Background: Cyanobacteria can utilize solar energy and convert carbon dioxide into biofuel molecules in one single biological system. Synechocystis sp. PCC 6803 is a model cyanobacterium for basic and applied research. Alkanes are the major constituents of gasoline, diesel and jet fuels. A two-step alkane biosynthetic pathway was identified in cyanobacteria recently. It opens a door to achieve photosynthetic production of alka(e)nes with high efficiency by genetically engineering cyanobacteria. Results: A series of Synechocystis sp. PCC6803 mutant strains have been constructed and confirmed. Overexpression of both acyl-acyl carrier protein reductase and aldehyde-deformylating oxygenase from several cyanobacteria strains led to a doubled alka(e)ne production. Redirecting the carbon flux to acyl- ACP can provide larger precursor pool for further conversion to alka(e)nes. In combination with the overexpression of alkane biosynthetic genes, alka(e)ne production was significantly improved in these engineered strains. Alka(e)ne content in a Synechocystis mutant harboring alkane biosynthetic genes over-expressed in both slr0168 and slr1556 gene loci (LX56) was 1.3% of cell dry weight, which was enhanced by 8.3 times compared with wildtype strain (0.14% of cell dry weight) cultivated in shake flasks. Both LX56 mutant and the wildtype strain were cultivated in column photo-bioreactors, and the alka(e)ne production in LX56 mutant was 26 mg/L (1.1% of cell dry weight), which was enhanced by 8 times compared with wildtype strain (0.13% of cell dry weight). Conclusions: The extent of alka(e)ne production could correlate positively with the expression level of alkane biosynthetic genes. Redirecting the carbon flux to acyl-ACP and overexpressing alkane biosynthetic genes simultaneously can enhance alka(e)ne production in cyanobacteria effectively.

Background: Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria. Results: Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent. Conclusions: Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.

Cyanobacteria are the oldest photosynthetic microorganisms with good environmental adaptability. They are ubiquitous in light-exposed habitats on Earth. In recent years, cyanobacteria have become an ideal platform for producing biofuels and biochemicals from solar energy and carbon dioxide. Alka(e)nes are the main constituents of gasoline, diesel, and jet fuels. Alka(e)ne biosynthesis pathways are present in all sequenced cyanobacteria. Most cyanobacteria biosynthesize long chain alka(e)nes via acyl-acyl-carrier proteins reductase (AAR) and aldehyde-deformylating oxygenase (ADO). Alka(e)nes can be biodegraded by a variety of cyanobacteria, which lack a β-oxidation pathway. However, the mechanisms of alka(e)ne biodegradation in cyanobacteria remain elusive. In this study, a cyanobacterial alka(e)ne biodegradation pathway was uncovered by in vitro enzyme assays. Under high light, alka(e)nes in the membrane can be converted into alcohols and aldehydes by ADO, and aldehyde dehydrogenase (ALDH) can then convert the aldehydes into fatty acids to maintain lipid homeostasis in cyanobacteria. As highly reduced molecules, alka(e)nes could serve as electron donors to further reduce partially reduced reactive oxygen species (ROS) in cyanobacteria under high light. Alka(e)ne biodegradation may serve as an emergency mechanism for responding to the oxidative stress generated by excess light exposure. This study will shed new light on the roles of alka(e)ne metabolism in cyanobacteria. It is important to reduce the content of ROS by optimization of cultivation and genetic engineering for efficient alka(e)ne biosynthesis in cyanobacteria.

Cyanobacteria are essential primary producers in aquatic ecosystems. Silver nanoclusters (Ag NCs) are emerging ultrasmall nanomaterials with broad applications; however, they inevitably enter aquatic environments. This study investigated the nanotoxicity of Ag NCs on model cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942), revealing concentration-dependent growth inhibition with a 72 h half-maximal effective concentration of 1.19 mg L⁻¹. Ag NCs internalized into Syn7942 cells severely impaired the photosynthetic machinery, evidenced by reduced chlorophyll a content and suppressed photochemical reactions, leading to impaired electron transport. This photosynthetic dysfunction resulted in substantial reactive oxygen species generation and lipid peroxidation, as indicated by elevated levels of malondialdehydes. Although the activities of key antioxidant enzymes were enhanced, oxidative damage ultimately overwhelmed the cellular defense capacity. Furthermore, quantitative analysis of silver ions (Ag⁺) release coupled with comparative toxicity assays of Ag⁺ and Ag NCs confirmed that the toxicity originates from the Ag NCs themselves, rather than from the released Ag⁺. Transcriptional level analysis revealed that Ag NCs altered the expression of genes involved in photosynthesis and metal detoxification. Collectively, this study reveals a unique toxicity mechanism for Ag NCs distinct from that for Ag NPs and Ag⁺, highlighting the potential ecological risks posed by ultrasmall nanomaterials.