Synthetic biology of cyanobacteria: unique challenges and opportunities

Abstract

Photosynthetic organisms, and especially cyanobacteria, hold great promise as sources of renewably-produced fuels, bulk and specialty chemicals, and nutritional products. Synthetic biology tools can help unlock cyanobacteria's potential for these functions, but unfortunately tool development for these organisms has lagged behind that for S. cerevisiae and E. coli. While these organisms may in many cases be more difficult to work with as "chassis" strains for synthetic biology than certain heterotrophs, the unique advantages of autotrophs in biotechnology applications as well as the scientific importance of improved understanding of photosynthesis warrant the development of these systems into something akin to a "green E. coli." In this review, we highlight unique challenges and opportunities for development of synthetic biology approaches in cyanobacteria. We review classical and recently developed methods for constructing targeted mutants in various cyanobacterial strains, and offer perspective on what genetic tools might most greatly expand the ability to engineer new functions in such strains. Similarly, we review what genetic parts are most needed for the development of cyanobacterial synthetic biology. Finally, we highlight recent methods to construct genome-scale models of cyanobacterial metabolism and to use those models to measure properties of autotrophic metabolism. Throughout this paper, we discuss some of the unique challenges of a diurnal, autotrophic lifestyle along with how the development of synthetic biology and biotechnology in cyanobacteria must fit within those constraints.

Keywords: biofuel; cyanobacteria; flux balance analysis; metabolic flux analysis; synthetic biology; systems biology.

Figure 1

Different methods for constructing cyanobacterial mutants. (A) shows the traditional method using double homologous recombination to insert a suicide vector into the genome at a neutral site (NS, gold) with upstream (US, orange) and downstream (DS, magenta) flanking regions in the vector. The insert contains an arbitrary sequence of interest (ATGCATG, green) and a selectable marker (SM, blue). (B) shows two methods of creating markerless mutants, either by selection-counterselection or by using a recombinase system such as FLP/FRT, The counter-selection method's first step is the same as for the method in panel a, except that the insert also contains a counter-selectable marker (CSM, purple) such as sacB. A second transformation is performed to create a markerless mutant. Alternatively, the insert can contain recombinase recognition sites (RRS, gray) that are controlled by an inducible recombinase at a second (or the same) site in the genome. While it erases the selectable marker, this method does leave a scar sequence behind. (C) shows genetic modification in trans via expression plasmids.

Figure 2

DNA assembly methods. (A) Traditionally in cyanobacterial synthetic biology, plasmids are assembled in vitro and then propagated in E. coli before being transformed into cyanobacteria. (B) More recently, methods have been developed for in vitro assembly and direct transformation via fusion PCR. (C) Another recent method has been developed for in vivo plasmid assembly via homologous recombination in yeast which may also be applicable in certain cyanobacterial strains.

Figure 3

Using fluxomics and genome scale models to link genotype to metabolic phenotype. From an annotated genome sequence, a stoichiometric model of metabolism can be constructed. That model can be solved via either prediction of an optimal flux phenotype (FBA) or measurement of actual flux phenotype (¹³C-MFA). These results can help suggest modifications for altering the phenotype of the cell in a desired manner. In this way, a synthetic biologist can design new strains, build them using genetic modification methods, and test their phenotypes before designing new modifications in an iterative fashion.