Cyanobacterial Cell Factories Research Articles

Perspectives of cyanobacterial cell factories.

Cyanobacteria are prokaryotic photosynthetic microorganisms that can generate, in addition to biomass, useful chemicals and proteins/enzymes, essentially from sunlight, carbon dioxide, and water. Selected aspects of cyanobacterial production (isoprenoids and high-value proteins) and scale-up methods suitable for product generation and downstream processing are addressed in this review. The work focuses on the challenge and promise of specialty chemicals and proteins production, with isoprenoid products and biopharma proteins as study cases, and the challenges encountered in the expression of recombinant proteins/enzymes, which underline the essence of synthetic biology with these microorganisms. Progress and the current state-of-the-art in these targeted topics are emphasized.

Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing

By directly converting solar energy and carbon dioxide into biobased products, cyanobacteria are promising chassis for photosynthetic biosynthesis. To make cyanobacterial photosynthetic biosynthesis technology economically feasible on industrial scales, exploring and engineering cyanobacterial chassis and cell factories with fast growth rates and carbon fixation activities facing environmental stresses are of great significance. To simplify and accelerate the screening for fast-growing cyanobacteria strains, a method called Individual Cyanobacteria Vitality Tests and Screening (iCyanVS) was established. We show that the 13C incorporation ratio of carotenoids can be used to measure differences in cell growth and carbon fixation rates in individual cyanobacterial cells of distinct genotypes that differ in growth rates in bulk cultivations, thus greatly accelerating the process screening for fastest-growing cells. The feasibility of this approach is further demonstrated by phenotypically and then genotypically identifying individual cyanobacterial cells with higher salt tolerance from an artificial mutant library via Raman-activated gravity-driven encapsulation and sequencing. Therefore, this method should find broad applications in growth rate or carbon intake rate based screening of cyanobacteria and other photosynthetic cell factories.

Engineering Cyanobacterial Cell Factories for Photosynthetic Production of Fructose.

Fructose is an important monosaccharide product widely applied in the food, medicine, and chemical industries. Currently, fructose is mainly manufactured with plant biomass-sourced polysaccharides through multiple steps of digestion, conversion, separation, and purification. The development of cyanobacterial metabolic engineering provides an attractive alternative route for the one-step direct production of fructose utilizing carbon dioxide and solar energy. In this work, we developed a paradigm for engineering cyanobacterial chassis cells into efficient cell factories for the photosynthetic production of fructose. In a representative cyanobacterial strain, Synechococcus elongatus PCC 7942, knockout of fructokinase effectively activated the synthesis and secretion of fructose in hypersaline conditions, independent of any heterologous transporters. The native sucrose synthesis pathway was identified as playing a primary role in fructose synthesis. Through combinatory optimizations on the levels of metabolism, physiology, and cultivation, the fructose yield of the Synechococcus cell factories was stepwise improved to 3.9 g/L. Such a paradigm was also adopted to engineer another Synechococcus strain, the marine species Synechococcus sp. PCC 7002, and facilitated an even higher fructose yield of over 6 g/L. Finally, the fructose synthesized and secreted by the cyanobacterial photosynthetic cell factories was successfully extracted and prepared from the culture broth in the form of products with 86% purity through multistep separation-purification operations. This work demonstrated a paradigm for systematically engineering cyanobacteria for photosynthetic production of desired metabolites, and it also confirmed the feasibility and potential of cyanobacterial photosynthetic biomanufacturing as a simple and efficient route for fructose production.

Conversion of carbon dioxide into valencene and other sesquiterpenes with metabolic engineered Synechocystis sp. PCC 6803 cell factories

AbstractValencene is a natural sesquiterpene with desirable bioactivity and aroma, making it a valuable ingredient in the food and cosmetics industries. Traditionally, valencene was extracted from the citrus fruits, and its applications were restricted by the low concentrations in natural sources and high costs for extraction. Photosynthetic biomanufacturing represents a promising route for efficient and stable production of valencene, while cyanobacteria have been considered one of the most promising platforms regarding biotechnological routes for the direct conversion of CO2. In this work, we engineered Synechocystis sp. PCC 6803 to synthesize valencene. By introducing a heterologous valencene synthase and modifying the native MEP pathway, we obtained an efficient cyanobacterial cell factory that produced 154 mg/L valencene during a semi‐continual cultivation, with an average productivity of 4.3 mg/L/day, and the cell factory exhibited robust growth and production in non‐sterilized conditions. We also achieved the production of other sesquiterpenes including bisabolene, amorpha‐4,11‐diene, farnesene, and nerolidol by engineered cyanobacteria with enhanced MEP pathway flux, showing promising potentials as a universal chassis.

