Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway

Abstract

The poly-I-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, I-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.Poly-p-hydroxybutyrate (PHB), a homopolymer of D-(-)-3-Hydroxybutyrate, is a storage material produced by a variety of bacteria in response to environmental stress. The presence of PHB in bacteria was first recognized by Lemoigne in 1926 (11), and it has since been identified in more than 20 bacterial genera, including Azotobacter, Bacillus, Beijerinckia, Alcaligenes, Pseudomonas, Rhizobium, and Rhodospirillum (4).The PHB pathway and its regulation have been studied extensively in Alcaligenes eutrophus H16 and Azotobacter beijerinckii (3,4,9,10,15,(18)(19)(20)(21)24). The pathway consists of a biosynthetic portion and a degradative portion and is made up of five enzymes. One of these enzymes, 0-ketothiolase, is both the entry point and the exit point of the cycle (4). In the biosynthetic part of the pathway, ,-ketothiolase catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. Acetoacetyl-CoA is subsequently reduced to D-(-)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase, and PHB is then produced by the polymerization of 0-hydroxybutyrylCoA via the action of PHB synthetase.Studies in A. eutrophus H16 and in Azotobacter beijerinckii have shown the PHB pathway to be regulated in response to several types of environmental limitation. These limitations include oxygen deprivation, nitrogen deprivation, sulfate limitation, and magnesium limitation (3,4,10,15,(18)(19)(20)(21)24). Under limiting environmental conditions, PHB may constitute as much as 80% of the dry cell weight. When limiting conditions are relaxed, PHB quantities decrease to preinduction levels (4). Induction studies in which 1-ketothiolase and acetoacetyl-CoA reductase were studied have revealed that both enzymatic activities increase markedly in response to PHB-stimulating limitations (4,10,15,19).These experiments indicate that the PHB pathway may exhibit a mode of transcriptional control that is similar to that of other metabolic pathways that are induced by environmental stress. Examples of such pathways include the heat shock regulon, the pho regulon, and the carbon starvati...

Effect of temperature and cycle length on microbial competition in PHB-producing sequencing batch reactor

“…Since the first discovery of PHA production in Bacillus megaterium by Lemoigne, 1926, over 250 different types of bacteria have been reported as natural PHA producers (Steinbü chel, 1991). In addition to natural producers, genetically-modified organisms are used for the industrial production of PHA (Slater et al, 1988;Braunegg et al, 1998). However, the use of sterile equipment, defined substrates and downstream processing contribute to the high costs of PHA production, which limits the industrial application of PHA (Keshavarz and Roy, 2010).…”

Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism

“…PHB biosynthesis from the central intermediate acetyl-CoA occurs in three steps and is mediated by a b-ketothiolase (PhaA), which catalyses the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA, an acetoacetyl-CoA reductase (PhaB), which reduces acetoacetylCoA to R-3-hydroxybutyryl-CoA, and finally the key enzyme PHB synthase (PhaC), which catalyses the polymerization of the hydroxyacyl moiety of R-3-hydroxybutyryl-CoA. The genes for the three enzymes are encoded by the phaCAB operon which was cloned during the 1980s (Schubert et al, 1988;Slater et al, 1988;Peoples & Sinskey, 1989). Since then, several additional genes involved in PHB metabolism have been identified.…”

Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae

“…The genes of A. eutrophus are organized in a single operon as phbC-A-B, which are genes of PHA synthase, ␤-ketothiolase, and NADPH-acetoacetyl-coenzyme A (CoA) reductase, respectively (26,31,34). Whereas in P. oleovorans, two structural genes of PHA synthases (phaC1 and phaC2) flanking a PHA depolymerase gene have been identified (14), PHA synthases of Chromatium vinosum (21) and Thiocapsa pfennigii (35) consist of two different types of subunits encoded by phbC and phbE in a single operon.…”