Marine Synechococcus sp. Strain WH7803 Shows Specific Adaptative Responses to Assimilate Nanomolar Concentrations of Nitrate

Marine Synechococcus, together with Prochlorococcus, contribute to a significant proportion of the primary production on Earth. The spatial distribution of these two groups of marine picocyanobacteria depends on different factors such as nutrient availability and temperature. Some Synechococcus ecotypes thrive in mesotrophic and moderately oligotrophic waters, where they exploit both oxidized and reduced forms of nitrogen. Here, we present a comprehensive study, which includes transcriptomic and proteomic analyses of the response of Synechococcus sp. strain WH7803 to nanomolar concentrations of nitrate, compared to micromolar ammonium or nitrogen starvation. We found that Synechococcus has a specific response to a nanomolar nitrate concentration that differs from the response shown under nitrogen starvation or the presence of standard concentrations of either ammonium or nitrate. This fact suggests that the particular response to the uptake of nanomolar concentrations of nitrate could be an evolutionary advantage for marine Synechococcus against Prochlorococcus in the natural environment. IMPORTANCE Marine Synechococcus are a very abundant group of photosynthetic organisms on our planet. Previous studies have shown blooms of these organisms when nanomolar concentrations of nitrate become available. We have assessed the effect of nanomolar nitrate concentrations by studying the transcriptome and proteome of Synechococcus sp. WH7803, together with some physiological parameters. We found evidence that Synechococcus sp. strain WH7803 does sense and react to nanomolar concentrations of nitrate, suggesting the occurrence of specific adaptive mechanisms to allow their utilization. Thus, very low concentrations of nitrate in the ocean seem to be a significant nitrogen source for marine picocyanobacteria

Towards renewable chemicals production in cyanobacteria

The intensive fossil fuel combustion by humanity resulted in the increase of atmospheric CO2 concentration creating the greenhouse effect, which in turn causes severe environmental problems. Thus, humanity should find solutions to decrease dependency on fossil hydrocarbons in order to lower CO2 emission into the atmosphere. Photosynthetic microorganisms, including cyanobacteria, exhibit great potential for this purpose, being able to fix and directly convert CO2 into organic chemicals using solar irradiation as an energy source. Establishing cyanobacteria as microbial cell factories enables the sustainable production of bulk chemicals and biofuels.   In this thesis, the production of industrially relevant chemicals, i.e. succinate and aromatic compounds by the cyanobacterium Synechocystis sp. PCC 6803 was explored. Succinate is naturally formed via the tricarboxylic acid cycle (TCA cycle) in cyanobacteria. Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in replenishing the oxaloacetate intermediate of the TCA cycle. Succinate production was investigated by introducing a new capacity for its synthesis via overexpression of heterologous glyoxylate shunt genes together with overexpression of native PEPc. The implementation of the glyoxylate shunt proved successful as succinate productivity was enhanced in certain conditions. Moreover, the formation of succinate during anaerobic darkness was explored. The enzyme L-aspartate oxidase was studied and the in vitro ability of this enzyme to reduce fumarate to succinate anaerobically was demonstrated, which contributes to the understanding of the cyanobacterial TCA cycle for future engineering purposes.  The production of the first intermediates of the plant phenylpropanoid pathway, trans-cinnamic and p-coumaric acid, which derive from the aromatic amino acids phenylalanine and tyrosine, was implemented by overexpression of phenylalanine- and tyrosine ammonia lyases in Synechocystis. The subsequent metabolic engineering, such as the elimination of competing pathways of tocopherol synthesis, demonstrated increased productivity for both target molecules. Moreover, laboratory evolution of Synechocystis was performed and several metabolic mutants were selected for their ability to secrete phenylalanine in the growth medium. The laboratory-evolved mutants provide an important basis for investigating pathway regulation of aromatic amino acid synthesis. In summary, the findings in this thesis contribute to the development of cyanobacteria as microbial cell factories for the sustainable production of renewable chemicals

Towards renewable chemicals production in cyanobacteria

The intensive fossil fuel combustion by humanity resulted in the increase of atmospheric CO2 concentration creating the greenhouse effect, which in turn causes severe environmental problems. Thus, humanity should find solutions to decrease dependency on fossil hydrocarbons in order to lower CO2 emission into the atmosphere. Photosynthetic microorganisms, including cyanobacteria, exhibit great potential for this purpose, being able to fix and directly convert CO2 into organic chemicals using solar irradiation as an energy source. Establishing cyanobacteria as microbial cell factories enables the sustainable production of bulk chemicals and biofuels.   In this thesis, the production of industrially relevant chemicals, i.e. succinate and aromatic compounds by the cyanobacterium Synechocystis sp. PCC 6803 was explored. Succinate is naturally formed via the tricarboxylic acid cycle (TCA cycle) in cyanobacteria. Phosphoenolpyruvate carboxylase (PEPc) is an essential enzyme in replenishing the oxaloacetate intermediate of the TCA cycle. Succinate production was investigated by introducing a new capacity for its synthesis via overexpression of heterologous glyoxylate shunt genes together with overexpression of native PEPc. The implementation of the glyoxylate shunt proved successful as succinate productivity was enhanced in certain conditions. Moreover, the formation of succinate during anaerobic darkness was explored. The enzyme L-aspartate oxidase was studied and the in vitro ability of this enzyme to reduce fumarate to succinate anaerobically was demonstrated, which contributes to the understanding of the cyanobacterial TCA cycle for future engineering purposes.  The production of the first intermediates of the plant phenylpropanoid pathway, trans-cinnamic and p-coumaric acid, which derive from the aromatic amino acids phenylalanine and tyrosine, was implemented by overexpression of phenylalanine- and tyrosine ammonia lyases in Synechocystis. The subsequent metabolic engineering, such as the elimination of competing pathways of tocopherol synthesis, demonstrated increased productivity for both target molecules. Moreover, laboratory evolution of Synechocystis was performed and several metabolic mutants were selected for their ability to secrete phenylalanine in the growth medium. The laboratory-evolved mutants provide an important basis for investigating pathway regulation of aromatic amino acid synthesis. In summary, the findings in this thesis contribute to the development of cyanobacteria as microbial cell factories for the sustainable production of renewable chemicals