Light-Driven Synthetic Biology: Progress in Research and Industrialization of Cyanobacterial Cell Factory.

Light-driven synthetic biology refers to an autotrophic microorganisms-based research platform that remodels microbial metabolism through synthetic biology and directly converts light energy into bio-based chemicals. This technology can help achieve the goal of carbon neutrality while promoting green production. Cyanobacteria are photosynthetic microorganisms that use light and CO2 for growth and production. They thus possess unique advantages as “autotrophic cell factories”. Various fuels and chemicals have been synthesized by cyanobacteria, indicating their important roles in research and industrial application. This review summarized the progresses and remaining challenges in light-driven cyanobacterial cell factory. The choice of chassis cells, strategies used in metabolic engineering, and the methods for high-value CO2 utilization will be discussed.

One cell-two wells bio-refinery: Demonstrating cyanobacterial chassis for co-production of heterologous and natural hydrocarbons

In order to improve the potential of cyanobacterial cell factories, Synechococcus sp. PCC7002 was engineered as ‘one cell-two wells bio-refinery’, for ethylene (‘heterologous’ hydrocarbon) and carotenoids (‘natural’ metabolites) production, and demonstrating its outdoor performance. Although the cultures showed better production outdoor, they experienced multiple collapses during scale-up. Hence, flux balance analysis was performed which predicted higher ethylene production with increase in carbon input under outdoor light conditions. Furthermore, FBA predicted that ethylene production will not increase beyond a threshold carbon input flux, owing to limitations on ribulose-1,5-bisphosphate regeneration. Hence, a bicarbonate-supplementation strategy was devised. Cultures grown outdoor at optimal bicarbonate concentration (20 g/L) resulted in improved growth (0.141/h) and ethylene productivity (1.88 mL/L.h) for > 10 days, with enhanced carotenoid titres (40.4 mg/L). In a 100 L air-lift photo-bioreactor; cultures exhibited efficient ethylene (2.464 mL/L.h) and biomass (0.3 g/L.d) productivities, and carotenoids titres (64.4 mg/L), establishing a significant step towards commercialization.

The transcriptional regulator RbcR controls ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes in the cyanobacterium Synechocystis sp. PCC 6803.

Oxygenic photosynthesis evolved in cyanobacteria, primary producers of striking ecological importance. Like plants, cyanobacteria use the Calvin-Benson-Bassham cycle for CO2 fixation, fuelled by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). In a competitive reaction this enzyme also fixes O2 which makes it rather ineffective. To mitigate this problem, cyanobacteria evolved a CO2 concentrating mechanism (CCM) to pool CO2 in the vicinity of RuBisCO. However, the regulation of these carbon (C) assimilatory systems is understood only partially. Using the model Synechocystis sp. PCC 6803 we characterized an essential LysR-type transcriptional regulator encoded by gene sll0998. Transcript profiling of a knockdown mutant revealed diminished expression of several genes involved in C acquisition, including rbcLXS, sbtA and ccmKL encoding RuBisCO and parts of the CCM, respectively. We demonstrate that the Sll0998 protein binds the rbcL promoter and acts as a RuBisCO regulator (RbcR). We propose ATTA(G/A)-N5 -(C/T)TAAT as the binding motif consensus. Our data validate RbcR as a regulator of inorganic C assimilation and define the regulon controlled by it. Biological CO2 fixation can sustain efforts to reduce its atmospheric concentrations and is fundamental for the light-driven production of chemicals directly from CO2 . Information about the involved regulatory and physiological processes is crucial to engineer cyanobacterial cell factories.

Engineering the glycogen metabolism in cyanobacterial photosynthetic cell factories: a review

Cyanobacteria are important photosynthetic autotrophic microorganisms and are considered as one of the most promising microbial chassises for photosynthetic cell factories. Glycogen is the most important natural carbon sink of cyanobacteria, playing important roles in regulating its intracellular carbon distributions. In order to optimize the performances of cyanobacterial photosynthetic cell factories and drive more photosynthetic carbon flow toward the synthesis of desired metabolites, many strategies and approaches have been developed to manipulate the glycogen metabolism in cyanobacteria. However, the disturbances on glycogen metabolism usually cause complex effects on the physiology and metabolism of cyanobacterial cells. Moreover, the effects on synthesis efficiencies of different photosynthetic cell factories usually differ. In this manuscript, we summarized the recent progress on engineering cyanobacterial glycogen metabolism, analyzed and compared the physiological and metabolism effects caused by engineering glycogen metabolism in different cyanobacteria species, and prospected the future trends of this strategy on optimizing cyanobacterial photosynthetic cell factories.

