SYNECHOCOCCUS SP. PCC 7002: A ROBUST AND VERSATILE CYANOBACTERIAL PLATFORM FOR BIOFUELS DEVELOPMENT
Open Access
- Author:
- Xu, Yu
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 12, 2010
- Committee Members:
- Donald Ashley Bryant, Dissertation Advisor/Co-Advisor
Donald Ashley Bryant, Committee Chair/Co-Chair
John H Golbeck, Committee Member
Joseph M Bollinger Jr., Committee Member
Stephan C Schuster, Committee Member
John Michael Regan, Committee Member - Keywords:
- carbohydrate
metabolic engineering
biofuel
cyanobacteria
hydrogen
gene expression
organic acid
co-culturing - Abstract:
- A cyanobacterial platform for biofuels or biofuel-related applications has been established in a marine cyanobacterium, Synechococcus sp. PCC 7002. To better understand H2-production mechanisms, the bidirectional [NiFe]-hydrogenase in Synechococcus sp. PCC 7002 was characterized with respect to its transcriptional and post-transcriptional regulation in response to several environmental cues, and its physiological roles in oxygenic photosynthetic conditions. To improve carbohydrate production, the glycogen biosynthesis pathway in Synechococcus sp. PCC 7002 was abolished by inactivating the genes encoding two glycogen synthases, GlgA1 and GlgA2, which led to accumulation and excretion of small sugars and osmoprotective carbohydrates (e. g. glucose, sucrose, glucosylglycerol), especially in osmotic stress conditions. Furthermore, a general expression system was developed to facilitate gene expression at any locus of the chromosome or endogenous plasmids in Synechococcus sp. PCC 7002. Particularly, the expression system pAQ1Ex, using the high-copy endogenous plasmid pAQ1, has been successfully constructed with a relatively strong, constitutive promoter PcpcBA for high-yield gene expression, or with an inducible, moderate strength promoter PnrtABCD for regulatable gene expression by changing the medium nitrogen source. These expression systems have greatly facilitated genetic and metabolic engineering studies in this cyanobacterium. For instance, overproduction of Synechococcus sp. PCC 7002 glbN gene encoding the hemoglobin helped to discover its protective functions against challenges from reactive nitrogen species (RNS) and reactive oxygen species (ROS); overexpression of genes encoding the Ralstonia eutropha NAD(P)+-reducing [NiFe]-hydrogenase led to the first successful overproduction of an active [NiFe]-hydrogenase and its maturation system in a heterologous cyanobacterial host. Further indications from hydrogenase overproduction studies suggest that reductant availability to hydrogenases is an important limiting factor for physiological H2 production. In addition, overproduction of Synechococcus sp. PCC 7002 lactate dehydrogenase (LdhA) resulted in high level organic acid production (lactate, acetate and formate) in a CO2-level-independent, continuous, and photoautotrophic manner.