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Engineering the fast growing and highly productive cyanobacterium Synechococcus sp. PCC 11901
No full textSynechococcus sp. PCC 11901 (PCC 11901) is a new cyanobacteria strain exhibiting
fast and sustained growth and biomass accumulation, making it an interesting and potentially
revolutionary host strain for biotechnology. At the time of starting the PhD project, very limited
information was available apart from a first report published in literature (Wlodarczyk et al.
2020). The overarching goal of my thesis is to develop PCC 11901 as a chassis strain for
cyanobacterial biotechnology.
In this thesis I first reviewed the current state of the art in cyanobacteria biotechnology,
with emphasis on where new fast-growing strains like PCC 11901 can excel and what
molecular and computational tools are needed to maximise their use towards carbon negative
emissions technologies (NETs). I highlight the potential of cyanobacterial biorefineries based
on these new strains in making NETs more cost-effective, as this has been the main bottleneck
in the uptake of cyanobacteria-based solutions by industry.
I next developed a CyanoGate-based synthetic biology toolkit which significantly
expanded our ability to engineer PCC 11901 by characterising new and existing standard
parts (neutral sites, constitutive and inducible promoters, transcriptional terminators). I
performed a proof-of-concept study of conditional knockdown of essential genes using
CRISPRi and a novel markerless genome editing strategy using CRISPR/Cas12a. This
extensive toolkit is a major milestone in engineering PCC 11901 and has been made available
through Addgene, an open vector repository for the research community. This toolkit chapter
is complemented by collaborative published papers (Mills et al., 2022; Mager et al., 2023).
I then performed an RNA-seq study of PCC 11901 to understand the transcriptomic
landscape of this fast-growing strain and find out differentially expressed genes across
different growth phases/densities in comparison to Synechococcus sp. PCC 7002, a strain
with 96% genome similarity but does not exhibit the maximum growth densities reached by
PCC 11901. In this chapter I generated the first RNA-seq dataset of PCC 11901 at different
growth densities, which will ultimately be a valuable resource to inform future engineering
work this strain.
Finally, to demonstrate the potential of PCC 11901 for biotechnology, I explored its
capability as a platform for the bioproduction of high-value plant-derived products. First is the
small, taste-modifying protein monellin, and second is the terpenoid α-bisabolene. I adapted
and developed a phycobiliprotein (cpcB) fusion strategy to improve protein expression, and
optimised growth conditions which led to increased protein and enzyme production. The
results in this chapter set the stage for PCC 11901 as a viable photoautotrophic host for
sustainable bioproduction
Development of Synechococcus sp. PCC 11901 as a biotechnology platform
Get PDFCyanobacteria are key organisms in the global ecosystem and potential renewable platforms for production of chemicals. Many aspects of cyanobacterial biology are unique to this subset of prokaryotes. Characterising cyanobacterial metabolism and physiology is key to understanding their environmental role and unlocking their potential for biotechnology. This thesis provides a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in the model organism, Synechocystis sp. PCC 6803 (PCC 6803). One of the main issues within cyanobacterial bioindustry has been the lack of sustained fast-growing strains. The newly discovered Synechococcus sp. PCC 11901 (PCC 11901) reportedly demonstrates the highest, most sustained growth of any known cyanobacterium. Knowledge of PCC 11901 biology, including the factors underlying the sustained fast growth, is limited, which hinders its potential for biotechnology. Genetic tools for generating unmarked mutants in PCC 11901 are not established. This thesis confirms that PCC 11901 displays faster growth than other model cyanobacteria. Comparative genomics between PCC 11901 and 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 efficient light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. Attempts to generate unmarked knockouts using two negative selectable markers were unsuccessful, suggesting that recombinase or CRISPR-based approaches may be necessary for the industry requirement of repeated genetic manipulation. To further cement PCC 11901 as a future industrial strain, biomass and optical density measurements were carried out over a range of light intensities to aid photo-mechanistic modelling of the strain. This thesis establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatic tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype
A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901
