Dataset Information

Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC.

ABSTRACT:

Background

PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon-neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~?15% per cell-dry-weight (CDW).

Results

We have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (?pirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic.

Conclusion

The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB.

SUBMITTER: Koch M

PROVIDER: S-EPMC7756911 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Publications

Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC.

Koch Moritz M Bruckmoser Jonas J Scholl Jörg J Hauf Waldemar W Rieger Bernhard B Forchhammer Karl K

Microbial cell factories 20201222 1

<h4>Background</h4>PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon-neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW).<h4>Results</h4>We have created a new strain that lacks the regulatory protein PirC (prod ...[more]

PMID: 33353555

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Project description:BackgroundPhenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis.ResultsWe eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout.ConclusionsHPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.

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

Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC.

Background

Results

Conclusion

Publications

Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC.

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