Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction

Carpine, Roberta; Du, Weian; Olivieri, Giuseppe; Pollio, Antonino; Hellingwerf, Klaas J.; Marzocchella, Antonio; Santos, Filipe Branco dos

doi:10.1016/j.algal.2017.05.013

Cited by 71 publications

(46 citation statements)

References 43 publications

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“…). The overproduction of RPS, strongly suggests a re‐direction of energy and carbon fluxes towards RPS production, with a negative impact on the growth rate, as commonly observed for Synechocystis overproducing other carbon‐rich compounds (Yoo et al ., ; Carpine et al ., ). In addition, no increase in the storage of other carbon reserves (e.g.…”

Section: Discussionmentioning

confidence: 97%

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

Flores

Santos

Pereira

et al. 2018

Environmental Microbiology

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Summary Cyanobacterial alternative sigma factors are crucial players in environmental adaptation processes, which may involve bacterial responses related to maintenance of cell envelope and control of secretion pathways. Here, we show that the Group 3 alternative sigma factor F (SigF) plays a pleiotropic role in Synechocystis sp. PCC 6803 physiology, with a major impact on growth and secretion mechanisms, such as the production of extracellular polysaccharides, vesiculation and protein secretion. Although ΔsigF growth was significantly impaired, the production of released polysaccharides (RPS) increased threefold to fourfold compared with the wild‐type. ΔsigF exhibits also impairment in formation of outer‐membrane vesicles (OMVs) and pili, as well as several other cell envelope alterations. Similarly, the exoproteome composition of ΔsigF differs from the wild‐type both in amount and type of proteins identified. Quantitative proteomics (iTRAQ) and an in silico analysis of SigF binding motifs revealed possible targets/pathways under SigF control. Besides changes in protein levels involved in secretion mechanisms, our results indicated that photosynthesis, central carbon metabolism and protein folding/degradation mechanisms are altered in ΔsigF. Overall, this work provided new evidences about the role of SigF on Synechocystis physiology and associates this regulatory element with classical and non‐classical secretion pathways.

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Section: Discussionmentioning

confidence: 97%

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

Flores

Santos

Pereira

et al. 2018

Environmental Microbiology

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“…1). 42 Since acetyl-CoA acts as the branching point for 1-butanol biosynthesis, acetate metabolism, and other competing metabolic pathways, the availability of acetyl-CoA may affect the carbon flux towards 1-butanol production. Therefore, in Module 3, we manipulated the flow of acetyl-CoA by deleting genes responsible for the acetate metabolism to save this building block for 1-butanol biosynthesis.…”

Section: View Article Onlinementioning

confidence: 99%

“…Overexpression of a PK enzyme was shown to lead to higher production of PHB in one study in Synechocystis. 42 PK genes and the corresponding enzymes, which have previously been experimentally verified and characterized in several filamentous fungi, two cyanobacteria and some prokaryotic heterotrophic bacteria (ESI, † Table S3), display significant differences in their substrate specificities and catalytic kinetics towards X5P and F6P. Therefore, nine PKs were chosen as candidates to investigate their distinct efficiencies towards 1-butanol production (ESI, † Table S3).…”

Section: Rewriting the Central Carbon Metabolism (Module 1 + 2 + 3 + 4)mentioning

confidence: 99%

Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO₂in cyanobacteria

Liu

Miao

Lindberg

et al. 2019

Energy Environ. Sci.

132

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Cyanobacteria are photoautotrophic microorganisms which can be engineered to directly convert CO 2 and water into biofuels and chemicals via photosynthesis using sunlight as energy. However, the product titers and rates are the main challenges that need to be overcome for industrial applications.Here we present systematic modular engineering of the cyanobacterium Synechocystis PCC 6803, enabling efficient biosynthesis of 1-butanol, an attractive commodity chemical and gasoline substitute.Through introducing and re-casting the 1-butanol biosynthetic pathway at the gene and enzyme levels, optimizing the 5 0 -regions of expression units for tuning transcription and translation, rewiring the carbon flux and rewriting the photosynthetic central carbon metabolism to enhance the precursor supply, and performing process development, we were able to reach a cumulative 1-butanol titer of 4.8 g L À1 with a maximal rate of 302 mg L À1 day À1 from the engineered Synechocystis. This represents the highest 1-butanol production from CO 2 reported so far. Our multi-level modular strategy for high-level production of chemicals and advanced biofuels represents a blue-print for future systematic engineering in photosynthetic microorganisms. Broader contextIn order to reduce the impact of human activities on climate change, we must address our dependence on fossil resources. Biological systems are able to make molecules identical to petroleum-derived compounds used in fuels and in the chemical industry. This has led to increasing attention on the field of metabolic engineering, where microorganisms are genetically engineered to produce compounds of industrial interest. Most such microbial systems employ heterotrophic organisms fed substrates generated from plant biomass. However, photosynthetic microorganisms could be used to perform direct production of fuels and chemicals from photosynthesis, enabling a more efficient conversion of solar energy and carbon dioxide to desirable products, thus leading to more sustainable processes. Engineered strains of photosynthetic cyanobacteria can make many different compounds on a proof-of-concept level, but few products so far show titers approaching those achieved in heterotrophic organisms. We have systematically engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce 1-butanol. The resulting titers are among the highest for any heterologous product from cyanobacteria, and we show that long-term productivity is feasible. The employed strategy can be used for other products when engineering photosynthetic microorganisms for direct production of solar chemicals and biofuels.

