Recombinant Cyanobacteria Research Articles

Towards the rate limit of heterologous biotechnological reactions in recombinant cyanobacteria

BackgroundCyanobacteria have emerged as highly efficient organisms for the production of chemicals and biofuels. Yet, the productivity of the cell has been low for commercial application. Cyanobacterial photobiotransformations utilize photosynthetic electrons to form reducing equivalents, such as NADPH-to-fuel biocatalytic reactions. These photobiotransformations are a measure to which extent photosynthetic electrons can be deviated toward heterologous biotechnological processes, such as the production of biofuels. By expressing oxidoreductases, such as YqjM from Bacillus subtilis in Synechocystis sp. PCC 6803, a high specific activity was obtained in the reduction of maleimides. Here, we investigated the possibility to accelerate the NAD(P)H-consuming redox reactions by addition of carbohydrates as exogenous carbon sources such as D-Glucose under light and darkness.ResultsA 1.7-fold increase of activity (150 µmol min−1 gDCW−1) was observed upon addition of D-Glucose at an OD750 = 2.5 (DCW = 0.6 g L−1) in the biotransformation of 2-methylmaleimide. The stimulating effect of D-Glucose was also observed at higher cell densities in light and dark conditions as well as in the reduction of other substrates. No increase in both effective photosynthetic yields of Photosystem II and Photosystem I was found upon D-Glucose addition. However, we observed higher NAD(P)H fluorescence when D-Glucose was supplemented, suggesting increased glycolytic activity. Moreover, the system was scaled-up (working volume of 200 mL) in an internally illuminated Bubble Column Reactor exhibiting a 2.4-fold increase of specific activity under light-limited conditions.ConclusionsResults show that under photoautotrophic conditions at a specific activity of 90 µmol min−1 gDCW−1, the ene-reductase YqjM in Synechocystis sp. PCC 6803 is not NAD(P)H saturated, which is an indicator that an increase of the rates of heterologous electron consuming processes for catalysis and biofuel production will require funnelling further reducing power from the photosynthetic chain toward heterologous processes.

Cover Feature: Design and Investigation of a Photocatalytic Setup for Efficient Biotransformations Within Recombinant Cyanobacteria in Continuous Flow (ChemSusChem 22/2022)

The Cover Feature shows a novel approach to bring photobiotransformations within recombinant cyanobacteria from batch to flow. The proposed setup consists of a coil reactor, shown in the right half of the image, that has been assembled in house via means of 3D printing and made of commercially available materials. Starting from the substrate shown in the upper half of the image, the presented approach proved not only to achieve increased productivity, but also to be a more sustainable solution for the production of the desired compound (shown to the bottom right. More information can be found in the Research Article by A. Valotta et al.

Design and Investigation of a Photocatalytic Setup for Efficient Biotransformations Within Recombinant Cyanobacteria in Continuous Flow.

Photo- and biocatalysis show many advantages as more sustainable solutions for the production of fine chemicals. In an effort to combine the benefits and the knowledge of both these areas, a continuous photobiocatalytic setup was designed and optimized to carry out whole-cell biotransformations within cells of the cyanobacterium Synechocystis sp. PCC 6803 expressing the gene of the ene-reductase YqjM from B. subtilis. The effect of the light intensity and flow rate on the specific activity in the stereoselective reduction of 2-methyl maleimide was investigated via a design-of-experiments approach. The cell density in the setup was further increased at the optimal operating conditions without loss in specific activity, demonstrating that the higher surface area/volume ratio in the coil reactor improved the illumination efficiency of the process. Furthermore, different reactor designs were compared, proving that the presented approach was the most cost- and time-effective solution for intensifying photobiotransformations within cyanobacterial cells.

Recent Advances in Cyanobacterial Biotransformations

AbstractLight‐driven biotransformations in recombinant cyanobacteria have been explored in recent years for the production of chiral compounds and platform chemicals. In particular, cyanobacteria harboring oxidoreductases proved to be sustainable hosts providing and recycling reducing equivalents and improving the atom economy of the reactions. However, the provision of light in photobioreactors is considered to be the major bottleneck for up‐scaling. In this review, genetic tools for generating recombinant cyanobacterial strains and up to date cyanobacterial biotransformations including redesigning of the photosynthetic electron transport chain to increase specific activities are presented. Finally, several photobioreactor geometries to circumvent the light limitation are discussed and some future perspectives are presented.

Biosensor-informed engineering of Cupriavidus necator H16 for autotrophic D-mannitol production

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations.

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h–1 L–1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

Scalable Cultivation of Engineered Cyanobacteria for Squalene Production from Industrial Flue Gas in a Closed Photobioreactor.

