Unlocking the potential of cyanobacteria: A high-throughput strategy for enhancing biocatalytic performance through genetic optimization
Abstract
Cyanobacteria show promise as hosts for whole-cell biocatalysis. Their photoautotrophic metabolism can be leveraged for a sustainable production process. Despite advancements, performance still lags behind heterotrophic hosts. A key challenge is the limited ability to overexpress recombinant enzymes, which also hinders their biocatalytic efficiency. To address this, we generated large-scale expression libraries and developed a high-throughput method combining fluorescence-activated cell sorting (FACS) and deep sequencing in Synechocystis sp. PCC 6803 (Syn. 6803) to screen and optimize its genetic background. We apply this approach to enhance expression and biocatalyst performance for three enzymes: the ketoreductase LfSDR1M50, enoate reductase YqjM, and Baeyer-Villiger monooxygenase (BVMO) CHMO mut. Diverse genetic combinations yielded significant improvements: optimizing LfSDR1M50 expression showed a 17-fold increase to 39.2 U g cell dry weight (CDW)-1. In vivo activity of Syn. YqjM was improved 16-fold to 58.7 U g CDW-1 and, for Syn. CHMO mut, a 1.5-fold increase to 7.3 U g CDW-1 was achieved by tailored genetic design. Thus, this strategy offers a pathway to optimize cyanobacteria as expression hosts, paving the way for broader applications in other cyanobacteria strains and larger libraries.
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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.
“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 [147]
Recent work combining fluorescence-activated cell sorting, deep sequencing and genetic optimisation in Synechocystis sp. PCC 6803 demonstrates how large variant libraries can be screened to improve enzyme expression and whole-cell biocatalytic performance.
“For example, recent work combining fluorescence-activated cell sorting, deep sequencing and genetic optimisation in Synechocystis sp. PCC 6803 demonstrates how large variant libraries can be screened to improve enzyme expression and whole-cell biocatalytic performance [147].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [147]
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He, H., Gao, K., Mao, B., Shi, G., Li, Y., & Deng, X. (2025) · Biotechnology Journal · doi:10.1002/biot.70162 · Cited by 4 · see citing works
Venero, O. M., Ndeh, R., Pan, A., Patrikainen, P., Eriksson, M., Akhgari, A., Kakko, L., Nedorezova, D., Mohamed, O., Thomson, L., et al. (2026) · BMC Biotechnology · doi:10.1186/s12896-025-01098-7 · Cited by 3 · see citing works
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“…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].”
- Authors
- Jodlbauer, J., Schmal, M., Waltl, C., Rohr, T., Mach-Aigner, A. R., Mihovilovic, M. D., & Rudroff, F.
- Affiliations
- TU Wien (AT)
- Corresponding author
- Rudroff, Florian
- ORCID
- Venue
- Trends in Biotechnology
- Publisher
- Elsevier BV
- Published
- 2024-12
- Volume
- 42
- Issue
- 12
- Pages
- 1795-1818
- ISSN
- 0167-7799 (ISSN-L)
- OpenAlex
- W4401985084
- Cited by
- 9
- References
- 49
- Citations by year
- 2026: 5 · 2025: 3 · 2024: 1
- FWCI
- 0.37
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Trends in biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Technische Universität Wien
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
- Keywords
- cyanobacteria, whole-cell biocatalysis, Synechocystis sp. PCC 6803, recombinant enzyme overexpression, genetic algorithm optimization, fluorescence-activated cell sorting, deep sequencing, expression libraries, Baeyer-Villiger monooxygenase
- MeSH terms
- Cyanobacteria, Bacterial Proteins, Flow Cytometry, Recombinant Proteins, Synechocystis, Biocatalysis, High-Throughput Screening Assays, High-Throughput Nucleotide Sequencing, Metabolic Engineering
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