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“…Cyanobacteria may accumulate considerable amounts of PHB as product of the wild metabolism and as results of the genetic engineering of strains by heterologous transformation with genes involved in the PHB pathway of R. eutropha [73]. In many cyanobacteria, PHB is a native polymer that stores carbon, produced via the polyhydroxyalkanoate (PHA) biosynthetic pathway [74]. The pathway from acetyl-CoA to PHB includes the following three catalytic steps: Genetic and metabolic engineering together with recombinant DNA technology have shown the ability to increase the PHB accumulation to produce a considerable amount of biomass [4].…”

Section: Genetic Engineering Approachmentioning

confidence: 99%

“…The PHB content reported for cultures carried out under photoautotrophic growth conditions using just CO 2 as carbon source was lower [78,79] than that measured for cultures with other carbon sources. The overexpression of the native sigE gene (encoding of the minor sigma factors of the organism) provided the integration of the latter in the Synechocystis chromosome, and increased the PHB content from 0.6% to 1.4% [74] when cells were grown in a N-deprived medium. Hondo et al [78] transformed Synechocystis cells with the vector pAM461c, harboring a PHA biosynthetic operon from Microcystis aeruginosa NIES-843, and reached a PHB content of about 7% in the N-deprived medium [74].…”

Section: Genetic Engineering Approachmentioning

confidence: 99%

Industrial Production of Poly-β-hydroxybutyrate from CO2: Can Cyanobacteria Meet this Challenge?

et al. 2020

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The increasing impact of plastic materials on the environment is a growing global concern. In regards to this circumstance, it is a major challenge to find new sources for the production of bioplastics. Poly-β-hydroxybutyrate (PHB) is characterized by interesting features that draw attention for research and commercial ventures. Indeed, PHB is eco-friendly, biodegradable, and biocompatible. Bacterial fermentation processes are a known route to produce PHB. However, the production of PHB through the chemoheterotrophic bacterial system is very expensive due to the high costs of the carbon source for the growth of the organism. On the contrary, the production of PHB through the photoautotrophic cyanobacterium system is considered an attractive alternative for a low-cost PHB production because of the inexpensive feedstock (CO2 and light). This paper regards the evaluation of four independent strategies to improve the PHB production by cyanobacteria: (i) the design of the medium; (ii) the genetic engineering to improve the PHB accumulation; (iii) the development of robust models as a tool to identify the bottleneck(s) of the PHB production to maximize the production; and (iv) the continuous operation mode in a photobioreactor for PHB production. The synergic effect of these strategies could address the design of the optimal PHB production process by cyanobacteria. A further limitation for the commercial production of PHB via the biotechnological route are the high costs related to the recovery of PHB granules. Therefore, a further challenge is to select a low-cost and environmentally friendly process to recover PHB from cyanobacteria.

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Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction

Cited by 71 publications

References 43 publications

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO₂in cyanobacteria

Industrial Production of Poly-β-hydroxybutyrate from CO2: Can Cyanobacteria Meet this Challenge?

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Genetic engineering of Synechocystis sp. PCC6803 for poly-β-hydroxybutyrate overproduction

Cited by 71 publications

References 43 publications

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

The alternative sigma factor SigF is a key player in the control of secretion mechanisms in Synechocystis sp. PCC 6803

Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO2in cyanobacteria

Industrial Production of Poly-β-hydroxybutyrate from CO2: Can Cyanobacteria Meet this Challenge?

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Modular engineering for efficient photosynthetic biosynthesis of 1-butanol from CO₂in cyanobacteria