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Cyanobacteria have promising potential as sustainable cell factories. However, one challenge that is still largely unreported in scaling-up cyanobacteria bioproduction is phenotypic instability, where the emergence and selection of nonproducing cells leading to loss in production has longer evolutionary timescales to take place in industrial-scale bioreactors. Quantifying phenotypic instability early on in strain development allows researchers to make informed decisions on whether to proceed with scalable designs, or if present, devise countermeasures to reduce instability. One particularly effective strategy to mitigate instability is the use of genome-scale metabolic models to design growth-coupled production strains. In silico studies have predicted that creating certain cofactor imbalances or removing recycling reactions in cyanobacteria can be exploited to stably produce a wide variety of metabolites., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A sustainable society will have to largely refrain from the use of fossil carbon deposits. In such a regime, renewable electricity can be harvested as a primary source of energy. However, as for the synthesis of carbon-based materials from bulk chemicals, an alternative is required. A sustainable approach towards this is the synthesis of commodity chemicals from CO 2 , water and sunlight. Multiple paths to achieve this have been designed and tested in the domains of chemistry and biology. In the latter, the use of both chemotrophic and phototrophic organisms has been advocated. 'Direct conversion' of CO 2 and H 2 O, catalyzed by an oxyphototroph, has excellent prospects to become the most economically competitive of these transformations, because of the relative ease of scale-up of this process. Significantly, for a wide range of energy and commodity products, a proof of principle via engineering of the corresponding production organism has been provided. In the optimization of a cyanobacterial production organism, a wide range of aspects has to be addressed. Of these, here we will put our focus on: (1) optimizing the (carbon) flux to the desired product; (2) increasing the genetic stability of the producing organism and (3) maximizing its energy conversion efficiency. Significant advances have been made on all these three aspects during the past 2 years and these will be discussed: (1) increasing the carbon partitioning to >50%; (2) aligning product formation with the growth of the cells and (3) expanding the photosynthetically active radiation region for oxygenic photosynthesis., (© 2019 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Cyanobacterial direct conversion of CO 2 to several commodity chemicals has been recognized as a potential contributor to support the much-needed sustainable development of human societies. However, the feasibility of this "green conversion" hinders on our ability to overcome the hurdles presented by the natural evolvability of microbes. The latter may result in the genetic instability of engineered cyanobacterial strains leading to impaired productivity. This challenge is general to any "cell factory" approach in which the cells grow for multiple generations, and based on several studies carried out in different microbial hosts, we could identify that three distinct strategies have been proposed to tackle it. These are (1) to reduce microbial evolvability by decreasing the native mutation rate, (2) to align product formation with cell growth/fitness, and, paradoxically, (3) to efficiently reallocate cellular resources to product formation by uncoupling it from growth. The implementation of either of these strategies requires an advanced synthetic biology toolkit. Here, we review the existing methods available for cyanobacteria and identify areas of focus in which specific developments are still needed. Furthermore, we discuss how potentially stabilizing strategies may be used in combination leading to further increases of productivity while ensuring the stability of the cyanobacterial-based direct conversion process.

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. Glycerol is an important commodity chemical. Introduction of phosphoglycerol phosphatase 2 from Saccharomyces cerevisiae into the model cyanobacterium Synechocystis sp. PCC6803 resulted in a mutant strain that produced a considerable amount of glycerol from light, water and COPhotosynthetic production . Mild salt stress (200 mM NaCl) on the cells led to an increase of the extracellular glycerol concentration of more than 20%. Under these conditions the mutant accumulated glycerol to an extracellular concentration of 14.3 mM after 17 days of culturing., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Microalgal biomass represents a very interesting biological feedstock to be converted into several high-value products in a biorefinery approach. In this study, the cyanobacterium Synechocystis sp. PCC6803 was used to obtain different classes of molecules: proteins, carotenoids and lipids by using a cascade approach. In particular, the protein extract showed a selective cytotoxicity towards cancer cells, whereas carotenoids were found to be active as antioxidants both in vitro and on a cell-based model. Finally, for the first time, lipids were recovered from Synechocystis biomass as the last class of molecules and were successfully used as an alternative substrate for the production of polyhydroxyalkanoate (PHA) by the native PHA producer Pseudomonas resinovorans. Taken together, our results lead to a significant increase in the valorization of Synechocystis sp. PCC6803 biomass, thus allowing a possible offsetting of the process costs. [ABSTRACT FROM AUTHOR]

