Engineering Cyanobacteria for Biofuel Production

Miao, Rui; Wegelius, Adam; Durall, Claudia; Liang, Feiyan; Khanna, Namita; Lindblad, Peter

doi:10.1007/978-3-319-46261-5_11

Cited by 8 publications

(5 citation statements)

References 174 publications

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“…CO 2 emissions from the use of fossil fuels has increased significantly since 1900 and has caused an urgent demand for renewable energy alternatives ( Hewitson et al, 2014 ). During the last decades, a lot of research has been focused on identifying environmentally sustainable methods to produce renewable biofuels for replacing traditional fossil fuels ( Machado and Atsumi, 2012 , Miao et al, 2017 ). Isobutanol is a strong candidate to be used as an alternative biofuel due to its high energy content (98% of energy content in gasoline) and lower vapor pressure compared to ethanol ( Sheehan, 2009 ).…”

Section: Introductionmentioning

confidence: 99%

Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

Miao

Liu

Englund

et al. 2017

Metabolic Engineering Communications

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Isobutanol is a flammable compound that can be used as a biofuel due to its high energy density and suitable physical and chemical properties. In this study, we examined the capacity of engineered strains of Synechocystis PCC 6803 containing the α-ketoisovalerate decarboxylase from Lactococcus lactis and different heterologous and endogenous alcohol dehydrogenases (ADH) for isobutanol production. A strain expressing an introduced kivd without any additional copy of ADH produced 3 mg L−1 OD750−1 isobutanol in 6 days. After the cultures were supplemented with external addition of isobutyraldehyde, the substrate for ADH, 60.8 mg L−1 isobutanol was produced after 24 h when OD750 was 0.8. The in vivo activities of four different ADHs, two heterologous and two putative endogenous in Synechocystis, were examined and the Synechocystis endogenous ADH encoded by slr1192 showed the highest efficiency for isobutanol production. Furthermore, the strain overexpressing the isobutanol pathway on a self-replicating vector with the strong Ptrc promoter showed significantly higher gene expression and isobutanol production compared to the corresponding strains expressing the same operon introduced on the genome. Hence, this study demonstrates that Synechocystis endogenous AHDs have a high capacity for isobutanol production, and identifies kivd encoded α-ketoisovalerate decarboxylase as one of the likely bottlenecks for further isobutanol production.

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Section: Introductionmentioning

confidence: 99%

Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

Miao

Liu

Englund

et al. 2017

Metabolic Engineering Communications

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“…Cyanobacteria are being explored as the next-generation hosts for the production of value-added chemicals due to their ability to perform oxygenic photosynthesis. Although cyanobacteria have been engineered for the production of a range of chemicals, the reported productivities have been low. − The ongoing efforts in metabolic engineering have been directed toward modulating the flux controlling steps of the pathway of interest. Therefore, the success of rational engineering of strains hinges heavily on the availability of synthetic biology tools including well-characterized DNA elements to be used as promoters.…”

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confidence: 99%

A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria

2020

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Cyanobacteria are emerging as hosts for various biotechnological applications. The ability to engineer these photosynthetic prokaryotes greatly depends on the availability of well-characterized promoters. Inducer-free promoters of a range of activities may be desirable for the eventual large-scale, outdoor cultivations. Further, several native promoters of cyanobacteria are repressed by high carbon dioxide or light, and it would be of interest to alter this property. We started with PrbcL and PcpcB, the well-characterized native promoters of the model cyanobacterium Synechococcus elongatus PCC 7942, found upstream of the two abundantly expressed genes, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and phycocyanin β-1 subunit, respectively. The library of 48 promoters created via error-prone PCR of these 300-bp-long native promoters showed 2 orders of magnitude dynamic range with activities that were both lower and higher than those of the wild-type promoters. A few mutants of the PrbcL showed greater strength than PcpcB, which is widely considered a superstrong promoter. A number of mutant promoters did not show repression by high CO2 or light, typically found for PrbcL and PcpcB, respectively. Further, the wild-type and mutant promoters showed comparable activities in the fast-growing and stress-tolerant strains S. elongatus PCC 11801 and PCC 11802, suggesting that the library can be used in different cyanobacteria. Interestingly, the majority of the promoters showed strong expression in E. coli, thus adding to the repertoire of inducer-free promoters for this heterotrophic workhorse. Our results have implications in the metabolic engineering of cyanobacteria and E. coli.

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“…Cyanobacteria are the oldest photosynthetic organisms on Earth and were responsible for raising O 2 levels when the atmosphere was anoxic [ 1 – 3 ]. Today, cyanobacteria are receiving increased attention as photosynthetic microbial cell factories [ 4 – 6 ]. They are relatively easy to grow and produce valuable native substances such as fatty acids and terpenoids, among others [ 7 , 8 ].…”

Section: Introductionmentioning

confidence: 99%

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

et al. 2021

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Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

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Engineering Cyanobacteria for Biofuel Production

Cited by 8 publications

References 174 publications

Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

Isobutanol production in Synechocystis PCC 6803 using heterologous and endogenous alcohol dehydrogenases

A Library of Tunable, Portable, and Inducer-Free Promoters Derived from Cyanobacteria

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

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