Pia Lindberg

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

SUMMARY Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.

Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions

Cyanobacteria are the only prokaryotes capable of using sunlight as their energy, water as an electron donor, and air as a source of carbon and, for some nitrogen-fixing strains, nitrogen. Compared to algae and plants, cyanobacteria are much easier to genetically engineer, and many of the standard biological parts available for Synthetic Biology applications in Escherichia coli can also be used in cyanobacteria. However, characterization of such parts in cyanobacteria reveals differences in performance when compared to E. coli, emphasizing the importance of detailed characterization in the cellular context of a biological chassis. Furthermore, cyanobacteria possess special characteristics (e.g., multiple copies of their chromosomes, high content of photosynthetically active proteins in the thylakoids, the presence of exopolysaccharides and extracellular glycolipids, and the existence of a circadian rhythm) that have to be taken into account when genetically engineering them. With this chapter, the synthetic biologist is given an overview of existing biological parts, tools and protocols for the genetic engineering, and molecular analysis of cyanobacteria for Synthetic Biology applications.

Photoproduction of H2 by wildtype Anabaena PCC 7120 and a hydrogen uptake deficient mutant: from laboratory experiments to outdoor culture.

Abstract We have analyzed the filamentous cyanobacterium Anabaena PCC 7120 (wildtype) containing one nitrogenase, one uptake hydrogenase and one bidirectional hydrogenase and its hydrogen uptake deficient mutant AMC 414 for their H2 production capacities. Anabaena PCC 7120 and AMC 414 had similar growth rates in turbidostat mode with increased growth rates at higher light intensity. Rates of C2H2 reduction were similar for both strains. In contrast to the wildtype, AMC 414 produced H2 in a PhotoBioReactor (PhBR) using air as the lifting gas. The rate of H2 production increased with light intensity and was not even saturated at 456 μE m −2 s −1 . H2 production increased significantly when replacing the air with argon. The maximal H2 production during outdoor conditions was recorded using AMC 414 with a peak at 14.9 ml H 2 h −1 l −1 . Despite the relatively high production, maximal efficiency of solar energy to H2 conversion was only 0.042%. A molecular method was developed to analyze the relative abundancies of weight and mutant in competition experiments in the PhBR.

A hydrogen-producing, hydrogenase-free mutant strain of Nostoc punctiforme ATCC 29133

Abstract The hupL gene, encoding the uptake hydrogenase large subunit, in Nostoc sp. strain ATCC 29133, a strain lacking a bidirectional hydrogenase, was inactivated by insertional mutagenesis. Recombinant strains were isolated and analysed, and one hupL − strain, NHM5, was selected for further study. Cultures of NHM5 were grown under nitrogen-fixing conditions and H 2 evolution under air was observed using an H 2 electrode.

Shewanella oneidensis : a new and efficient System for Expression and Maturation of heterologous [Fe-Fe] Hydrogenase from Chlamydomonas reinhardtii

BackgroundThe eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii.ResultsBased on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (ΔhydAΔhyaB) transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase.ConclusionIn the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 – 0.5 mg l-1(OD600 = 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of ~700 μmol H2 mg-1 min-1.

Production of Squalene in Synechocystis sp. PCC 6803

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.

Design, Engineering, and Construction of Photosynthetic Microbial Cell Factories for Renewable Solar Fuel Production

There is an urgent need to develop sustainable solutions to convert solar energy into energy carriers used in the society. In addition to solar cells generating electricity, there are several options to generate solar fuels. This paper outlines and discusses the design and engineering of photosynthetic microbial systems for the generation of renewable solar fuels, with a focus on cyanobacteria. Cyanobacteria are prokaryotic microorganisms with the same type of photosynthesis as higher plants. Native and engineered cyanobacteria have been used by us and others as model systems to examine, demonstrate, and develop photobiological H2 production. More recently, the production of carbon-containing solar fuels like ethanol, butanol, and isoprene have been demonstrated. We are using a synthetic biology approach to develop efficient photosynthetic microbial cell factories for direct generation of biofuels from solar energy. Present progress and advances in the design, engineering, and construction of such cyanobacterial cells for the generation of a portfolio of solar fuels, e.g., hydrogen, alcohols, and isoprene, are presented and discussed. Possibilities and challenges when introducing and using synthetic biology are highlighted.

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

ABSTRACT Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H2), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O2, CO2, N2, and H2 was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO2 and O2. The amount of H2 produced per molecule of N2 fixed was found to vary with light conditions, high light giving a greater increase in H2 production than N2 fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H2 produced per molecule of N2 fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO2, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)−1 h−1 to 9 μmol (mg of chlorophyll a)−1 h−1 after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.

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

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.

Terpenoids and Their Biosynthesis in Cyanobacteria

Terpenoids, or isoprenoids, are a family of compounds with great structural diversity which are essential for all living organisms. In cyanobacteria, they are synthesized from the methylerythritol-phosphate (MEP) pathway, using glyceraldehyde 3-phosphate and pyruvate produced by photosynthesis as substrates. The products of the MEP pathway are the isomeric five-carbon compounds isopentenyl diphosphate and dimethylallyl diphosphate, which in turn form the basic building blocks for formation of all terpenoids. Many terpenoid compounds have useful properties and are of interest in the fields of pharmaceuticals and nutrition, and even potentially as future biofuels. The MEP pathway, its function and regulation, and the subsequent formation of terpenoids have not been fully elucidated in cyanobacteria, despite its relevance for biotechnological applications. In this review, we summarize the present knowledge about cyanobacterial terpenoid biosynthesis, both regarding the native metabolism and regarding metabolic engineering of cyanobacteria for heterologous production of non-native terpenoids.