Correction: Direct carbon capture for the production of high-performance biodegradable plastics by cyanobacterial cell factories

Correction for ‘Direct carbon capture for the production of high-performance biodegradable plastics by cyanobacterial cell factories’ by Chunlin Tan et al., Green Chem., 2022, https://doi.org/10.1039/d1gc04188f.

Direct carbon capture for the production of high-performance biodegradable plastics by cyanobacterial cell factories

The engineered cyanobacterial cell factory was used to produce biodegradable polylactic acid directly from CO2.

The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories.

Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis, a solar-driven process which allows them to obtain electrons from water to reduce and finally assimilate carbon dioxide. Consequently, they are in the spotlight of biotechnology as photoautotrophic cell factories to generate a large variety of chemicals and biofuels in a sustainable way. Recent progress in synthetic biology has enlarged the molecular toolset to genetically engineer the metabolism of cyanobacteria, mainly targeting common model strains, such as Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, Synechococcus sp. PCC 7002, or Anabaena sp. PCC 7120. Nevertheless, the accessibility and flexibility of engineering cyanobacteria is still somewhat limited and less predictable compared to other biotechnologically employed microorganisms.This chapter gives a broad overview of currently available methods for the genetic modification of cyanobacterial model strains as well as more recently discovered and promising species, such as Synechococcus elongatus PCC 11801. It comprises approaches based on homologous recombination, replicative broad-host-range or strain-specific plasmids, CRISPR/Cas, as well as markerless selection. Furthermore, common and newly introduced molecular tools for gene expression regulation are presented, comprising promoters, regulatory RNAs, genetic insulators like transcription terminators, ribosome binding sites, CRISPR interference, and the utilization of heterologous RNA polymerases. Additionally, potential DNA assembly strategies, like modular cloning, are described. Finally, considerations about post-translational control via protein degradation tags and heterologous proteases, as well as small proteins working as enzyme effectors are briefly discussed.

Potential of Producing Flavonoids Using Cyanobacteria As a Sustainable Chassis.

Numerous plant secondary metabolites have remarkable impacts on both food supplements and pharmaceuticals for human health improvement. However, higher plants can only generate small amounts of these chemicals with specific temporal and spatial arrangements, which are unable to satisfy the expanding market demands. Cyanobacteria can directly utilize CO2, light energy, and inorganic nutrients to synthesize versatile plant-specific photosynthetic intermediates and organic compounds in large-scale photobioreactors with outstanding economic merit. Thus, they have been rapidly developed as a "green" chassis for the synthesis of bioproducts. Flavonoids, chemical compounds based on aromatic amino acids, are considered to be indispensable components in a variety of nutraceutical, pharmaceutical, and cosmetic applications. In contrast to heterotrophic metabolic engineering pioneers, such as yeast and Escherichia coli, information about the biosynthesis flavonoids and their derivatives is less comprehensive than that of their photosynthetic counterparts. Here, we review both benefits and challenges to promote cyanobacterial cell factories for flavonoid biosynthesis. With increasing concerns about global environmental issues and food security, we are confident that energy self-supporting cyanobacteria will attract increasing attention for the generation of different kinds of bioproducts. We hope that the work presented here will serve as an index and encourage more scientists to join in the relevant research area.

Rapidly Improving High Light and High Temperature Tolerances of Cyanobacterial Cell Factories Through the Convenient Introduction of an AtpA-C252F Mutation.

Photosynthetic biomanufacturing is a promising route for green production of biofuels and biochemicals utilizing carbon dioxide and solar energy. Cyanobacteria are important microbial platforms for constructing photosynthetic cell factories. Toward scaled outdoor cultivations in the future, high light and high temperature tolerances of cyanobacterial chassis strains and cell factories would be determinant properties to be optimized. We proposed a convenient strategy for rapidly improving high light and high temperature tolerances of an important cyanobacterial chassis Synechococcus elongatus PCC 7942 and the derived cell factories. Through introduction and isolation of an AtpA-C252F mutation, PCC 7942 mutants with improved high light and high temperature tolerances could be obtained in only 4 days with an antibiotics-free mode. Adopting this strategy, cellular robustness and sucrose synthesizing capacities of a PCC 7942 cell factory were successfully improved.

