Light-optimized growth of cyanobacterial cultures: Growth phases and productivity of biomass and secreted molecules in light-limited batch growth.
ABSTRACT: Cyanobacteria are photosynthetic microorganisms whose metabolism can be modified through genetic engineering for production of a wide variety of molecules directly from CO2, light, and nutrients. Diverse molecules have been produced in small quantities by engineered cyanobacteria to demonstrate the feasibility of photosynthetic biorefineries. Consequently, there is interest in engineering these microorganisms to increase titer and productivity to meet industrial metrics. Unfortunately, differing experimental conditions and cultivation techniques confound comparisons of strains and metabolic engineering strategies. In this work, we discuss the factors governing photoautotrophic growth and demonstrate nutritionally replete conditions in which a model cyanobacterium can be grown to stationary phase with light as the sole limiting substrate. We introduce a mathematical framework for understanding the dynamics of growth and product secretion in light-limited cyanobacterial cultures. Using this framework, we demonstrate how cyanobacterial growth in differing experimental systems can be easily scaled by the volumetric photon delivery rate using the model organisms Synechococcus sp. strain PCC7002 and Synechococcus elongatus strain UTEX2973. We use this framework to predict scaled up growth and product secretion in 1L photobioreactors of two strains of Synechococcus PCC7002 engineered for production of l-lactate or L-lysine. The analytical framework developed in this work serves as a guide for future metabolic engineering studies of cyanobacteria to allow better comparison of experiments performed in different experimental systems and to further investigate the dynamics of growth and product secretion.
Project description:Many of the cyanobacterial species found in marine and saline environments have a gene encoding a putative nitrite transporter of the formate/nitrite transporter (FNT) family. The presumed function of the gene (designated nitM) was confirmed by functional expression of the gene from the coastal marine species Synechococcus sp. strain PCC7002 in the nitrite-transport-less mutant (NA4) of the freshwater cyanobacterium Synechococcus elongatus strain PCC7942. The NitM-mediated nitrite uptake showed an apparent Km (NO2-) of about 8 ?M and was not inhibited by nitrate, cyanate or formate. Of the nitM orthologs from the three oceanic cyanobacterial species, which are classified as ?-cyanobacteria on the basis of the occurrence of Type 1a RuBisCO, the one from Synechococcus sp. strain CC9605 conferred nitrite uptake activity on NA4, but those from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313 did not. A strongly conserved hydrophilic amino acid sequence was found at the C-termini of the deduced NitM sequences from ?-cyanobacteria, with a notable exception of the Synechococcus sp. strain CC9605 NitM protein, which entirely lacked the C-terminal amino acids. The C-terminal sequence was not conserved in the NitM proteins from ?-cyanobacteria carrying the Type 1b RuBisCO, including the one from Synechococcus sp. strain PCC7002. Expression of the truncated nitM genes from Synechococcus sp. strain CC9311 and Prochlorococcus marinus strain MIT9313, encoding the proteins lacking the conserved C-terminal region, conferred nitrite uptake activity on the NA4 mutant, indicating that the C-terminal region of ?-cyanobacterial NitM proteins inhibits the activity of the transporter.
Project description:As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO2-concentrating mechanism (CCM) to impose a high-CO2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. We showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 108 CFU.
Project description:Cyanobacteria possess a highly effective CO(2)-concentrating mechanism that elevates CO(2) concentrations around the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase). This CO(2)-concentrating mechanism incorporates light-dependent, active uptake systems for CO(2) and HCO(-)(3). Through mutant studies in a coastal marine cyanobacterium, Synechococcus sp. strain PCC7002, we identified bicA as a gene that encodes a class of HCO(-)(3) transporter with relatively low transport affinity, but high flux rate. BicA is widely represented in genomes of oceanic cyanobacteria and belongs to a large family of eukaryotic and prokaryotic transporters presently annotated as sulfate transporters or permeases in many bacteria (SulP family). Further gain-of-function experiments in the freshwater cyanobacterium Synechococcus PCC7942 revealed that bicA expression alone is sufficient to confer a Na(+)-dependent, HCO(3)(-) uptake activity. We identified and characterized three cyanobacterial BicA transporters in this manner, including one from the ecologically important oceanic strain, Synechococcus WH8102. This study presents functional data concerning prokaryotic members of the SulP transporter family and represents a previously uncharacterized transport function for the family. The discovery of BicA has significant implications for understanding the important contribution of oceanic strains of cyanobacteria to global CO(2) sequestration processes.
