Abstract
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.
Highlights
Isoprenoids, called terpenoids, are a large family of compounds including carotenoids, tocopherol, phytol, sterols and hormones
The putative squalene hopene cyclase (Shc) amino acid sequence is homologous to other Shcs in the databases, with identity/similarity of 43%/58% to the structurally known Shc from A. acidocaldarius (PDB: 2SQC_A), and contains known conserved motifs such as the catalytic aspartate identified in A. acidocaldarius [21], a DXDD motif in the active site cavity important for the activity of the enzyme [23], and repeated QW-motifs [24]
Shc does not appear to be universally present in cyanobacteria
Summary
Isoprenoids, called terpenoids, are a large family of compounds including carotenoids, tocopherol, phytol, sterols and hormones. In algae, and in plant plastids, isoprenoids can be produced via the methyl-erythritol-4-phosphate (MEP) pathway ([1], see Fig. 1). This pathway was first characterized in Escherichia coli, and uses pyruvate and glyceraldehyde 3-phosphate as substrates [2] to form, in several steps, the products isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (see Fig. 1). A few studies have investigated this pathway in cyanobacteria [5,6,7,8], despite the fact that it is the origin of many interesting and potentially useful compounds. We are interested in investigating if it is possible to use cyanobacteria for generation of longer-chain isoprenoid hydrocarbons. Using cyanobacteria for direct production of a biofuel is advantageous, since they can grow photosynthetically on minimal media, fixing carbon dioxide from air and using sunlight as an energy source to generate the product
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