Impatti, adattamento e vulnerabilita Le cause e le responsabilita dei cambiamenti climatici sono state trattate sul numero di ottobre della rivista Cda. Approfondiamo l’argomento presentando il documento: “Cambiamenti climatici 2007: impatti, adattamento e vulnerabilita” votato ad aprile 2007 dal secondo gruppo di lavoro del Comitato Intergovernativo sui Cambiamenti Climatici (Intergovernmental Panel on Climate Change). Si tratta del secondo di tre documenti che compongono il quarto rapporto sui cambiamenti climatici.

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Climate Change 2007: Impacts, Adaptation and Vulnerability.

Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO 2

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https://www.cell.com/trends/biotechnology/pdf/S0167-7799(15)00071-2.pdf

Metabolic engineering of cyanobacteria for the synthesis of commodity products

Engineering cyanobacteria for direct biofuel production from CO2

Role of terpenes and isoprenoids has been pivotal in the survival and evolution of higher plants in various ecoregions. These products find application in the pharmaceutical, flavor fragrance, and biofuel industries. Fitness of plants in a wide range of environmental conditions entailed (i) evolution of secondary metabolic pathways enabling utilization of photosynthate for the synthesis of a variety of biomolecules, thereby facilitating diverse eco-interactive functions, and (ii) evolution of structural features for the sequestration of such compounds away from the mainstream primary metabolism to prevent autotoxicity. This review summarizes features and applications of terpene and isoprenoid compounds, comprising the largest class of secondary metabolites. Many of these terpene and isoprenoid biomolecules happen to be high-value bioproducts. They are essential components of all living organisms that are chemically highly variant. They are constituents of primary (quinones, chlorophylls, carotenoids, steroids) as well as secondary metabolism compounds with roles in signal transduction, reproduction, communication, climatic acclimation, defense mechanisms and more. They comprise single to several hundreds of repetitive five-carbon units of isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, there are two pathways that lead to the synthesis of terpene and isoprenoid precursors, the cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. The diversity of terpenoids can be attributed to differential enzyme and substrate specificities and to secondary modifications acquired by terpene synthases. The biological role of secondary metabolites has been recognized as pivotal in the survival and evolution of higher plants. Terpenes and isoprenoids find application in pharmaceutical, nutraceutical, synthetic chemistry, flavor fragrance, and possibly biofuel industries.

Terpenes and isoprenoids: a wealth of compounds for global use.

For effective metabolic engineering, a toolbox of genetic components that enables predictable control of gene expression is needed. Here we present a systematic study of promoters and ribosome binding sites in the unicellular cyanobacterium Synechocystis sp. PCC 6803. A set of metal ion inducible promoters from Synechocystis were compared to commonly used constitutive promoters, by measuring fluorescence of a reporter protein in a standardized setting to allow for accurate comparisons of promoter activity. The most versatile and useful promoter was found to be PnrsB, which from a relatively silent expression could be induced almost 40-fold, nearly up to the activity of the strong psbA2 promoter. By varying the concentrations of the two metal ion inducers Ni2+ and Co2+, expression from the promoter was highly tunable, results that were reproduced with PnrsB driving ethanol production. The activities of several ribosomal binding sites were also measured, and tested in parallel in Synechocystis and Escherichia coli. The results of the study add useful information to the Synechocystis genetic toolbox for biotechnological applications.

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

Forskolin is a high value diterpenoid with a broad range of pharmaceutical applications, naturally found in root bark of the plant Coleus forskohlii. Because of its complex molecular structure, chemical synthesis of forskolin is not commercially attractive. Hence, the labor and resource intensive extraction and purification from C. forskohlii plants remains the current source of the compound. We have engineered the unicellular cyanobacterium Synechocystis sp. PCC 6803 to produce the forskolin precursor 13R-manoyl oxide (13R-MO), paving the way for light driven biotechnological production of this high value compound. In the course of this work, a new series of integrative vectors for use in Synechocystis was developed and used to create stable lines expressing chromosomally integrated CfTPS2 and CfTPS3, the enzymes responsible for the formation of 13R-MO in C. forskohlii. The engineered strains yielded production titers of up to 0.24 mg g(-1) DCW 13R-MO. To increase the yield, 13R-MO producing strains were further engineered by introduction of selected enzymes from C. forskohlii, improving the titer to 0.45 mg g(-1) DCW. This work forms a basis for further development of production of complex plant diterpenoids in cyanobacteria.

Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide

https://escholarship.org/content/qt2jd3r4qd/qt2jd3r4qd.pdf?t=qr01mg

Biofuels for a sustainable future.

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.

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Elias Englund

Papers

Evaluation of promoters and ribosome binding sites for biotechnological applications in the unicellular cyanobacterium Synechocystis sp. PCC 6803

Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide

Biofuels for a sustainable future.

Production of squalene in Synechocystis sp. PCC 6803.

Engineered cyanobacteria with enhanced growth show increased ethanol production and higher biofuel to biomass ratio