Daniel C. Ducat

Engineering cyanobacteria to generate high-value products.

Although many microorganisms have been used for the bioindustrial generation of valuable metabolites, the productive potential of cyanobacterial species has remained largely unexplored. Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements, tolerance of marginal agricultural environments, rapid genetics, and carbon-neutral applications that could be leveraged to address global climate change concerns. Here, we review recent research involving the engineering of cyanobacterial species for the production of valuable bioindustrial compounds, including natural cyanobacterial products (e.g. sugars and isoprene), biofuels (e.g. alcohols, alkanes and hydrogen), and other commodity chemicals. Biological and economic obstacles to scaled cyanobacterial production are highlighted, and methods for increasing cyanobacterial production efficiencies are discussed.

Rerouting Carbon Flux To Enhance Photosynthetic Productivity

ABSTRACT The bioindustrial production of fuels, chemicals, and therapeutics typically relies upon carbohydrate inputs derived from agricultural plants, resulting in the entanglement of food and chemical commodity markets. We demonstrate the efficient production of sucrose from a cyanobacterial species, Synechococcus elongatus, heterologously expressing a symporter of protons and sucrose (cscB). cscB-expressing cyanobacteria export sucrose irreversibly to concentrations of >10 mM without culture toxicity. Moreover, sucrose-exporting cyanobacteria exhibit increased biomass production rates relative to wild-type strains, accompanied by enhanced photosystem II activity, carbon fixation, and chlorophyll content. The genetic modification of sucrose biosynthesis pathways to minimize competing glucose- or sucrose-consuming reactions can further improve sucrose production, allowing the export of sucrose at rates of up to 36.1 mg liter−1 h illumination−1. This rate of production exceeds that of previous reports of targeted, photobiological production from microbes. Engineered S. elongatus produces sucrose in sufficient quantities (up to ∼80% of total biomass) such that it may be a viable alternative to sugar synthesis from terrestrial plants, including sugarcane.

Rewiring hydrogenase-dependent redox circuits in cyanobacteria

Hydrogenases catalyze the reversible reaction 2H+ + 2e-↔H2 with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner. To examine the possibility of increasing the photobiological production of hydrogen within cyanobacterial cultures, we expressed the [FeFe] hydrogenase, HydA, from Clostridium acetobutylicum in the non-nitrogen-fixing cyanobacterium Synechococcus elongatus sp. 7942. We demonstrate that the heterologously expressed hydrogenase is functional in vitro and in vivo, and that the in vivo hydrogenase activity is connected to the light-dependent reactions of the electron transport chain. Under anoxic conditions, HydA activity is capable of supporting light-dependent hydrogen evolution at a rate > 500-fold greater than that supported by the endogenous [NiFe] hydrogenase. Furthermore, HydA can support limited growth solely using H2 and light as the source of reducing equivalents under conditions where Photosystem II is inactivated. Finally, we demonstrate that the addition of exogenous ferredoxins can modulate redox flux in the hydrogenase-expressing strain, allowing for greater hydrogen yields and for dark fermentation of internal energy stores into hydrogen gas.

Insulation of a synthetic hydrogen metabolism circuit in bacteria

BackgroundThe engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued.ResultsHere we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds.ConclusionsThrough the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.

Functional role of oligomerization for bacterial and plant SWEET sugar transporter family

Significance SWEET sugar transporter homologs from bacteria were identified and named SemiSWEETs. They are small proteins with only three transmembrane domains (TMs); they are too small to create pores by themselves, but likely, they assemble multiple 3-TMs into a complex. SemiSWEETs are related to SWEETs, which play important roles in intercellular and interorgan sugar translocation in plants, and they are found in animals. SWEETs have fused two 3-TM units through a linker. However, SWEETs seem to be too small to transport sugars on their own. Here, we show that SWEET function requires assembly into oligomers, indicating that a pore requires at least an SWEET dimer. Eukaryotic sugar transporters of the MFS and SWEET superfamilies consist of 12 and 7 α-helical transmembrane domains (TMs), respectively. Structural analyses indicate that MFS transporters evolved from a series of tandem duplications of an ancestral 3-TM unit. SWEETs are heptahelical proteins carrying a tandem repeat of 3-TM separated by a single TM. Here, we show that prokaryotes have ancestral SWEET homologs with only 3-TM and that the Bradyrhizobium japonicum SemiSWEET1, like Arabidopsis SWEET11, mediates sucrose transport. Eukaryotic SWEETs most likely evolved by internal duplication of the 3-TM, suggesting that SemiSWEETs form oligomers to create a functional pore. However, it remains elusive whether the 7-TM SWEETs are the functional unit or require oligomerization to form a pore sufficiently large to allow for sucrose passage. Split ubiquitin yeast two-hybrid and split GFP assays indicate that Arabidopsis SWEETs homo- and heterooligomerize. We examined mutant SWEET variants for negative dominance to test if oligomerization is necessary for function. Mutation of the conserved Y57 or G58 in SWEET1 led to loss of activity. Coexpression of the defective mutants with functional A. thaliana SWEET1 inhibited glucose transport, indicating that homooligomerization is necessary for function. Collectively, these data imply that the basic unit of SWEETs, similar to MFS sugar transporters, is a 3-TM unit and that a functional transporter contains at least four such domains. We hypothesize that the functional unit of the SWEET family of transporters possesses a structure resembling the 12-TM MFS structure, however, with a parallel orientation of the 3-TM unit.

