Developing a Cas9-based tool to engineer native plasmids in Synechocystis sp. PCC 6803.

The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.

Diurnal Regulation of Cellular Processes in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Insights from Transcriptomic, Fluxomic, and Physiological Analyses.

UNLABELLED: Synechocystis sp. strain PCC 6803 is the most widely studied model cyanobacterium, with a well-developed omics level knowledgebase. Like the lifestyles of other cyanobacteria, that of Synechocystis PCC 6803 is tuned to diurnal changes in light intensity. In this study, we analyzed the expression patterns of all of the genes of this cyanobacterium over two consecutive diurnal periods. Using stringent criteria, we determined that the transcript levels of nearly 40% of the genes in Synechocystis PCC 6803 show robust diurnal oscillating behavior, with a majority of the transcripts being upregulated during the early light period. Such transcripts corresponded to a wide array of cellular processes, such as light harvesting, photosynthetic light and dark reactions, and central carbon metabolism. In contrast, transcripts of membrane transporters for transition metals involved in the photosynthetic electron transport chain (e.g., iron, manganese, and copper) were significantly upregulated during the late dark period. Thus, the pattern of global gene expression led to the development of two distinct transcriptional networks of coregulated oscillatory genes. These networks help describe how Synechocystis PCC 6803 regulates its metabolism toward the end of the dark period in anticipation of efficient photosynthesis during the early light period. Furthermore, in silico flux prediction of important cellular processes and experimental measurements of cellular ATP, NADP(H), and glycogen levels showed how this diurnal behavior influences its metabolic characteristics. In particular, NADPH/NADP(+) showed a strong correlation with the majority of the genes whose expression peaks in the light. We conclude that this ratio is a key endogenous determinant of the diurnal behavior of this cyanobacterium. IMPORTANCE: Cyanobacteria are photosynthetic microbes that use energy from sunlight and CO2 as feedstock. Certain cyanobacterial strains are amenable to facile genetic manipulation, thus enabling synthetic biology and metabolic engineering applications. Such strains are being developed as a chassis for the sustainable production of food, feed, and fuel. To this end, a holistic knowledge of cyanobacterial physiology and its correlation with gene expression patterns under the diurnal cycle is warranted. In this report, a genomewide transcriptional analysis of Synechocystis PCC 6803, the most widely studied model cyanobacterium, sheds light on the global coordination of cellular processes during diurnal periods. Furthermore, we found that, in addition to light, the redox level of NADP(H) is an important endogenous regulator of diurnal entrainment of Synechocystis PCC 6803.

Microbial production of isoprenoids enabled by synthetic biology.

Microorganisms transform inexpensive carbon sources into highly functionalized compounds without toxic by-product generation or significant energy consumption. By redesigning the natural biosynthetic pathways in an industrially suited host, microbial cell factories can produce complex compounds for a variety of industries. Isoprenoids include many medically important compounds such as antioxidants and anticancer and antimalarial drugs, all of which have been produced microbially. While a biosynthetic pathway could be simply transferred to the production host, the titers would become economically feasible when it is rationally designed, built, and optimized through synthetic biology tools. These tools have been implemented by a number of research groups, with new tools pledging further improvements in yields and expansion to new medically relevant compounds. This review focuses on the microbial production of isoprenoids for the health industry and the advancements though synthetic biology.