Project description:Many efforts have focused on the adsorption of metals from contaminated water by microbes. <i>Synechococcus</i> PCC7002, a major marine cyanobacteria, is widely applied to remove metals from the ocean's photic zone. However, its ability to adsorb cesium (Cs) nuclides has received little attention. In this study, the biosorption behavior of Cs(I) from ultrapure distilled water by living <i>Synechococcus</i> PCC7002 was investigated based on kinetic and isotherm studies, and the biosorption mechanism was characterized by Fourier-transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectrometry, and three-dimensional excitation emission matrix fluorescence spectroscopy. <i>Synechococcus</i> PCC7002 showed extremely high tolerance to Cs ions and its minimal inhibitory concentration was 8.6 g/L. Extracellular polymeric substances (EPS) in <i>Synechococcus</i> PCC7002 played a vital role in this tolerance. The biosorption of Cs by <i>Synechococcus</i> PCC7002 conformed to a Freundlich-type isotherm model and pseudo-second-order kinetics. The binding of Cs(I) was primarily attributed to the extracellular proteins in EPS, with the amino, hydroxyl, and phosphate groups on the cell walls contributing to Cs adsorption. The biosorption of Cs involved two mechanisms: Passive adsorption on the cell surface at low Cs concentrations and active intracellular adsorption at high Cs concentrations. The results demonstrate that the behavior and mechanism of Cs adsorption by <i>Synechococcus</i> PCC7002 differ based on the Cs ions concentration.
Project description:Synechococcus elongatus strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium, Synechococcus elongatus PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.
Project description:The photosynthetic capabilities of cyanobacteria make them interesting candidates for industrial bioproduction. One obstacle to large-scale implementation of cyanobacteria is their limited growth rates as compared to industrial mainstays. Synechococcus UTEX 2973, a strain closely related to Synechococcus PCC 7942, was recently identified as having the fastest measured growth rate among cyanobacteria. To facilitate the development of 2973 as a model organism we developed in this study the genome-scale metabolic model iSyu683. Experimental data were used to define CO2 uptake rates as well as the biomass compositions for each strain. The inclusion of constraints based on experimental measurements of CO2 uptake resulted in a ratio of the growth rates of Synechococcus 2973 to Synechococcus 7942 of 2.03, which nearly recapitulates the in vivo growth rate ratio of 2.13. This identified the difference in carbon uptake rate as the main factor contributing to the divergent growth rates. Additionally four SNPs were identified as possible contributors to modified kinetic parameters of metabolic enzymes and candidates for further study. Comparisons against more established cyanobacterial strains identified a number of differences between the strains along with a correlation between the number of cytochrome c oxidase operons and heterotrophic or diazotrophic capabilities.
Project description:Eutrophication and deoxygenation possibly occur in coastal waters due to excessive nutrients from agricultural and aquacultural activities, leading to sulfide accumulation. Cyanobacteria, as photosynthetic prokaryotes, play significant roles in carbon fixation in the ocean. Although some cyanobacteria can use sulfide as the electron donor for photosynthesis under anaerobic conditions, little is known on how they interact with sulfide under aerobic conditions. In this study, we report that Synechococcus sp. strain PCC7002 (PCC7002), harboring an sqr gene encoding sulfide:quinone oxidoreductase (SQR), oxidized self-produced sulfide to S0, present as persulfide and polysulfide in the cell. The Δsqr mutant contained less cellular S0 and had increased expression of key genes involved in photosynthesis, but it was less competitive than the wild type in cocultures. Further, PCC7002 with SQR and persulfide dioxygenase (PDO) oxidized exogenous sulfide to tolerate high sulfide levels. Thus, SQR offers some benefits to cyanobacteria even under aerobic conditions, explaining the common presence of SQR in cyanobacteria.IMPORTANCE Cyanobacteria are a major force for primary production via oxygenic photosynthesis in the ocean. A marine cyanobacterium, PCC7002, is actively involved in sulfide metabolism. It uses SQR to detoxify exogenous sulfide, enabling it to survive better than its Δsqr mutant in sulfide-rich environments. PCC7002 also uses SQR to oxidize endogenously generated sulfide to S0, which is required for the proper expression of key genes involved in photosynthesis. Thus, SQR has at least two physiological functions in PCC7002. The observation provides a new perspective for the interplays of C and S cycles.
Project description:Cyanobacterial HCO3(-) transporters BCT1, SbtA and BicA are important components of cyanobacterial CO2-concentration mechanisms. They also show potential in applications aimed at improving photosynthetic rates and yield when expressed in the chloroplasts of C3 crop species. The present study investigated the feasibility of using Escherichia coli to assess function of a range of SbtA and BicA transporters in a heterologous expression system, ultimately for selection of transporters suitable for chloroplast expression. Here, we demonstrate that six ?-forms of SbtA are active in E. coli, although other tested bicarbonate transporters were inactive. The sbtA clones were derived from Synechococcus sp. WH5701, Cyanobium sp. PCC7001, Cyanobium sp. PCC6307, Synechococcus elongatus PCC7942, Synechocystis sp. PCC6803, and Synechococcus sp. PCC7002. The six SbtA homologs varied in bicarbonate uptake kinetics and sodium requirements in E. coli. In particular, SbtA from PCC7001 showed the lowest uptake affinity and highest flux rate and was capable of increasing the internal inorganic carbon pool by more than 8 mM relative to controls lacking transporters. Importantly, we were able to show that the SbtB protein (encoded by a companion gene near sbtA) binds to SbtA and suppresses bicarbonate uptake function of SbtA in E. coli, suggesting a role in post-translational regulation of SbtA, possibly as an inhibitor in the dark. This study established E. coli as a heterologous expression and analysis system for HCO3(-) transporters from cyanobacteria, and identified several SbtA transporters as useful for expression in the chloroplast inner envelope membranes of higher plants.
Project description:Cyanobacteria are an integral part of Earth's biogeochemical cycles and a promising resource for the synthesis of renewable bioproducts from atmospheric CO<sub>2</sub> Growth and metabolism of cyanobacteria are inherently tied to the diurnal rhythm of light availability. As yet, however, insight into the stoichiometric and energetic constraints of cyanobacterial diurnal growth is limited. Here, we develop a computational framework to investigate the optimal allocation of cellular resources during diurnal phototrophic growth using a genome-scale metabolic reconstruction of the cyanobacterium <i>Synechococcus elongatus</i> PCC 7942. We formulate phototrophic growth as an autocatalytic process and solve the resulting time-dependent resource allocation problem using constraint-based analysis. Based on a narrow and well-defined set of parameters, our approach results in an ab initio prediction of growth properties over a full diurnal cycle. The computational model allows us to study the optimality of metabolite partitioning during diurnal growth. The cyclic pattern of glycogen accumulation, an emergent property of the model, has timing characteristics that are in qualitative agreement with experimental findings. The approach presented here provides insight into the time-dependent resource allocation problem of phototrophic diurnal growth and may serve as a general framework to assess the optimality of metabolic strategies that evolved in phototrophic organisms under diurnal conditions.
Project description:Cyanobacteria are serving as promising microbial platforms for development of photosynthetic cell factories. For enhancing the economic competitiveness of the photosynthetic biomanufacturing technology, comprehensive improvements on industrial properties of the cyanobacteria chassis cells and engineered strains are required. Cellular morphology engineering is an up-and-coming strategy for development of microbial cell factories fitting the requirements of industrial application. In this work, we performed systematic evaluation of potential genes for cyanobacterial cellular morphology engineering. Twelve candidate genes participating in cell morphogenesis of an important model cyanobacteria strain, Synechococcus elongatus PCC7942, were knocked out/down and overexpressed, respectively, and the influences on cell sizes and cell shapes were imaged and calculated. Targeting the selected genes with potentials for cellular morphology engineering, the controllable cell lengthening machinery was also explored based on the application of sRNA approaches. The findings in this work not only provided many new targets for cellular morphology engineering in cyanobacteria, but also helped to further understand the cell division process and cell elongation process of Synechococcus elongatus PCC7942.