Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria
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Abstract
Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO2 capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO2, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO2, avoiding fossil carbon or competition with food production.
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Recent advances include the development of computationally designed RBS libraries and modular expression systems, which have significantly improved the ability to control protein expression in cyanobacterial hosts.
“Recent advances include the development of computationally designed RBS libraries and modular expression systems, which have significantly improved the ability to control protein expression in cyanobacterial hosts [100,106].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [100]
The Results section describes a modular assembly platform and promoter and RBS libraries that provide a wide range of expression strengths in Synechocystis. But it explicitly says the authors chose a degenerate, rather than computationally designed, RBS library because computational design tools had limited predictive ability in cyanobacteria. The claim is therefore supported for modular expression control, not for computationally designed RBS libraries. (LLM, conf=0.90)
Experimental — accuracy not yet measuredThe successful implementation of such pathways demands careful consideration of various factors, including gene expression levels, enzyme activity, metabolic flux distribution, and the availability of cellular cofactors.
“The successful implementation of such pathways demands careful consideration of various factors, including gene expression levels, enzyme activity, metabolic flux distribution, and the availability of cellular cofactors [79,100].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [100]
The Introduction (page unavailable) supports considering enzyme expression levels when optimizing pathways, and the Results section on precursor supply (page unavailable) discusses how precursor availability and possible changes in flux affect lycopene accumulation. The copy does not establish enzyme activity or the availability of cellular cofactors as factors that must be considered for successful pathway implementation; its Discussion mentions cofactors as essential products whose synthesis competes with terpenoid production. (LLM, conf=0.86)
Experimental — accuracy not yet measuredHigh-level expression of multiple heterologous proteins competes with native protein synthesis for ribosomes, amino acids, and ATP, diverting resources from photosynthetic maintenance and generating selective pressure for pathway-silencing mutations during prolonged cultivation.
“At the level of host fitness, high-level expression of multiple heterologous proteins competes with native protein synthesis for ribosomes, amino acids, and ATP, diverting resources from photosynthetic maintenance and generating selective pressure for pathway-silencing mutations during prolonged cultivation [100,147].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [100]
Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.
Experimental — accuracy not yet measuredSynthetic promoters are especially useful in metabolic engineering contexts where careful adjustment of multiple gene expression levels is necessary for efficient pathway function.
“Synthetic promoters are especially useful in metabolic engineering contexts where careful adjustment of multiple gene expression levels is necessary for efficient pathway function [100,101].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [100]
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- Authors
- Taylor, G. M., Hitchcock, A., & Heap, J. T.
- Affiliations
- Imperial College London (GB)
- University of Sheffield (GB)
- University of Nottingham (GB)
- Corresponding author
- Heap, John T
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2021-09-23
- Volume
- 49
- Issue
- 21
- Pages
- e123-e123
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC8643660
- OpenAlex
- W3201549100
- Cited by
- 38
- References
- 88
- Citations by year
- 2026: 8 · 2025: 7 · 2024: 6 · 2023: 6 · 2022: 7 · 2021: 2 · 2020: 2
- FWCI
- 1.65
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Repository@Nottingham (University of Nottingham) (publishedVersion, other-oa)
- White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
- Repository@Nottingham (University of Nottingham) (submittedVersion, cc-by-nc-nd)
- Repository@Nottingham (University of Nottingham) (submittedVersion, cc-by)
- Funders
- Imperial College London
- BBSRC (BB/M002454/1, BB/M011321/1)
- Royal Society University Research Fellowship (URF\R1\191548)
- University of Nottingham
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
- Keywords
- cyanobacteria, Synechocystis sp. PCC 6803, metabolic pathway engineering, genomic instability, synthetic promoters, ribosome binding site, lycopene production, terpenoid biosynthesis, CO2 fixation, CO2 capture
- MeSH terms
- Lycopene, Promoter Regions, Genetic, Gene Library, Synechocystis, Metabolic Networks and Pathways, Metabolic Engineering
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