A Synthetic Biological Engineering Approach to Secretion- Based Recovery of Polyhydroxyalkanoates and Other Cellular Products

• Main Author: Linton, Elisabeth
• Format: Others
• Published: DigitalCommons@USU 2010
• Subjects: BioBrick; Green Fluorescent Protein; NMR; Polyhyroxyalkanoates; Secretion; Synthetic Biology; Analytical Chemistry; Biomedical Engineering and Bioengineering; Molecular Biology
• Online Access: https://digitalcommons.usu.edu/etd/678; https://digitalcommons.usu.edu/cgi/viewcontent.cgi?article=1674&context=etd

The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion-based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, and PelB) that operate via type I and II secretory machinery. GFP translocation was investigated in parallel due to its relative ease of monitoring to gather information about the functionality of signal peptide sequences. The translocation of phasin was investigated because of its physical binding interaction with the PHA granule surface. Genetic fusion of phasin with targeting signal peptides creates a PHA-phasin-signal peptide complex that can then be potentially used for cellular export. An important design aspect of this investigation is that synthetic biological engineering principles and standardized technical formats BBF RFC 10 and BBF RFC 23 were applied for more efficient construction of genetic devices. As an additional part of this study, an 1H NMR-based PHA quantification method was developed to facilitate analysis of intracellular PHAs. Overall, this study demonstrated that the BioBrick model can be used to construct functional devices that promote secretion of cellular compounds. The information gathered from this work can be further optimized and applied to more complex cellular manufacturing systems.