The plasticity of cyanobacterial metabolism supports direct CO2 conversion to ethylene

Xiong, Wei; Morgan, John A.; Ungerer, Justin; Wang, Bo; Maness, Pin‐Ching; Yu, Jianping

doi:10.1038/nplants.2015.53

Cited by 138 publications

(161 citation statements)

References 30 publications

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“…S1 in the supplemental material). A recently published report on the overproduction of ethylene in Synechocystis PCC 6803 (43) estimated that ϳ10% of fixed carbon was diverted into ethylene with no negative effect on photosynthetic growth rates. If we assume a biomass yield of 2.4 g biomass/g C and a cellular protein content of 36% (g/g), and we also assume that 20% of total cellular protein is EYFP in the strain with P cpc560 in NSP1 (from Fig.…”

Section: Resultsmentioning

confidence: 99%

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Berla

Pakrasi

2015

Appl Environ Microbiol

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Cyanobacteria are photosynthetic cell factories that use solar energy to convert CO 2 into useful products. Despite this attractive feature, the development of tools for engineering cyanobacterial chassis has lagged behind that for heterotrophs such as Escherichia coli or Saccharomyces cerevisiae. Heterologous genes in cyanobacteria are often integrated at presumptively "neutral" chromosomal sites, with unknown effects. We used transcriptome sequencing (RNA-seq) data for the model cyanobacterium Synechocystis sp. strain PCC 6803 to identify neutral sites from which no transcripts are expressed. We characterized the two largest such sites on the chromosome, a site on an endogenous plasmid, and a shuttle vector by integrating an enhanced yellow fluorescent protein (EYFP) expression cassette expressed from either the P cpc560 or the P trc1O promoter into each locus. Expression from the endogenous plasmid was as much as 14-fold higher than that from the chromosome, with intermediate expression from the shuttle vector. The expression characteristics of each locus correlated predictably with the promoters used. These findings provide novel, characterized tools for synthetic biology and metabolic engineering in cyanobacteria.C yanobacteria are oxygenic photosynthetic prokaryotes that use solar energy to fix CO 2 , converting it into biomass and valuable products. Since these organisms do not require fixed carbon feedstocks, they have great potential for synthetic biology and metabolic engineering applications (1-4). Several strains of cyanobacteria have been engineered to act as microbial cellular factories for the production of fuels and chemicals, such as isobutanol, 2,3-butanediol, free fatty acids, and D-lactate (5-7). Despite these advances, none of these engineered strains has been able to achieve industrially relevant levels of productivity. A lack of effective and well-characterized tools for synthetic biology in cyanobacteria has limited progress relative to that with other established microbial chassis, such as Escherichia coli or Saccharomyces cerevisiae (1). Synechocystis sp. strain PCC 6803 is a naturally transformable cyanobacterial chassis for synthetic biology. This strain carries several endogenous plasmids in addition to its single chromosome (8-11). Since there are a limited number of self-replicating exogenous plasmids for the expression of heterologous genes in Synechocystis PCC 6803 (12) and other cyanobacteria, these endogenous plasmids may prove to be attractive parts for synthetic biology (13). Additionally, these plasmids increase their copy numbers during the transition from the exponential-growth phase to the stationary phase (14). Thus, we have hypothesized that the expression of heterologous genes from sites in these plasmids could be autoinduced by such a growth transition. This induction could be advantageous in a production system that first yields a dense culture of light-harvesting cyanobacteria and later uses those microbial cell factories to churn out a product of interest.One strat...

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Section: Resultsmentioning

confidence: 99%

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Berla

Pakrasi

2015

Appl Environ Microbiol

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“…To produce high titer of bioproducts in microbes, high-flux pathways (17) are desirable to direct sufficient carbon into target compounds. A few studies have implemented primary metabolic pathways to reach a high yield of bioproducts in cyanobacteria (3,18,19). In contrast, the MEP pathway is a secondary metabolic pathway with a low carbon partition, believed to be 1% or less (1).…”

Section: Significancementioning

confidence: 99%

“…Earlier approaches often involved overexpressing pathway enzymes to enhance carbon flux, but these approaches were hindered by the limited understanding of metabolic network and its regulation. In particular, many low-flux pathways (e.g., terpene biosynthesis) impede carbon partition due to metabolic rigidity (2,3). Moreover, the importance and requirement of energy balance in improving photosynthetic productivity (4), is often neglected in engineering efforts.…”