Get PDFSynechococcus 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 characterized 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% to 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
MBP-11901 Inhibits Tumor Growth of Hepatocellular Carcinoma through Multitargeted Inhibition of Receptor Tyrosine Kinases
No full textHepatocellular carcinomas (HCCs) are aggressive tumors with a poor prognosis. Approved first-line treatments include sorafenib, lenvatinib, and a combination of atezolizumab and bevacizumab; however, they do not cure HCC. We investigated MBP-11901 as a drug candidate for HCC. Cell proliferation and cytotoxicity were evaluated using normal and cancer human liver cell lines, while Western blotting and flow cytometry evaluated apoptosis. The anticancer effect of MBP-11901 was verified in vitro through migration, invasion, colony formation, and JC-1 MMP assays. In mouse models, the tumor volume, tumor weight, and bodyweight were measured, and cancer cell proliferation and apoptosis were analyzed. The toxicity of MBP-11901 was investigated through GOT/GPT and histological analyses in the liver and kidney. The signaling mechanism of MBP-11901 was investigated through kinase assays, phosphorylation analysis, and in silico docking simulations. Results. MBP-11901 was effective against various human HCC cell lines, leading to the disappearance of most tumors when administered orally in animal models. This effect was dose-dependent, with no differences in efficacy according to administration intervals. MBP-11901 induced anticancer effects by targeting the signaling mechanisms of FLT3, VEGFR2, c-KIT, and PDGFRβ. MBP-11901 is suggested as a novel therapeutic agent for the treatment of advanced or unresectable liver cancer
Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901
Get PDFSynechococcus 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
Development of a biotechnology platform for the fast-growing cyanobacterium Synechococcus sp. PCC 11901
Get PDFSynechococcus 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
Astaxanthin production from newly discovered Synechococcus sp. PCC 11901 in a continuous photobioreactor
No full textreservedSynechococcus sp. PCC 11901 is a newly discovered cyanobacterial species with interesting features like a fast growth rate and a remarkable biomass accumulation even under high irradiances. The aim of this study is to characterize the effect of cultivating conditions on the biomass productivity and pigments accumulation in a continuous photobioreactor. In particular, the effect of light intensity and residence time was assessed. As Synechococcus sp. PCC 11901 is a promising species for genetic engineering purposes, the cultivation of a GMO strain producing astaxanthin, i.e. a high-value antioxidant, was also assessed, as kindly provided by University of Verona. The astaxanthin production was compared to the one obtained from Haematococcus pluvialis, which is the most common species commercially applied for the production of this pigment.Synechococcus sp. PCC 11901 is a newly discovered cyanobacterial species with interesting features like a fast growth rate and a remarkable biomass accumulation even under high irradiances. The aim of this study is to characterize the effect of cultivating conditions on the biomass productivity and pigments accumulation in a continuous photobioreactor. In particular, the effect of light intensity and residence time was assessed. As Synechococcus sp. PCC 11901 is a promising species for genetic engineering purposes, the cultivation of a GMO strain producing astaxanthin, i.e. a high-value antioxidant, was also assessed, as kindly provided by University of Verona. The astaxanthin production was compared to the one obtained from Haematococcus pluvialis, which is the most common species commercially applied for the production of this pigment
Newly discovered Synechococcus sp. PCC 11901 is a robust cyanobacterial strain for high biomass production
Get PDFCyanobacteria, which use solar energy to convert carbon dioxide into biomass, are potential solar biorefineries for the sustainable production of chemicals and biofuels. However, yields obtained with current strains are still uncompetitive compared to existing heterotrophic production systems. Here we report the discovery and characterization of a new cyanobacterial strain, Synechococcus sp. PCC 11901, with promising features for green biotechnology. It is naturally transformable, has a short doubling time of ≈2 hours, grows at high light intensities and in a wide range of salinities and accumulates up to ≈33 g dry cell weight per litre when cultured in a shake-flask system using a modified growth medium − 1.7 to 3 times more than other strains tested under similar conditions. As a proof of principle, PCC 11901 engineered to produce free fatty acids yielded over 6 mM (1.5 g L−1), an amount comparable to that achieved by similarly engineered heterotrophic organisms
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