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.

Background: Our team discovered thatProchlorococcuscan take up glucose, in a process that changes the transcriptional pattern of several genes involved in glucose metabolization. We have also shown thatglcHencodes a very high affinity glucose transporter, and that glucose is taken up by naturalProchlorococcuspopulations. We demonstrated that the kinetic parameters of glucose uptake show significant diversity in differentProchlorococcusandSynechococcusstrains. Here, we tested whether the transcriptional response ofglcHto 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 determineglcHexpression in fourProchlorococcusand twoSynechococcusstrains.Results: In all studied strainsglcHwas 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,SynechococcusBL107 showed the maximum glucose uptake at 5 nM glucose. Darkness induced a strong decrease inglcHexpression, especially remarkable inProchlorococcusMIT9313.Discussion: Our results suggest that marine picocyanobacteria are actively monitoring the availability of glucose, to upregulateglcHexpression 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 inglcHexpression in all studied strains, which fits with previous results on glucose uptake inProchlorococcus. Overall, this work reinforces the importance of mixotrophy for marine picocyanobacteria.

Numerical optimization solves problems that are analytically intractable at the cost of arriving at a sufficiently good but rarely optimal solution. To maximize the result, optimization algorithms are run with the guidance and supervision of a human, usually an expert in the problem. Recent advances in deep reinforcement learning motivate interest in an artificial agent capable of learning to do the expert’s task. Specifically, we present a proximal policy optimization agent that learns to optimize in a real case study such as the modeling of the photo-fenton disinfection process, which involves a number of parameters that have to be adjusted to minimize the error of the model with respect to the experimental data collected in several trials. The expert spends an average of 4 h to find a suitable set of parameters. On the other hand, the agent we present does not require a human expert to guide or validate the optimization procedure and achieves similar results in $$2.5\times$$2.5 × less time.

The fast-growing cyanobacterium Synechococcus sp. PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.

The fast-growing cyanobacterium Synechococcus sp . PCC 11901 is emerging as a promising chassis for photosynthetic biomanufacturing. Here we report recombinant protein production in PCC 11901 via signal peptide-mediated secretion, enabling direct recovery of target proteins from the culture medium without cell disruption. Seven signal peptides spanning both Sec and Tat pathways are screened using eYFP as a reporter, with secretion quantified daily over seven days by fluorescence measurements. FutA, belonging to the Tat pathway from Synechocystis sp. PCC 6803, achieves 92.2% extracellular export by day 7, substantially outperforming all Sec candidates, including the best Sec signal peptide thermitase from Cyanobacterium aponinum PCC 10605 (55.7%). Signal peptide-bearing strains exhibit growth reductions of up to 26% relative to the wild-type, with FutA most affected, indicating a general metabolic cost correlated with secretion efficiency. The best-performing signal peptides from both pathways, FutA and thermitase, are validated with secretion of lichenase. Notably, the rank order of signal peptide performance is reversed for lichenase: thermitase demonstrates 2.6-fold higher extracellular activity than FutA, indicating that optimal signal peptide selection is cargo-dependent. These results establish PCC 11901 as a secretion-competent chassis and provide a rational framework for matching signal peptide pathways to target protein properties.