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confidence: 99%

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations

Wang

Liu

Xin

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

144

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Terpenes are the major secondary metabolites produced by plants, and have diverse industrial applications as pharmaceuticals, fragrance, solvents, and biofuels. Cyanobacteria are equipped with efficient carbon fixation mechanism, and are ideal cell factories to produce various fuel and chemical products. Past efforts to produce terpenes in photosynthetic organisms have gained only limited success. Here we engineered the cyanobacterium Synechococcus elongatus PCC 7942 to efficiently produce limonene through modeling guided study. Computational modeling of limonene flux in response to photosynthetic output has revealed the downstream terpene synthase as a key metabolic flux-controlling node in the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway-derived terpene biosynthesis. By enhancing the downstream limonene carbon sink, we achieved over 100-fold increase in limonene productivity, in contrast to the marginal increase achieved through stepwise metabolic engineering. The establishment of a strong limonene flux revealed potential synergy between photosynthate output and terpene biosynthesis, leading to enhanced carbon flux into the MEP pathway. Moreover, we show that enhanced limonene flux would lead to NADPH accumulation, and slow down photosynthesis electron flow. Fine-tuning ATP/NADPH toward terpene biosynthesis could be a key parameter to adapt photosynthesis to support biofuel/ bioproduct production in cyanobacteria.photosynthesis | limonene | advanced biofuel | terpene | MEP E fficient carbon partition into desired molecules is a major scientific challenge in producing chemicals in photosynthetic organisms (1). Earlier approaches often involved overexpressing pathway enzymes to enhance carbon flux, but these approaches were hindered by the limited understanding of metabolic network and its regulation. In particular, many low-flux pathways (e.g., terpene biosynthesis) impede carbon partition due to metabolic rigidity (2, 3). Moreover, the importance and requirement of energy balance in improving photosynthetic productivity (4), is often neglected in engineering efforts. Recently, a few studies have demonstrated the possibility of producing terpenes in cyanobacteria, but the productivity is rather low (2, 5-7). Enhancing carbon flux into a low-flux terpene pathway could provide intuitive insight to both carbon partition and photosynthesis regulations. Through computational modeling, we show that downstream limonene synthase is a key flux-controlling node in the 2-C-methyl-Derythritol 4-phosphate (MEP)-derived limonene biosynthesis in cyanobacteria. Overcoming this metabolic bottleneck led to a record limonene productivity in the engineered cyanobacteria. Moreover, we show that enhanced limonene production led to redox change and energy imbalance, which ultimately limit photosynthesis capacity. The study demonstrates a successful strategy to enhance carbon partition into MEP-derived terpene biosynthesis, and reveals key photosynthesis regulations in providing ATP/NADPH to support terpene production. Stepwise M...

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“…phaseolicola ethylene-forming enzyme (PsEFE) have been developed to ripen fruit as an alternative to the use of synthetic ethylene (15,16). Ethylene-forming enzymes are being explored for biocatalysis in cyanobacteria (17)(18)(19). PsEFE-catalyzed ethylene production is 2OG-dependent and is stimulated by the addition of L-arginine (L-Arg), which is also converted by PsEFE into pyrroline-5-carboxylate (P5C; Fig.…”

mentioning

confidence: 99%

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

Zhang

Smart

Choi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the fourelectron oxidation of 2-oxoglutarate to give ethylene in an argininedependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of L-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.ethylene-forming enzyme | 2-oxoglutarate-dependent oxygenases | hydroxylase | plant development | oxidoreductase E thylene is of industrial importance and is a vital signaling molecule in plants, where it has roles in germination, senescence, and stress responses (1). Commercial manipulation of the natural ethylene response is agriculturally important in controlling fruit ripening (2). In higher plants, ethylene is produced from methionine, via oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) in an unusual reaction catalyzed by the Fe(II)-dependent ACC oxidase (ACCO) (3, 4), which is part of the 2-oxoglutarate (2OG)-dependent oxygenase superfamily, although it does not use a 2OG cosubstrate (Fig. 1A) (5-7). Ethylene is also produced in some microorganisms by oxidation of 2-oxo-4-methylthiobutyric acid in a reaction not directly enzyme catalyzed (8,9).In work aimed at producing industrial ethylene by biocatalysis, Pseudomonas strains, including plant pathogens, were shown to produce large amounts of ethylene (10)(11)(12)(13)(14). Bacteria engineered to produce ethylene using the Pseudomonas syringae pv. phaseolicola ethylene-forming enzyme (PsEFE) have been developed to ripen fruit as an alternative to the use of synthetic ethylene (15, 16). Ethylene-forming enzymes are being explored for biocatalysis in cyanobacteria (17-19). PsEFE-catalyzed ethylene production is 2OG-dependent and is stimulated by the addition of L-arginine (L-Arg), which is also converted by PsEFE into pyrroline-5-carboxylate (P5C; Fig. 1B) (13, 20). In contrast to the consensus 2OG oxygenase mechanism, which involves sequential binding of 2OG, substrate, and then oxygen, an unprecedented "dual circuit" mechanism is proposed for PsEFE (13).We describe biochemical, structural, and modeling studies supporting a branched mechanistic pathway for PsEFE that can lead either to ethylene via oxidative fragmentation of 2OG or to succinate via a more typical 2OG oxygenase reaction, which sometimes results in P5C formation (Fig....

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The plasticity of cyanobacterial metabolism supports direct CO2 conversion to ethylene

Cited by 138 publications

References 30 publications

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Fine-Tuning of Photoautotrophic Protein Production by Combining Promoters and Neutral Sites in the Cyanobacterium Synechocystis sp. Strain PCC 6803

Enhanced limonene production in cyanobacteria reveals photosynthesis limitations

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate

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