Environmental Engineering Reference
In-Depth Information
the provision of reactive sites for applications such as adhesives and coatings. Representative
side chains that have been incorporated into 3HA include unsaturated [8, 28], halogenated
[10], branched [29], and aromatic [30] moieties.
The addition of inhibitors, reducing the function of metabolic pathways that compete
with PHA synthesis, is another feeding strategy that has been employed to improve PHA
yield and/or to facilitate incorporation of longer monomers. In one example, acrylate was
used to inhibit β-oxidation in R. eutropha, such that the microbe accumulated a copolymer of
both short- and medium-chain length monomers rather than exclusively short chain-length
monomers [12].
3.1.2. Genetic engineering
. Extensive investigations into PHA synthesis has led to the
identification, cloning, and sequencing of approximately 40 responsible genes from various
gram-positive and gram-negative bacteria, providing a great deal of material to support efforts
in genetic engineering [31].
Among the most commonly-used heterologous hosts, including Ralstonia eutropha,
Pseudomonas putida, Pseudomonas oleovorans, and Escherichia coli, the latter deserves
special mention for its advantages as a host. It has an extremely thoroughly understood
physiology; it is not a native PHA producer, with the result that productivity is not limited by
natural regulation; it harbors no native machinery for PHA degradation; and its cells are
easily disrupted, facilitating PHA recovery [12].
One straightforward approach to increasing PHA synthesis is simply increasing gene
dosage or providing additional copies of the PHA synthetic enzymes. While this has shown
success in efforts with Aeromonas punctata and R. eutropha, not all attempts have been
successful. This shows that the effectiveness of gene dosage depends on the limiting factor
for polymer synthesis in each individual case.
A more involved but increasingly popular approach is the expression of heterologous
genes for polymer precursor production in a desirable host, with the goals of facilitating
synthesis of polymers that would not naturally accumulate and/or that might have desirable
structures and properties, as well as facilitating use of simple, inexpensive carbon sources for
production of the desired polymers. In one encouraging example, the incorporation of three
enzymes into a recombinant Salmonella allowed the synthesis of propionate, an expensive but
previously necessary substrate for the synthesis of PHB-co-PHV, from succinyl-CoA—an
intermediate in the TCA cycle. The microbe was then able to synthesize PHB-co-PHV from
glycerol, a significantly less expensive carbon source. A number of similar efforts have also
been successful [12].
In the realm of pathway engineering, competing pathways can also be eliminated and
regulatory systems can be altered to facilitate PHA synthesis. Related to the example above
involving propionate, propionate-degrading enzymes have been deleted, and propionate
synthesis machinery has been placed under the control of an IPTG-inducible promoter,
allowing the composition of PHB-co-PHV polymers to be adjusted at will [12].
Finally, PHA biosynthetic enzymes are amenable to protein engineering, and PHA
synthase in particular is the subject of an on-going effort to develop a complete understanding
of its structural and mechanical characteristics. In the case of the Pseudomonas 61-3 PHA
synthase, error-prone PCR mutagenesis revealed two primary sites that affected PHB
accumulation, allowing subsequent site-directed mutagenesis to test all possible amino acid
combinations at those sites. The optimal combination of amino acids at those sites yielded a
synthase that promoted the accumulation of up to 400-fold more PHB in the microbe [12].
