Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound

• Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain. 

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Carbon flux

Cyanobacteria

Isoprene

MEP pathway

Metabolic engineering

Metabolic modeling

Enzymes

Lipids

Metabolism

Strain

Carbon fluxes

Commercial productions

Cyanobacterium synechocystis

MEP pathways

Reporter molecules

Synechocystis pcc 6803

bacterial enzyme

enzyme Dxs

enzyme Ipi

enzyme IspD

isoprene synthase

methylerythritol 4 phosphate

phosphate

synthetase

terpene derivative

terpenoid

unclassified drug

Agrobacterium tumefaciens

Article

bacterial genome

bacterial growth

bacterial strain

biotechnological production

Botryococcus braunii

Coleus

Coleus forskohlii

controlled study

Deinococcus radiodurans

enzyme activity

Escherichia coli

gene expression level

gene overexpression

gene targeting

glycolysis

heterologous expression

molecular cloning

nonhuman

operon

priority journal

protein analysis

protein expression level

Synechocystis sp. PCC 6803

upregulation

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)