA MCL formation.Searches of the E. coli nu...

SummaryThe production of polyhydroxybutyrate (PHB) involves a multigene pathway consisting of thiolase, reductase and synthase genes. In order to simplify this pathway for plant-based expression, a library of thiolase and reductase gene fusions was generated by randomly ligating a short core linker DNA sequence to create in-frame fusions between the thiolase and reductase genes. The resulting fusion constructs were screened for PHB formation in Escherichia coli . This screen identified a polymer-producing candidate in which the thiolase and reductase genes were fused via a 26-amino-acid linker. This gene fusion, designated phaA-phaB , represents an active gene fusion of two homotetrameric enzymes. Expression of phaA-phaB in E. coli and Arabidopsis yielded a fusion protein observed to be the expected size by Western blotting techniques. The fusion protein exhibited thiolase and reductase enzyme activities in crude extracts of recombinant E. coli that were three-fold and nine-fold less than those of the individually expressed thiolase and reductase enzymes, respectively. When targeted to the plastid, and coexpressed with a plastid-targeted polyhydroxyalkanoate (PHA) synthase, the fusion protein enabled PHB formation in Arabidopsis , yielding roughly half the PHB formed in plants expressing individual thiolase, reductase and synthase enzymes. This work represents a first step towards simplifying the expression of the PHB biosynthetic pathway in plants.