Improved production of poly(hydroxybutyrate) and related enzymes
Polyhydroxyalkanoates (PHAs) are biodegradable, thermoplastic polymers that are produced by a wide range of bacteria in response to nutrient limitation and function as carbon and energy storage molecules. They have a wide range of potential applications ranging from direct replacements for traditional plastics in packaging, industry and agriculture to new markets such as drug delivery, tissue engineering and environmental remediation. This thesis begins with a review of PHA metabolism and uses, in which they are compared to other carbon storage compounds. The most common method of production at present is within bacteria, particularly transgenic Escherichia coli. However, bacterial production is expensive and limits the range of monomers that can be incorporated. This makes it difficult to fine-tune the physical properties to the desired applications. This thesis aims to address both of these problems, using polyhydroxybutyrate as a simple model. To simplify the purification of the polymer and to broaden the range of potential monomers, PHA can be produced in vitro. To achieve this on a commercial scale would require the production of large quantities of the polymerase enzyme, PhaCRe. Therefore, a novel Escherichia coli cell factory, which can enter a non-growing but metabolically active quiescent state, was tested for high-efficiency PhaCRe production. Although the cells produced more PhaCRe, the majority of the enzyme was accumulated in insoluble and inactive deposits. Therefore, chaperone proteins were co-expressed with PhaCRe to facilitate its correct folding. This resulted in an approximately three-fold increase in the yield of PhaCRe. Another strategy for controlling the physical properties of PHAs is to blend them with other polymers. E. coli typically produces very high molecular weight PHA, so smallchain alcohols were investigated for their efficiency in reducing the molecular weight to allow easier blending. Methanol and ethanol were shown to reduce the molecular weight by up to 70% at concentrations that are non-toxic to the cells. Both are cheap and readily available chemicals, and could therefore be easily used in commercial-scale production. Finally, to understand better the mechanisms that take place within bacterial cells during PHA production, a new electron microscopy technique was investigated: wet scannningtransmission electron microscopy allows the interior features of unfixed, unstained and hydrated cells to be quickly and easily viewed at high resolution, with a limit of 15– 20 nm. This was the first time the technique had been used to view the cytoplasmic contents of bacteria and demonstrated its potential as an intermediate between light microscopy and more damaging (but higher resolution) electron microscopy techniques. The technique was also used to view triacylglyceride and polyphosphate inclusions bodies within cells.
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