Expanding the Biotechnological Potential of the Model Cyanobacterium Synechococcus sp. strain PCC 7002
Open Access
- Author:
- Perez, Adam Alberto
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 03, 2016
- Committee Members:
- Donald Ashley Bryant, Dissertation Advisor/Co-Advisor
Donald Ashley Bryant, Committee Chair/Co-Chair
John H Golbeck, Committee Member
Ming Tien, Committee Member
Manish Kumar, Outside Member - Keywords:
- Cyanobacteria
Photosynthesis
Biotechnology
Photosystem I - Abstract:
- The marine cyanobacterium Synechococcus sp. PCC 7002 has been extensively developed as a platform for biotechnological applications, with much emphasis in biofuel-related research. A significant limitation to this strain is its obligate requirement for exogenous vitamin B12 (cobalamin). This study demonstrated that the limitation in methionine biosynthesis occurs because cobalamin is a coenzyme for conversion of L-homocysteine to L-methionine via the methionine synthase enzyme MetH. Complementation of Synechococcus sp. PCC 7002 with a cobalamin-independent methionine synthase isozyme, MetE, from Synechococcus sp. PCC 73109 was eliminated the cobalamin auxotrophy. This new strain will enable large-scale growth of Synechococcus sp. PCC 7002 without the addition of cobalamin, which will reduce the cost of cultivation. Additionally, the MetE enzyme can be employed as a selective marker by auxotrophic complementation that is useful as a non-antibiotic selection marker. The leader sequence of MetE contains a putative cobalamin riboswitch that was utilized to develop a cobalamin-repressible system as an addition to the expanding genetic toolbox of Synechococcus sp. PCC 7002. Since this organism does not produce the essential cobalamin coenzyme de novo, it must be taken up from an exogenous source. An active transport ATP-Binding Cassette (ABC) transporter system for cobalamin uptake was identified in silico and verified in vivo by knock-out mutagenesis. The MetE riboswitch fused to the yellow fluorescent protein (YFP) protein served as a fluorescence reporter to assess intracellular uptake of cobalamin in the ABC transporter deletion mutants. This work corrected a previous misannotation of the genome, in which the genes for cobalamin uptake were previously assigned to uptake of an iron siderophore.. In addition, it was discovered that this operon is under the control of the sole cobalamin riboswitch in Synechococcus sp. PCC 7002 To expand the genetic toolbox for industrial application of Synechococcus sp. PCC 7002, a Zn++-inducible expression system was developed utilizing the methallothionein (smtA) promoter of Synechococcus elongatus PCC 7942 and co-expressing its native repressor StmB. When combined with a zurA mutant background in Synechococcus sp. PCC 7002, this system provides a tightly regulated induction system that utilizes M concentrations of Zn++ as a low-cost inducer in comparison to more traditional agents such as isopropyl -D-thiogalactopyranoside (IPTG) utilized in the lac operon. These tools and strains were designed to assist in the genetic manipulation of Synechococcus sp. PCC 7002 and further increase its value as a versatile cyanobacterial model. This study also focused on in vivo implementation of photosystem I (PS I) as a light-driven module for activating exogenous redox-active catalysts (e.g., hydrogenase for H2 production). A strain capable of producing PS I complexes with a variant stromal PsaC subunit poised to tether thiolated compounds was constructed. The strain harboring the variant PS I complexes was still capable of photoautotrophic growth, despite the observation that a novel iron-sulfur cluster resides in the FB site of PsaC, and acts as the terminal electron acceptor in the PS I electron transfer chain. Spectroscopic analyses that the PsaCC14G assembles with a functional [3Fe-4S] cluster instead of the canonical [4Fe-4S] cluster found normally associated with functional PS I.