Development of the Marine Cyanobacterium Synechococcus sp. PCC 7002 as a Scalable Low-Cost Photoautotrophic Protein Production Platform

Open Access
Author:
Curtis, Erik M
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Wayne Roger Curtis, Thesis Supervisor
  • Wayne Roger Curtis, Honors Advisor
  • Andrew Zydney, Faculty Reader
Keywords:
  • Cyanobacteria
  • Synechococcus
  • Cloning
  • CRISPR
  • Cas9
  • Protein Expression
  • Protein Production
Abstract:
This work describes the development of a novel protein expression platform which allows for low-capital, high-level expression of functional proteins in the marine cyanobacteria Synechococcus sp. PCC 7002. While significant research has focused on high-level expression of genes in cyanobacteria, the platform cannot effectively be scaled due to the need to maintain antibiotic selection pressure to prevent loss of transformed genes. In order to improve this expression platform, this work employs the CRISPR/Cas9 genome editing system to selectively cause double stranded breaks (DSBs) in untransformed copies of a native high copy number expression plasmid, thereby providing selection pressure against the native plasmid to facilitate its elimination. Cloning genes of interest and the CRISPR system into separate endogenous plasmids provides for a platform approach to generate genetically stable, high level expression strains of cyanobacteria. Once the native plasmid is eliminated, these strains can be used for large-scale protein production without the presence of antibiotic selection pressure. This platform can in turn be implemented as a value-added approach to production of biomass for conversion to hydrocarbon biofuels; as well as facilitate low-capital production of high-value proteins. This work details the development of a CRISPR/Cas9 complex capable of providing selection pressure against the native plasmid, as well as progress towards integrating this complex into a cyanobacterial cloning vector.