Improving heterologous protein expression in Synechocystis sp. PCC 6803 for alpha-bisabolene production
Open accessAbstract
Cyanobacterial biofuels have the potential to reduce the cost and climate impacts of biofuel production because primary carbon fixation and conversion to fuel are completed together in the cultivation of the cyanobacteria. Cyanobacterial biofuels, therefore, do not rely on costly organic carbon feedstocks that heterotrophs require, which reduces competition for agricultural resources such as arable land and freshwater. However, the published product titer achieved for most molecules of interest using cyanobacteria lag behind what has been achieved using yeast and Escherichia coli (E. coli) cultures. In Synechocystis sp. PCC 6803 (S. 6803), we attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement. We tested 19 strains of genetically modified S. 6803 with five different codon usage sequences of the bisabolene synthase from the grand fir tree (Abies grandis). At least three ribosome binding sites (most designed using the RBS Calculator) were tested for each codon usage sequence. We also tested strains with and without the farnesyl pyrophosphate synthase gene from E. coli. Bisabolene titers after five days of growth in continuous light ranged from un-detected to 7.8 mg/L. Bisabolene synthase abundance was measured and found to be well correlated with titer. Select strains were also tested in 12:12 light:dark cycles, where similar titers were reached after the same amount of light exposure time. One engineered strain was also tested in photobioreactors exposed to a simulated outdoor light pattern with maximum light intensity of 1600 μmol photons m−2 s−1. Here, the bisabolene titer reached 22.2 mg/L after 36 days of growth. Dramatic improvements in our ability to control gene expression in cyanobacteria such as S. 6803, and the co-utilization of additional metabolic engineering methods, are needed in order for these titers to improve to the levels reported for engineered E. coli.
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In Synechocystis sp. PCC 6803, systematic optimisation of heterologous expression for α-bisabolene production showed that codon optimisation, expression-cassette design and translational tuning can strongly influence protein accumulation and product titres.
“In Synechocystis sp. PCC 6803, systematic optimisation of heterologous expression for α-bisabolene production showed that codon optimisation, expression-cassette design and translational tuning can strongly influence protein accumulation and product titres [165].”
From Cyanobacteria as a Photosynthetic Chassis for Metabolic Pathway Engineering with Heterologous Gene Expression (2026) · cites this paper as [165]
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Metabolic engineering of Synechocystis sp. PCC 6803 for improved bisabolene production
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“In Synechocystis sp. PCC 6803, systematic optimisation of heterologous expression for α -bisabolene production showed that codon optimisation, expression-cassette design and translational tuning can strongly influence protein accumulation and product titres [165].”
- Authors
- Sebesta, J., & Peebles, C. A.
- Affiliations
- Colorado State University (US)
- Corresponding author
- Peebles, Christie AM.
- Venue
- Metabolic Engineering Communications
- Publisher
- Elsevier BV
- Published
- 2020-06
- Volume
- 10
- Pages
- e00117
- ISSN
- 2214-0301 (ISSN-L)
- PMC
- PMC6940699
- OpenAlex
- W2995566875
- Cited by
- 46
- References
- 46
- Citations by year
- 2026: 6 · 2025: 6 · 2024: 3 · 2023: 3 · 2022: 9 · 2021: 14 · 2020: 5
- FWCI
- 2.32
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Metabolic Engineering Communications (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Science Foundation
- 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
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
- Keywords
- Synechocystis sp. PCC 6803, α-bisabolene, Abies grandis, codon optimization, ribosome binding site, farnesyl diphosphate synthase, heterologous expression, sesquiterpenes, bisabolanes, diesel replacement, photobioreactors, light-dark cycle, product titer
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