Attacking the plastic waste problem: a two-pronged approach
Faced with the issues of plastic waste accumulation and environmental pollution, a two-pronged approach with the potential to solve these problems has been developed. Firstly, biologically produced plastics such as polyhydroxyalkanoates (PHAs) are superior to petroleum-based plastics because they are both biodegradable and biocompatible. By focusing on modulating the ratio of two PHA monomers, 3-hydroxybutyrate and 4-hydroxybutyrate, the copolymer poly(3HB-co-4HB) can be created featuring increased elasticity and utility over any particular PHA monomer. Secondly, a novel polyethylene-degradation pathway is being engineered based on the oxidation of long-chain alkanes by alkane monooxygenase LadA. The region inhibiting the binding and catalysis of polyethylene has been computationally identified and site-directed mutagenesis is being conducted at this region to yield a mutant of LadA that oxidizes polyethylene and thereby increases its biodegradability. The combination of the production of an eco-friendly bioplastic with the degradation of petroleum-based plastics is a promising method of waste reduction.
Regulation of the synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutryate) - Experimental
Polyhydroxyalkanoic acids (PHA), naturally occurring storage polymers found in a variety of bacteria, have received increased attention for their potential use as bioplastics that are both biodegradable and reduce reliance on petroleum-based plastics. In particular, the copolymer poly(3-hyroxybutyrate-co-4-hydroxybutyrate), or poly(3HB-co-4HB), which combines the 3HB and 4HB polymers from different bacteria, has elastic properties ideal for a wide range of thermoplastic applications. The high cost of PHA, however, is the biggest impediment to widespread use of bioplastics. Moreover, poly(3HB-co-4HB) pathways developed so far in E. coli have yielded undesirably low and unpredictable 4HB-to-3HB ratios.
Thus, this project aims to develop a more efficient biopathway for poly(3HB-co-4HB) while increasing the 4HB monomer composition predictably. It was hypothesized that optimizing codon permutations of the phaC gene would greatly increase affinity of PHA synthase to the 4HB monomer. To date, the phaCAB and cat2 operons have been cloned into pUC19, PCR Blunt II-TOPO, and pASK vectors for independent production of the 3HB and 4HB polymers. Transformation of the genes required for both 3HB (phaA, phaB, and phaC) and 4HB (cat2 and phaC) production into E. coli as several different recombinant constructs is being completed and will allow for engineering of the poly(3HB-co4HB) biopathway. Furthermore, 3HB polymer has been successfully isolated from the pASK bacteria and characterized by H1 NMR spectroscopy.
Future directions will test the hypothesis to see if phaC can be manipulated to increase 4HB-to-3HB composition in poly(3HB-co-4HB) and to increase efficient production of the bioplastic by engineering the FtsZ cell division protein to allow for cells to accumulate larger quantities of PHA granules before dividing. Ultimately, once an optimal biopathway is found, the goal would be to explore a model for mass production of PHA bioplastics so that novel applications of bioplastics can be feasible economically.
Rational Bioengineering of LadA Monooxygenase in a Polyethylene Biodegradation Pathway
Polyethylene, a stable and common commercial plastic, presents a costly and persistent environmental problem. We are currently engineering a polyethylene biodegradation pathway by modifying an alkane degradation pathway in which a monooxygenase enzyme, LadA, performs the initial terminal oxidation step converting the alkane into an alcohol. The alcohol can later be processed into a fatty acid to be consumed for energy in a natural fatty acid metabolic pathway. Using Molegro Virtual Docker, we computationally analyzed the substrate specificity of LadA and identified a sub-region of Insertion Region 4 (IS4), comprising of residues 300-349, as the primary residues responsible for hindering polyethylene binding. We have isolated the wild-type gene from Geobacillus thermodenitrificans and are currently engineering a LadA mutant capable of binding to polyethylene through a combination of rational design and directed evolution. A large library of mutant LadA genes, that vary in the IS4 subregion, will be synthesized, spliced into bacteriophage vector and amplified in E. coli BLT5403 host to screen for a polyethylene-binding mutant through a phage display assay. In further research, the selected mutant will be combined with enzymes in the natural alkane biodegradation pathway to form an in vitro process that degrades polyethylene.