A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria, and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

Mixotrophy in cyanobacteria.

Cyanobacteria evolved the oxygenic photosynthesis to generate organic matter from CO2 and sunlight, and they were responsible for the production of oxygen in the Earth's atmosphere. This made them a model for photosynthetic organisms, since they are easier to study than higher plants. Early studies suggested that only a minority among cyanobacteria might assimilate organic compounds, being considered mostly autotrophic for decades. However, compelling evidence from marine and freshwater cyanobacteria, including toxic strains, in the laboratory and in the field, has been obtained in the last decades: by using physiological and omics approaches, mixotrophy has been found to be a more widespread feature than initially believed. Furthermore, dominant clades of marine cyanobacteria can take up organic compounds, and mixotrophy is critical for their survival in deep waters with very low light. Hence, mixotrophy seems to be an essential trait in the metabolism of most cyanobacteria, which can be exploited for biotechnological purposes.

Integrated Proteomic and Metabolomic Analyses Show Differential Effects of Glucose Availability in Marine Synechococcus and Prochlorococcus.

We compared changes induced by the addition of 100 nM and 5 mM glucose on the proteome and metabolome complements in Synechococcus sp. strains WH8102, WH7803, and BL107 and Prochlorococcus sp. strains MED4, SS120, and MIT9313, grown either under standard light conditions or in darkness. Our results suggested that glucose is metabolized by these cyanobacteria, using primarily the oxidative pentoses and Calvin pathways, while no proof was found for the involvement of the Entner-Doudoroff pathway in this process. We observed differences in the effects of glucose availability, both between genera and between Prochlorococcus MED4 and SS120 strains, which might be related to their specific adaptations to the environment. We found evidence for fermentation in Prochlorococcus sp. strain SS120 and Synechococcus sp. strain WH8102 after 5 mM glucose addition. Our results additionally suggested that marine cyanobacteria can detect nanomolar glucose concentrations in the environment and that glucose might be used to sustain metabolism under darkness. Furthermore, the KaiB and KaiC proteins were also affected in Synechococcus sp. WH8102, pointing to a direct link between glucose assimilation and circadian rhythms in marine cyanobacteria. In conclusion, our study provides a wide overview on the metabolic effects induced by glucose availability in representative strains of the diverse marine picocyanobacteria, providing further evidence for the importance of mixotrophy in marine picocyanobacteria. IMPORTANCE Glucose uptake by marine picocyanobacteria has been previously described and strongly suggests they are mixotrophic organisms (capable of using energy from the sun to make organic matter, but also to directly use organic matter from the environment when available). However, a detailed analysis of the effects of glucose addition on the proteome and metabolome of these microorganisms had not been carried out. Here, we analyzed three Prochlorococcus sp. and three Synechococcus sp. strains which were representative of several marine picocyanobacterial clades. We observed differential features in the effects of glucose availability, depending on both the genus and strain; our study illuminated the strategies utilized by these organisms to metabolize glucose and showed unexpected links to other pathways, such as circadian regulation. Furthermore, we found glucose addition had profound effects in the microbiome, favoring the growth of coexisting heterotrophic bacteria.

Production, homology modeling and mutagenesis studies on GlcH glucose transporter from Prochlorococcus sp. strain SS120.

The marine cyanobacterium Prochlorococcus is one of the main primary producers on Earth, which can take up glucose by using the high affinity, multiphasic transporter GlcH. We report here the overexpression of glcH from Prochlorococcus marinus strain SS120 in Escherichia coli. Modeling studies of GlcH using the homologous MelB melibiose transporter from Salmonella enterica serovar Typhimurium showed high conservation at the overall fold. We observed that an important structural interaction, mediated by a strong hydrogen bond between D8 and R141, is conserved in Prochlorococcus, although the corresponding amino acids in MelB from Salmonella are different. Biased docking studies suggested that when glucose reaches the pocket of the transporter and interacts with D8 and R141, the hydrogen bond network in which these residues are involved could be disrupted, favoring a conformational change with the subsequent translocation of the glucose molecule towards the cytoplasmic region of the pmGlcH structure. Based on these theoretical predictions and on the conservation of N117 and W348 in other MelB structures, D8, N117, R141 and W348 were mutated to glycine residues. Their key role in glucose transport was evaluated by glucose uptake assays. N117G and W348G mutations led to 17 % decrease in glucose uptake, while D8G and R141G decreased the glucose transport by 66 % and 92 % respectively. Overall, our studies provide insights into the Prochlorococcus 3D-structure of GlcH, paving the way for further analysis to understand the features which are involved in the high affinity and multiphasic kinetics of this transporter.

Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation of PCC 11901 are not established. Here, we demonstrate that PCC 11901 shows faster growth than other model cyanobacteria, including the fast-growing species Synechococcuselongatus UTEX 2973, under optimal growth conditions for UTEX 2973. Comparative genomics between PCC 11901 and Synechocystis sp. PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light harvesting complex. This may underlie its superior light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. To aid biotechnology applications, we developed a vitamin B12 auxotrophic mutant but were unable to generate unmarked knockouts using two negative selectable markers, suggesting that recombinase- or CRISPR-based approaches may be required for repeated genetic manipulation. Overall, this study establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatics tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype.

Differential expression of the glucose transporter gene glcH in response to glucose and light in marine picocyanobacteria.

BACKGROUND: Our team discovered that Prochlorococcus can take up glucose, in a process that changes the transcriptional pattern of several genes involved in glucose metabolization. We have also shown that glcH encodes a very high affinity glucose transporter, and that glucose is taken up by natural Prochlorococcus populations. We demonstrated that the kinetic parameters of glucose uptake show significant diversity in different Prochlorococcus and Synechococcus strains. Here, we tested whether the transcriptional response of glcH to several glucose concentrations and light conditions was also different depending on the studied strain. METHODS: Cultures were grown in the light, supplemented with five different glucose concentrations or subjected to darkness, and cells harvested after 24 h of treatment. qRT-PCR was used to determine glcH expression in four Prochlorococcus and two Synechococcus strains. RESULTS: In all studied strains glcH was expressed in the absence of glucose, and it increased upon glucose addition to cultures. The changes differed depending on the strain, both in the magnitude and in the way cells responded to the tested glucose concentrations. Unlike the other strains, Synechococcus BL107 showed the maximum glucose uptake at 5 nM glucose. Darkness induced a strong decrease in glcH expression, especially remarkable in Prochlorococcus MIT9313. DISCUSSION: Our results suggest that marine picocyanobacteria are actively monitoring the availability of glucose, to upregulate glcH expression in order to exploit the presence of sugars in the environment. The diverse responses observed in different strains suggest that the transcriptional regulation of glucose uptake has been adjusted by evolutive selection. Darkness promotes a strong decrease in glcH expression in all studied strains, which fits with previous results on glucose uptake in Prochlorococcus. Overall, this work reinforces the importance of mixotrophy for marine picocyanobacteria.