Computational Analysis of Dynamic Light Exposure of Unicellular Algal Cells in a Flat-Panel Photobioreactor to Support Light-Induced CO2 Bioprocess Development.

Cyanobacterial cell factories trace a vibrant pathway to climate change neutrality and sustainable development owing to their ability to turn carbon dioxide-rich waste into a broad portfolio of renewable compounds, which are deemed valuable in green chemistry cross-sectorial applications. Cell factory design requires to define the optimal operational and cultivation conditions. The paramount parameter in biomass cultivation in photobioreactors is the light intensity since it impacts cellular physiology and productivity. Our modeling framework provides a basis for the predictive control of light-limited, light-saturated, and light-inhibited growth of the Synechocystis sp. PCC 6803 model organism in a flat-panel photobioreactor. The model here presented couples computational fluid dynamics, light transmission, kinetic modeling, and the reconstruction of single cell trajectories in differently irradiated areas of the photobioreactor to relate key physiological parameters to the multi-faceted processes occurring in the cultivation environment. Furthermore, our analysis highlights the need for properly constraining the model with decisive qualitative and quantitative data related to light calibration and light measurements both at the inlet and outlet of the photobioreactor in order to boost the accuracy and extrapolation capabilities of the model.

Advances of polymer-monomer production by cyanobacterial cell factory

Cyanobacteria is one of the promising microbial chassis in synthetic biology, which serves as a typical host for light-driven production. With the gradual depletion of fossil resources and intensification of global warming, the research on cyanobacterial cell factory using CO2 as carbon resource is ushering in a new wave. For a long time, research focus on cyanobacterial cell factory has mainly been the production of energy products, such as liquid fuels and hydrogen. One of the critical bottlenecks occurring in cyanobacterial cell factory is the poor economic performance, which is mainly caused by the inherent inefficiency of cyanobacteria. The problem is particularly prominent for these extremely cost-sensitive energy products. As an indispensable basis for modern industry, polymer monomers belong to the bulk chemicals with high added value. Therefore, increasing attention has been focused on polymer monomers which are superior in overcoming the economic barrier in commercialization of cyanobacterial cell factories. Here, we systematically review the progress on the production of polymer monomers using cyanobacteria, including the strategies for improving production, and the related technologies for the application of this important microbial cell factory. Finally, we summarize several issues in cyanobacterial synthetic biology and proposed future developing trends in this field.

Absence of KpsM (Slr0977) Impairs the Secretion of Extracellular Polymeric Substances (EPS) and Impacts Carbon Fluxes in Synechocystis sp. PCC 6803.

Many cyanobacteria produce extracellular polymeric substances (EPS), composed mainly of heteropolysaccharides, that play a variety of physiological roles, being crucial for cell protection, motility, and biofilm formation. However, due to their complexity, the EPS biosynthetic pathways as well as their assembly and export mechanisms are still far from being fully understood. Here, we show that the absence of a putative EPS-related protein, KpsM (Slr0977), has a pleiotropic effect on Synechocystis sp. strain PCC 6803 physiology, with a strong impact on the export of EPS and carbon fluxes. The kpsM mutant exhibits a significant reduction of released polysaccharides and a smaller decrease of capsular polysaccharides, but it accumulates more polyhydroxybutyrate (PHB) than the wild type. In addition, this strain shows a light/cell density-dependent clumping phenotype and exhibits an altered protein secretion capacity. Furthermore, the most important structural component of pili, the protein PilA, was found to have a modified glycosylation pattern in the mutant compared to the wild type. Proteomic and transcriptomic analyses revealed significant changes in the mechanisms of energy production and conversion, namely, photosynthesis, oxidative phosphorylation, and carbon metabolism, in response to the inactivation of slr0977 Overall, this work shows for the first time that cells with impaired EPS secretion undergo transcriptomic and proteomic adjustments, highlighting the importance of EPS as a major carbon sink in cyanobacteria. The accumulation of PHB in cells of the mutant, without affecting significantly its fitness/growth rate, points to its possible use as a chassis for the production of compounds of interest.IMPORTANCE Most cyanobacteria produce extracellular polymeric substances (EPS) that fulfill different biological roles depending on the strain/environmental conditions. The interest in the cyanobacterial EPS synthesis/export pathways has been increasing, not only to optimize EPS production but also to efficiently redirect carbon flux toward the production of other compounds, allowing the implementation of industrial systems based on cyanobacterial cell factories. Here, we show that a Synechocystis kpsM (slr0977) mutant secretes less EPS than the wild type, accumulating more carbon intracellularly, as polyhydroxybutyrate. Further characterization showed a light/cell density-dependent clumping phenotype, altered protein secretion, and modified glycosylation of PilA. The proteome and transcriptome of the mutant revealed significant changes, namely, in photosynthesis and carbon metabolism. Altogether, this work provides a comprehensive overview of the impact of kpsM disruption on Synechocystis physiology, highlighting the importance of EPS as a carbon sink and showing how cells adapt when their secretion is impaired, and the redirection of the carbon fluxes.