Background: Mannitol 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. Results: Here, 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. Conclusions: Mannitol 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. [ABSTRACT FROM AUTHOR]

Synechocystis gathered momentum in modelling studies and biotechnological applications owing to multiple factors like fast growth, ability to fix carbon dioxide into valuable products, and the relative ease of genetic manipulation. Synechocystis physiology and metabolism, and consequently, the productivity of Synechocystisbased photobioreactors (PBRs), are heavily light modulated. Here, we set up a turbidostat-controlled lab-scale cultivation system in order to study the influence of varying orange-red light intensities on Synechocystis growth characteristics and photosynthetic activity. Synechocystis growth and photosynthetic activity were found to raise as supplied light intensity increased up to 500 mmol photons m-2 s-1 and to enter the photoinhibition state only at 800 mmol photons m-2 s-1. Interestingly, reverting the light to a non-photo-inhibiting intensity unveiled Synechocystis to be able to promptly recover. Furthermore, our characterization displayed a clear correlation between variations in growth rate and cell size, extending a phenomenon previously observed in other cyanobacteria. Further, we applied a modelling approach to simulate the effects produced by varying the incident light intensity on its local distribution within the PBR vessel. Our model simulations suggested that the photosynthetic activity of Synechocystis could be enhanced by finely regulating the intensity of the light incident on the PBR in order to prevent cells from experiencing light-induced stress and induce their exploitation of areas of different local light intensity formed in the vessel. In the latter case, the heterogeneous distribution of the local light intensity would allow Synechocystis for an optimized usage of light. [ABSTRACT FROM AUTHOR]

The biosynthesis of poly-β-hydroxybutyrate (PHB) directly from carbon dioxide is a sustainable alternative for non-renewable, petroleum-based polymer production. Synechocystis sp. PCC6803 can naturally accumulate PHB using CO 2 as the sole carbon source, particularly when major nutrients such as nitrogen become limiting. Many previous studies have tried to genetically engineer PHB overproduction; mostly by increasing the expression of enzymes directly involved in its biosynthesis pathway. Here, we have instead concentrated on engineering the central carbon metabolism of Synechocystis such that ( i ) the PHB synthesis pathway becomes deregulated, and/or ( ii ) the levels of its substrate, acetyl-CoA, were increased. Seven different mutants were constructed harboring, separately or in combination, three different genetic modifications to Synechocystis ' metabolic network. These were the deletions of phosphotransacetylase (Pta) and acetyl-CoA hydrolase (Ach), and the expression of a heterologous phosphoketolase (XfpK) from Bifidobacterium breve . The wild type Synechocystis and the derivative strains were compared in terms of biomass and the PHB production capability during photoautotrophic growth. This was performed in a photobioreactor exposed to a diel light/dark rhythm and using standard BG 11 as the growth medium. We found that the strain that combined all three genetic modifications, i.e. xfpk overexpression in a double pta and ach deletion background, showed the highest levels of PHB production from all the strains tested here. Encouragingly, the production levels obtained: 232 mg L − 1 , ~ 12% (w/w) of the dry biomass weight, and a productivity of 7.3 mg L − 1 d − 1 ; are to the best of our knowledge, the highest ever reported for PHB production directly from CO 2 . [ABSTRACT FROM AUTHOR]