Expression of phenylalanine ammonia lyases in Synechocystis sp. PCC 6803 and subsequent improvements of sustainable production of phenylpropanoids

Background Phenylpropanoids represent a diverse class of industrially important secondary metabolites, synthesized in plants from phenylalanine and tyrosine. Cyanobacteria have a great potential for sustainable production of phenylpropanoids directly from CO2, due to their photosynthetic lifestyle with a fast growth compared to plants and the ease of generating genetically engineered strains. This study focuses on photosynthetic production of the starting compounds of the phenylpropanoid pathway, trans-cinnamic acid and p-coumaric acid, in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Results A selected set of phenylalanine ammonia lyase (PAL) enzymes from different organisms was overexpressed in Synechocystis, and the productivities of the resulting strains compared. To further improve the titer of target compounds, we evaluated the use of stronger expression cassettes for increasing PAL protein levels, as well as knock-out of the laccase gene slr1573, as this was previously reported to prevent degradation of the target compounds in the cell. Finally, to investigate the effect of growth conditions on the production of trans-cinnamic and p-coumaric acids from Synechocystis, cultivation conditions promoting rapid, high density growth were tested. Comparing the different PALs, the highest specific titer was achieved for the strain AtC, expressing PAL from Arabidopsis thaliana. A subsequent increase of protein level did not improve the productivity. Production of target compounds in strains where the slr1573 laccase had been knocked out was found to be lower compared to strains with wild type background, and the Delta slr1573 strains exhibited a strong phenotype of slower growth rate and lower pigment content. Application of a high-density cultivation system for the growth of production strains allowed reaching the highest total titers of trans-cinnamic and p-coumaric acids reported so far, at around 0.8 and 0.4 g L-1, respectively, after 4 days. Conclusions Production of trans-cinnamic acid, unlike that of p-coumaric acid, is not limited by the protein level of heterologously expressed PAL in Synechocystis. High density cultivation led to higher titres of both products, while knocking out slr1573 did not have a positive effect on production. This work contributes to capability of exploiting the primary metabolism of cyanobacteria for sustainable production of plant phenylpropanoids

Laboratory evolution of Synechocystis sp. PCC 6803 for phenylpropanoid production

Cyanobacteria are promising as a biotechnological platform for production of various industrially relevant compounds, including aromatic amino acids and their derivatives, phenylpropanoids. In this study, we have generated phenylalanine resistant mutant strains (PRMs) of the unicellular cyanobacterium Synechocystis sp. PCC 6803, by laboratory evolution under the selective pressure of phenylalanine, which inhibits the growth of wild type Synechocystis. The new strains of Synechocystis were tested for their ability to secrete phenylalanine in the growth medium during cultivation in shake flasks as well as in a high-density cultivation (HDC) system. All PRM strains secreted phenylalanine into the culture medium, with one of the mutants, PRM8, demonstrating the highest specific production of 24.9 & PLUSMN; 7 mg L-1.OD750-1 or 610 & PLUSMN; 196 mg L-1 phenylalanine after four days of growth in HDC. We further overexpressed phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) in the mutant strains in order to determine the potential of PRMs for production of trans-cinnamic acid (tCA) and para-coumaric acid (pCou), the first intermediates of the plant phenylpropanoid pathway. Pro-ductivities of these compounds were found to be lower in the PRMs compared to respective control strains, except for PRM8 under HDC conditions. The PRM8 background strain in combination with PAL or TAL expression demonstrated a specific production of 52.7 & PLUSMN; 15 mg L- 1.OD750-1 tCA and 47.1 & PLUSMN; 7 mg L- 1.OD750 -1 pCou, respectively, with a volumetric titer reaching above 1 g L-1 for both products after four days of HDC cultivation. The genomes of PRMs were sequenced in order to identify which mutations caused the phenotype. Interestingly, all of the PRMs contained at least one mutation in their ccmA gene, which encodes DAHP synthase, the first enzyme of the pathway for aromatic amino acids biosynthesis. Altogether, we demonstrate that the combination of laboratory-evolved mutants and targeted metabolic engineering can be a powerful tool in cyanobacterial strain development

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

Background: Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results: We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions: Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle