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Synthetic metabolic pathways for conversion of CO2 into secreted short-to medium-chain hydrocarbons using cyanobacteria
Imperial Coll London, Dept Life Sci, London SW7 2AZ, England..
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.ORCID iD: 0000-0003-4105-7567
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.ORCID iD: 0000-0003-2911-6886
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2022 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 72, p. 14-23Article in journal (Refereed) Published
Abstract [en]

The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to mediumchain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of reaction ingredients, it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the reaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis even in the Synechocystis strains expressing GmLOX1. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioesterases ('CaFatB3.5 and 'ChoFatB2.2). When UndB instead was combined with a C12-specific 'UcFatB1 thioesterase, this resulted in a ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Preliminary optimization of the RBS, promoter and gene order in some of the synthetic operons resulted in improved 1-alkene productivity, reaching a titer of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was implemented with several different metabolic systems. Optimal productivity was observed with UndB and a C12 chain-length specific thioesterase, although further optimization of the entire biosynthetic system is still possible.

Place, publisher, year, edition, pages
Elsevier BV , 2022. Vol. 72, p. 14-23
Keywords [en]
Propane, Propene, Gasoline, Renewable, Biofuels, CRISPRi, dCas9, Cyanobacteria, Metabolic engineering, Hydrocarbon
National Category
Biochemistry Molecular Biology Organic Chemistry Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:kth:diva-313048DOI: 10.1016/j.ymben.2022.01.017ISI: 000793786700002PubMedID: 35134557Scopus ID: 2-s2.0-85124596602OAI: oai:DiVA.org:kth-313048DiVA, id: diva2:1662114
Note

QC 20220531

Available from: 2022-05-31 Created: 2022-05-31 Last updated: 2025-02-20Bibliographically approved

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Anfelt, JosefineSporre, EmilMiao, RuiHudson, Elton P.

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