Economically feasible photosynthetic cultivation of microalgal and cyanobacterial strains is crucial for the biological conversion of CO2 and potential CO2 mitigation to challenge global warming. To overcome the economic barriers, the production of value-added chemicals was desired by compensating for the overall processing cost. Here, we engineered cyanobacteria for photosynthetic squalene production and cultivated them in a scalable photobioreactor using industrial flue gas. First, an inducer-free gene expression system was developed for the cyanobacteria to lower production const. Then, the recombinant cyanobacteria were cultivated in a closed photobioreactor (100 L) using flue gas (5% CO2) as the sole carbon source under natural sunlight as the only energy source. Seasonal light intensities and temperatures were analyzed along with cyanobacterial cell growth and squalene production in August and October 2019. As a result, the effective irradiation hours were the most critical factor for the large-scale cultivation of cyanobacteria. Thus, an automated photobioprocess system will be developed based on the regional light sources.

Stereoselective Biotransformations of Cyclic Imines in Recombinant Cells of Synechocystis sp. PCC 6803

AbstractLight‐driven biotransformations in recombinant cyanobacteria allow to employ photosynthetic water‐splitting for cofactor‐regeneration and thus, to save the use of organic electron donors. The genes of three recombinant imine reductases (IREDs) were expressed in the cyanobacterium Synechocystis sp. PCC 6803 and eight cyclic imine substrates were screened in whole‐cell biotransformations. While initial reactions showed low to moderate rates, optimization of the reaction conditions in combination with promoter engineering allowed to alleviate toxicity effects and achieve full conversion of prochiral imines with initial rates of up to 6.3 mM h−1. The high specific activity of up to 22 U gCDW−1 demonstrates that recombinant cyanobacteria can provide large amounts of NADPH during whole cell reactions. The excellent optical purity of the products with up to >99 %ee underlines the usefulness of cyanobacteria for the stereoselective synthesis of amines.

Lichtgetriebene Ganzzellbiotransformation mit rekombinanten Cyanobakterien

Photosynthetic microorganisms have received considerable attention as production organisms for chemicals. An investigation of the photosynthetic NADPH supply for enantioselective biotransformations with recombinant cyanobacteria showed that the specific activity of the cells is comparable to heterotrophic organisms. Light-dispersion of the cells, however, limits the approach. A wide use as production organisms requires an improvement of several magnitudes.

Development of stable recombinant cyanobacteria for economically competent solar-fuel-factories: ethylene production

Development of stable recombinant cyanobacteria for economically competent solar-fuel-factories: ethylene production

Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria

Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.

Antibiotics-free stable polyhydroxyalkanoate (PHA) production from carbon dioxide by recombinant cyanobacteria

A practical antibiotics-free plasmid expression system in cyanobacteria was developed by using the complementation of cyanobacterial recA null mutation with the EscherichiacolirecA gene on the plasmid. This system was applied to the production of polyhydroxyalkanoate (PHA), a biodegradable plastic, and the transgenic cyanobacteria stably maintained the pha genes for PHA production in the antibiotics-free medium, and accumulated up to 52% cell dry weight of PHA.

Simulation of Ethylene Production by Recombinant Cyanobacteria

Kinetic studies were carried out for ethylene production by recombinant cyano bacteria which harbor a gene for the ethylene-forming enzyme of Pseudomonas syringae. The production rate depended on light intensity and cell concentration. Experimental data obtained at low cell concentrations were used to predict the production rate at high cell concentrations, as a function of light intensity. The computed production rate agreed well with measured data. The results are expected to be useful for optimization of the process.

Evaluation of four fresh-water unicellular cyanobacteria as potential hosts for mosquitocidal toxins

For biocontrol of mosquitoes, mosquitocidal toxin genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus have been cloned into a number of cyanobacteria. However, little is known about the persistence of such recombinant cyanobacteria in mosquito larval habitats. Four fresh water unicellular cyanobacteria, Synechococcus PCC6301, PCC7425, PCC7942 and Synechocystis PCC 6803, were evaluated under laboratory conditions related to mosquito breeding environments. Results indicated that Synechococcus PCC6301 was potentially the most suitable organism for use in the natural mosquito habitat as it could tolerate a wide range of temperatures, salinities, and biological and chemical insecticides. Moreover, strain PCC6301 could be ingested and digested by Culex quinquefasciatus larvae and could support the development of larvae to full insect maturity.

Pseudo-continuous culture of marine recombinant cyanobacteria under a light dark cycle

Pseudo-continuous culture of recombinant marine cyanobacteria was carried out under a light dark cycle. As a model foreign gene, the structural gene of chloramphenicol acetyl transferase (CAT) was used. Under a light dark alternate conditions, a steady state cell concentration was observed. However, the level of CAT expression did not show a stationary phase, but oscillated with the light dark cycle. This may be due to the lack of energy for foreign peptide synthesis during dark incubation. This result also indicates the possibility of regulation of foreign gene expression by controlling light illumination.

Isoamyl phenylacetate

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.