Improving Carbon Fixation Pathways

A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that novel pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials.

Regulation of Microtubule Assembly and Organization in Mitosis by the AAA+ ATPase Pontin

To identify novel proteins important for microtubule assembly in mitosis, we have used a centrosome-based complementation assay to enrich for proteins with mitotic functions. An RNA interference (RNAi)-based screen of these proteins allowed us to uncover 13 novel mitotic regulators. We carried out in-depth analyses of one of these proteins, Pontin, which is known to have several functions in interphase, including chromatin remodeling, DNA repair, and transcription. We show that reduction of Pontin by RNAi resulted in defects in spindle assembly in Drosophila S2 cells and in several mammalian tissue culture cell lines. Further characterization of Pontin in Xenopus egg extracts demonstrates that Pontin interacts with the gamma tubulin ring complex (gamma-TuRC). Because depletion of Pontin leads to defects in the assembly and organization of microtubule arrays in egg extracts, our studies suggest that Pontin has a mitosis-specific function in regulating microtubule assembly.

Engineering cyanobacteria as photosynthetic feedstock factories

Carbohydrate feedstocks are at the root of bioindustrial production and are needed in greater quantities than ever due to increased prioritization of renewable fuels with reduced carbon footprints. Cyanobacteria possess a number of features that make them well suited as an alternative feedstock crop in comparison to traditional terrestrial plant species. Recent advances in genetic engineering, as well as promising preliminary investigations of cyanobacteria in a number of distinct production regimes have illustrated the potential of these aquatic phototrophs as biosynthetic chassis. Further improvements in strain productivities and design, along with enhanced understanding of photosynthetic metabolism in cyanobacteria may pave the way to translate cyanobacterial theoretical potential into realized application.

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster.

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of -370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical S-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells.

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

BackgroundMicrobial consortia composed of autotrophic and heterotrophic species abound in nature, yet examples of synthetic communities with mixed metabolism are limited in the laboratory. We previously engineered a model cyanobacterium, Synechococcus elongatus PCC 7942, to secrete the bulk of the carbon it fixes as sucrose, a carbohydrate that can be utilized by many other microbes. Here, we tested the capability of sucrose-secreting cyanobacteria to act as a flexible platform for the construction of synthetic, light-driven consortia by pairing them with three disparate heterotrophs: Bacillus subtilis, Escherichia coli, or Saccharomyces cerevisiae. The comparison of these different co-culture dyads reveals general design principles for the construction of robust autotroph/heterotroph consortia.ResultsWe observed heterotrophic growth dependent upon cyanobacterial photosynthate in each co-culture pair. Furthermore, these synthetic consortia could be stabilized over the long-term (weeks to months) and both species could persist when challenged with specific perturbations. Stability and productivity of autotroph/heterotroph co-cultures was dependent on heterotroph sucrose utilization, as well as other species-independent interactions that we observed across all dyads. One destabilizing interaction we observed was that non-sucrose byproducts of oxygenic photosynthesis negatively impacted heterotroph growth. Conversely, inoculation of each heterotrophic species enhanced cyanobacterial growth in comparison to axenic cultures. Finally, these consortia can be flexibly programmed for photoproduction of target compounds and proteins; by changing the heterotroph in co-culture to specialized strains of B. subtilis or E. coli we demonstrate production of alpha-amylase and polyhydroxybutyrate, respectively.ConclusionsEnabled by the unprecedented flexibility of this consortia design, we uncover species-independent design principles that influence cyanobacteria/heterotroph consortia robustness. The modular nature of these communities and their unusual robustness exhibits promise as a platform for highly-versatile photoproduction strategies that capitalize on multi-species interactions and could be utilized as a tool for the study of nascent symbioses. Further consortia improvements via engineered interventions beyond those we show here (i.e., increased efficiency growing on sucrose) could improve these communities as production platforms.