Deciphering and engineering high-light tolerant cyanobacteria for efficient photosynthetic cell factories

Development and utilization of “liquid sunshine” could be one of key solutions to deal with the issues of fossil fuel depletion and increasing carbon dioxide. Cyanobacteria are the only prokaryotes capable of performing oxygenic photosynthesis, and their activity accounts for ~25% of the total carbon fixation on earth. More importantly, besides their traditional roles as primary producers, cyanobacteria could be modified as “photosynthetic cell factories” to produce renewable fuels and chemicals directly from CO 2 driven by solar energy, with the aid of cutting-edging synthetic biology technology. Towards their large-scale biotechnological application in the future, many challenges still need to be properly addressed, among which is cyanobacterial cell factories inevitably suffer from high light (HL) stress during large-scale outdoor cultivation, resulting in photodamage and even cell death, limiting their productivity. In this review, we critically summarized recent progress on deciphering molecular mechanisms to HL and developing HL-tolerant chassis in cyanobacteria, aiming at facilitating construction of HL-resistant chassis and promote the future application of the large-scale outdoor cultivation of cyanobacterial cell factories. Finally, the future directions on cyanobacterial chassis engineering were discussed.

Using osmotic stress to stabilize mannitol production in Synechocystis sp. PCC6803

BackgroundMannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p, in short: a ‘mannitol cassette’), proved to be genetically unstable. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC6803 as a model cyanobacterium.ResultsHere, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild-type Synechocystis, complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production.ConclusionsMannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.

Exploiting Day- and Night-Time Metabolism of Synechocystis sp. PCC 6803 for Fitness-Coupled Fumarate Production around the Clock.

Cyanobacterial cell factories are widely researched for the sustainable production of compounds directly from CO2. Their application, however, has been limited for two reasons. First, traditional approaches have been shown to lead to unstable cell factories that lose their production capability when scaled to industrial levels. Second, the alternative approaches developed so far are mostly limited to growing conditions, which are not always the case in industry, where nongrowth periods tend to occur (e.g., darkness). We tackled both by generalizing the concept of growth-coupled production to fitness coupling. The feasibility of this new approach is demonstrated for the production of fumarate by constructing the first stable dual-strategy cell factory. We exploited circadian metabolism using both systems and synthetic biology tools, resulting in the obligatorily coupling of fumarate to either biomass or energy production. Resorting to laboratory evolution experiments, we show that this engineering approach is more stable than conventional methods.

Systematic identification of a neutral site on chromosome of Synechococcus sp. PCC7002, a promising photosynthetic chassis strain

Cyanobacteria based photosynthetic biomanufacturing is supposed to be one of the alternative routes for sustainable production of biofuels and biochemicals. Synechococcus sp. PCC 7002 is a promising cyanobacterial chassis strain possessing desired properties for scaled cultivation in future. Development of cyanobacterial cell factories requires modifications of PCC7002 chromosome to expand the photosynthesis-based metabolism networks. Therefore, mining genomic neutral sites for stable integration of heterologous genes and pathways would be of great significance for expanding the toolbox for PCC7002 genome engineering. Here we demonstrate a paradigm for identification of potential neutral sites from chromosome of PCC7002 based on genomic and transcriptomics data. By refining the massive omics information from database, 51 putative sites with no significant physiological or metabolism effects were extracted as candidates. Combining genomic context analysis, a locus termed as NS0027 between two neighboring putative neutral genes was selected and evaluated as a neutral site for genetic integration. In addition, an ethanol synthesis pathway was introduced into the NS0027 site to assess the functionality of this site. The sites we identified and the strategy we adopted in this work would benefit the development of effective genetic toolbox and efficient photosynthetic cell factories based on PCC7002.