Douglas Dennis

Polyhydroxybutyrate (PHB), a high molecular weight polyester, is accumulated as a storage carbon in many species of bacteria and is a biodegradable thermoplastic. To produce PHB by genetic engineering in plants, genes from the bacterium Alcaligenes eutrophus that encoded the two enzymes required to convert acetoacetyl-coenzyme A to PHB were placed under transcriptional control of the cauliflower mosaic virus 35S promoter and introduced into Arabidopsis thaliana. Transgenic plant lines that contained both genes accumulated PHB as electron-lucent granules in the cytoplasm, nucleus, and vacuole; the size and appearance of these granules were similar to the PHB granules that accumulate in bacteria.

The poly-beta-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, beta-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.

An Escherichia coli strain has been constructed that produces the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) P(HB-co-HV). This has been accomplished by placing the PHB biosynthetic genes from Alcaligenes eutrophus into an E. coli fadR atoC(Con) mutant and culturing the strain in M9 minimal medium containing glucose and propionate. 3-Hydroxyvalerate incorporation is absolutely dependent on the presence of both glucose and propionate, and 3-hydroxybutyrate-3-hydroxyvalerate ratios in the copolymer can be manipulated by altering the propionate concentration and/or the glucose concentration in the culture. P(HB-co-HV) production can be accomplished by using a wide variety of feeding regimens, but the most efficient is to allow the culture to grow to late log phase in minimal medium containing acetate and then add glucose and propionate to initiate copolymer production. A broad range of propionate concentrations can be used in the culture to stimulate 3-hydroxyvalerate incorporation; however, the most efficient utilization of propionate occurs at concentrations below 10 mM. 3-Hydroxyvalerate molar percentages in the copolymer are relatively constant over the course of growth. The copolymer has been purified and confirmed to be P(HB-co-HV) by gas chromatography/mass spectrometry and differential scanning calorimetry.

The cloned poly-3-hydroxybutyrate (PHB) synthesis pathway from Alcaligenes eutrophus has been introduced into sucrose-utilizing strains of Escherichia coli, Klebsiella aerogenes, and Klebsiella oxytoca. The plasmid-borne genes were well expressed in these environments and were able to mediate the production of significant amounts of PHB when the bacteria were grown with sucrose as the sole carbon source. The molecular weight of the PHB polymer made in K. aerogenes and E. coli was approximately 1 x 10(6) to 2 x 10(6). Sucrose uptake in K. aerogenes was measured and found to be similar to that found for other Klebsiella strains, but sucrose uptake in the E. coli strain was not detectable. K. aerogenes is able to utilize sugarcane molasses as the sole carbon source to accumulate PHB at the rate of approximately 1 g of PHB per liter of culture fluid per h. A K. oxytoca fadR strain was able to incorporate 3-hydroxyvalerate into a poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-V) polymer to levels as high as 56 mol% when grown in a medium containing propionate. Total PHB-co-V levels could be enhanced by adding propionate at the beginning of stationary phase rather than at the time of inoculation.