Dataset Information

Novel allosteric inhibition of phosphoribulokinase identified by ensemble kinetic modeling of Synechocystis sp. PCC 6803 metabolism.

ABSTRACT: The present study attempted a computer simulation of the metabolism of a model cyanobacteria, Synechocystis sp. PCC 6803 (PCC 6803) to predict allosteric inhibitions that are likely to occur in photoautotrophic and mixotrophic conditions as well as in a metabolically engineered strain. PCC 6803 is a promising host for direct biochemical production from CO₂; however, further investigation of allosteric regulation is required for rational metabolic engineering to produce target compounds. Herein, ensemble modeling of microbial metabolism was applied to build accurate predictive models by synthesizing the results of multiple models with different parameter sets into a single score to identify plausible allosteric inhibitions. The data driven-computer simulation using metabolic flux, enzyme abundance, and metabolite concentration data successfully identified candidates for allosteric inhibition. The enzyme assay experiment using the recombinant protein confirmed isocitrate was a non-competitive inhibitor of phosphoribulokinase as a novel allosteric regulation of cyanobacteria metabolism.

SUBMITTER: Nishiguchi H

PROVIDER: S-EPMC7721636 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Novel allosteric inhibition of phosphoribulokinase identified by ensemble kinetic modeling of Synechocystis sp. PCC 6803 metabolism.

Nishiguchi Hiroki H Liao James J Shimizu Hiroshi H Matsuda Fumio F

Metabolic engineering communications 20201127

The present study attempted a computer simulation of the metabolism of a model cyanobacteria, Synechocystis sp. PCC 6803 (PCC 6803) to predict allosteric inhibitions that are likely to occur in photoautotrophic and mixotrophic conditions as well as in a metabolically engineered strain. PCC 6803 is a promising host for direct biochemical production from CO2; however, further investigation of allosteric regulation is required for rational metabolic engineering to produce target c ...[more]

PMID: 33312875

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Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Project description:BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

Dataset Information

Novel allosteric inhibition of phosphoribulokinase identified by ensemble kinetic modeling of Synechocystis sp. PCC 6803 metabolism.

Publications

Novel allosteric inhibition of phosphoribulokinase identified by ensemble kinetic modeling of <i>Synechocystis</i> sp. PCC 6803 metabolism.

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