The growth rate and physiology of photoautotrophic bacteria are dependent on the incident light color and intensity. Here we report a widely applicable and straightforward method for light-limited batch cultivation of phototrophic bacteria at different, yet constant, growth rates. We illustrate its usage with Synechocystis sp. PCC6803, a model cyanobacterium used as a chassis for sustainable cell-factories and capable of turning CO 2 into commodity products. The cultivation method we developed resembles a ‘photonfluxostat’. It enables the setting of the growth rate of phototrophs during batch cultivation by adjustment of the illumination intensity (‘photon dosing’). Using this method to study the growth-rate response of Synechocystis , we found that while the cell volume increased, the chlorophyll a content and the PSI/PSII ratio decreased, as growth rate increased. This method allows for a quantitative and controlled study of the light-dependent physiology of phototropic bacteria, a highly relevant group of bacteria for modern biotechnology. [ABSTRACT FROM AUTHOR]

When subjected to mild salt stress, the cyanobacterium Synechocystis sp. PCC 6803 produces small amounts of glycerol through an as of yet unidentified pathway. Here, we show that this glycerol is a degradation product of the main osmolyte of this organism, glucosylglycerol (GG). Inactivation of ggpS, encoding the first step of GG-synthesis, abolished de novo synthesis of glycerol, while the ability to hydrolyze exogenously supplied glucoslylglycerol was unimpaired. Inactivation of glpK, encoding glycerol kinase, had no effect on glycerol synthesis. Inactivation of slr1670, encoding a GHL5-type putative glycoside hydrolase, abolished de novo synthesis of glycerol, as well as hydrolysis of GG, and led to increased intracellular concentrations of this osmolyte. Slr1670 therefore presumably displays GG hydrolase activity. A gene homologous to the one encoded by slr1670 occurs in a wide range of cyanobacteria, proteobacteria, and archaea. In cyanobacteria, it co-occurs with genes involved in GG-synthesis. [ABSTRACT FROM AUTHOR]

Cyanobacterial thylakoid membranes are known to host photosynthetic and respiratory complexes. This hampers a straight forward interpretation of the highly dynamic fluorescence originating from photosynthetic units. The present study focuses on dark-to-light transitions in whole cells of a PSI-deficient mutant of the cyanobacterium Synechocystis sp. PCC 6803. The time-dependent cellular fluorescence spectrum has been measured, while having previously exposed the cells to different conditions that affect respiratory activity. The analysis method used allows the detected signal to be decomposed in a few components that are then assigned to functional emitting species. Additionally, we have worked out a minimal mathematical model consisting of sensible postulated species to interpret the recorded data. We conclude that the following two functional complexes play a major role: a phycobilisome antenna complex coupled to a PSII dimer with either two or no closed reaction centers. Crucially, we present evidence for an additional species capable of strongly quenching fluorescence, whose formation requires the presence of oxygen. [ABSTRACT FROM AUTHOR]

Decoupling growth from product synthesis is a promising strategy to increase carbon partitioning and maximize productivity in cell factories. However, reduction in both substrate uptake rate and metabolic activity in the production phase are an underlying problem for upscaling. Here, we used CRISPR interference to repress growth in lactate-producing Synechocystis sp. PCC 6803. Carbon partitioning to lactate in the production phase exceeded 90%, but CO 2 uptake was severely reduced compared to uptake during the growth phase. We characterized strains during the onset of growth arrest using transcriptomics and proteomics. Multiple genes involved in ATP homeostasis were regulated once growth was inhibited, which suggests an alteration of energy charge that may lead to reduced substrate uptake. In order to overcome the reduced metabolic activity and take advantage of increased carbon partitioning, we tested a novel production strategy that involved alternating growth arrest and recovery by periodic addition of an inducer molecule to activate CRISPRi. Using this strategy, we maintained lactate biosynthesis in Synechocystis for 30 days in a constant light turbidostat cultivation. Cumulative lactate titers were also increased by 100% compared to a constant growth-arrest regime, and reached 1 g/L. Further, the cultivation produced lactate for 30 days, compared to 20 days for the non-growth arrest cultivation. Periodic growth arrest could be applicable for other products, and in cyanobacteria, could be linked to internal circadian rhythms that persist in constant light. • CRISPRi to arrest growth and activate lactate synthesis. • Growth-arrested cells show decreased metabolic rate. • Cycling repression increases production rate and duration. • ATP imbalance implicated in reduced metabolic activity. [ABSTRACT FROM AUTHOR]