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Effects of over-expressing Calvin–Benson–Bassham Cycle carbon flux control enzymes in an isobutanol producing strain of Synechocystis sp. 6803

Fuller, Samantha Ankara (2022) MOBN02 20211
Degree Projects in Molecular Biology
Popular Abstract
Supply Chain Management inside the Cell Factory

Climate change and environmental sustainability goals decades ago led to an examination of bio-based solutions as possible alternatives to using fossil fuels. The use of modern genetic engineering and gene swapping between organisms allow modern microbiologists to create bespoke organisms that operate as cell factories, the most well-known of which produce insulin and rheumatoid drugs. Advances in molecular science and synthetic biology, along with parallel discoveries of microbial virtuosity in manipulating chemical compounds without polluting their habitats, has enabled design of advanced biofuels. Researchers have turned to cyanobacteria as possible low carbon cell factories. These... (More)
Supply Chain Management inside the Cell Factory

Climate change and environmental sustainability goals decades ago led to an examination of bio-based solutions as possible alternatives to using fossil fuels. The use of modern genetic engineering and gene swapping between organisms allow modern microbiologists to create bespoke organisms that operate as cell factories, the most well-known of which produce insulin and rheumatoid drugs. Advances in molecular science and synthetic biology, along with parallel discoveries of microbial virtuosity in manipulating chemical compounds without polluting their habitats, has enabled design of advanced biofuels. Researchers have turned to cyanobacteria as possible low carbon cell factories. These bacteria are photo synthesisers requiring minimal inputs, bypass the food vs fuel debate, and have the benefit of highly malleable genomes which puts them at the forefront of potential low carbon production chains.

Cell factories contain many enzymes (workers) that are sensitive to abundance or scarcity of substrates. Consequently, stockpiling and placement of ‘key workers’ seems to affect product output. Careful mapping of internal production pathways enables engineers to knockout other enzymes that could use the same raw materials, reduce competition for limited resources. Or engineers could remove potential bottlenecks by “doubling up”, overexpressing, “workers”. Cyanobacteria photosynthesising apparatus pulls in atmospheric C02 and prepares carbon chains as raw materials. Previous researchers demonstrated that overexpressing the critical enzymes in the synthesis production line could
could substantially increase ethanol production (a two-carbon fuel). In order for a cyanobacteria to produce isobutanol (a four-carbon fuel) a specialised gene from other species must be inserted, the most successful of which is kivd from bacteria Lactococcus lactis.

The research aim of this project was to discover if adding more “key workers” at the carbon supply chain could result in more isobutanol production compared to no extra workers. This experiment designed seven combinations of three specific key workers (simplified as: R, F, T, R&F, R&T, F&T, T&F). The genes were constructed on a small genetic ring, a plasmid, and after confirmation were introduced to a modified cyanobacteria that could produce isobutanol. The plasmid was designed to unwrap and insert itself and its payload of genes into the main DNA chromosome. Despite endless repetition and reviewing technical aspects of the genetic construction only one construct was fully confirmed.

Two colonies of construct F were put into isobutanol production and daily growth of the cell cultures measured via optical density, with alternative day measurement of isobutanol using gas chromatography. The constructs were compared to basic strains with no extra workers. Strain one produced +141% more isobutanol with healthy growth till day 13. Strain two produced +23% more isobutanol but died sooner, at the same rate as basic strain. The different growth and product outputs can possibly be explained by genetic complexity. Unusually, cyanobacteria can have over 100 copies of the chromosome. It is possible one strain had fewer of the genetic inserts as cells copied their DNA and divided in growth. This is a well debated and documented phenomenon in cyanobacteria with implications for industrial application. The remaining constructs need to be created and tested and deeper investigations should delve into chromosomal stability. But as proof of concept, it is clear each research step advances the future of biofuels from bacteria.

Master’s Degree Project in Molecular Microbiology 45 credits 2021
Department of Biology, Lund University
Supervisors: Professor Peter Lindblad & PhD student Hao Xie
Department of Chemistry, Microbial Chemistry, Uppsala University (Less)
Please use this url to cite or link to this publication:
author
Fuller, Samantha Ankara
supervisor
organization
course
MOBN02 20211
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9075596
date added to LUP
2022-02-18 11:02:05
date last changed
2022-02-18 11:02:05
@misc{9075596,
  author       = {{Fuller, Samantha Ankara}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Effects of over-expressing Calvin–Benson–Bassham Cycle carbon flux control enzymes in an isobutanol producing strain of Synechocystis sp. 6803}},
  year         = {{2022}},
}