Photosynthetic production of α-farnesene by engineered Synechococcus elongatus UTEX 2973 from carbon dioxide.

Cyanobacteria are the prospective biosolar cell factories to produce a range of bioproducts through CO2 sequestration. Farnesene is a sesquiterpene with an array of applications in biofuels, pest management, cosmetics, flavours and fragrances. This is the first time a codon-optimized farnesene synthase (AFS) gene is engineered into the genomic neutral site of Synechococcus elongatus UTEX 2973 for farnesene synthesis through its endogenous methylerythritol phosphate (MEP) pathway, rendering UTEX AFS strain. Similarly, bottleneck gene(s) of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and/or fusion of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase (idispA) were engineered engendering UTEX AFS::dxs, UTEX AFS::idispA and UTEX AFS::dxs::idispA strains. UTEX AFS::dxs::idispA achieves farnesene productivity of 2.57 mg/L/day, the highest among engineered cyanobacterial strains studied so far. It demonstrates farnesene production, which is 31.3-times higher than the UTEX AFS strain. Moreover, the engineered strains show similar productivity over a three-month period, stipulating the genetic stability of the strains.

Enhancement of isoprene production in engineered Synechococcus elongatus UTEX 2973 by metabolic pathway inhibition and machine learning-based optimization strategy.

An engineered Synechococcus elongatus UTEX 2973-IspS.IDI is used to enhance isoprene production through geranyl diphosphate synthase (CrtE) inhibition and process parameters (light intensity, NaHCO3 and growth temperature) optimization approach. A cumulative isoprene production of 1.21 mg/gDCW was achieved with productivity of 12.6 µg/gDCW/h in culture supplemented with 20 µg/mL alendronate. This inhibition strategy improvises the cumulative isoprene production 5.76-fold in presence of alendronate. The maximum cumulative production of isoprene is observed to be 5.22 and 6.20 mg/gDCW (54.4 and 64.6 µg/gDCW/h) at statistical and artificial neural network genetic algorithm (ANN-GA) optimized conditions, respectively. The overall increase of isoprene production is found to be 29.52-fold using an integrated approach of inhibition and ANN-GA optimization in comparison to unoptimized cultures without alendronate. This study reveals that alendronate use as a potential inhibitor and machine learning based optimization is a better approach in comparison to statistical optimization to enhance the isoprene production.

Ganga river water quality assessment using combined approaches: physico-chemical parameters and cyanobacterial toxicity detection with special reference to microcystins and molecular characterization of microcystin synthetase (mcy) genes carrying cyanobacteria.

Water quality assessment relies mostly on physico-chemical-based characterization; however, eutrophication and climate change advocate the abundance of toxic microcystins (MCs) producing cyanobacteria as emerging bio-indicator. In the present study, a spatial-temporal analysis was carried out at ten sampling sites of Prayagraj and Varanasi during June 2017 and March 2018 to determine the Ganga River water quality using physico-chemical parameters, cyanobacteria diversity, detection of MCs producing strains and MC-LR equivalence. Coliform bacteria, COD, NO3-N, and phosphate are the significant contaminated parameters favoring the growth of putative MCs producing cyanobacteria. National Sanitation Foundation WQI (NSFWQI) indicates water quality, either bad or medium category at sampling points. The morphological analysis confirms the occurrence of diverse cyanobacterial genera such as Microcystis, Anabaena, Oscillatoria, and Phormidium. PCR amplification affirmed the presence of toxic microcystin (mcy) genes in uncultured cyanobacteria at all the sampling sites. The concentration of MC-LR equivalence in water samples by protein phosphatase 1 inhibition assay (PPIA) and high-performance liquid chromatography (HPLC) methods was observed in the range of 23.4-172 ng/L and 13.2-97.5 ng/L respectively which is lower than the harmful exposure limit by World Health Organization (WHO). Ganga isolate 1 was identified as Microcystis based on partial 16S rDNA sequence and its toxicity was confirmed due to presence of mcy genes and MCs production potential. These findings suggest the presence of MCs producers as new emerging parameter to monitor water quality index and identification up to species level will be valuable for restoration strategies of river Ganga.

Approaches in the photosynthetic production of sustainable fuels by cyanobacteria using tools of synthetic biology.

Cyanobacteria, photosynthetic prokaryotic microorganisms having a simple genetic composition are the prospective photoautotrophic cell factories for the production of a wide range of biofuel molecules. The simple genetic composition of cyanobacteria allows effortless genetic manipulation which leads to increased research endeavors from the synthetic biology approach. Various unicellular model cyanobacterial strains like Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been successfully engineered for biofuels generation. Improved development of synthetic biology tools, genetic modification methods and advancement in transformation techniques to construct a strain that can contain multiple foreign genes in a single operon have vastly expanded the functions that can be used for engineering photosynthetic cyanobacteria for the generation of various biofuel molecules. In this review, recent advancements and approaches in synthetic biology tools used for cyanobacterial genome editing have been discussed. Apart from this, cyanobacterial productions of various fuel molecules like isoprene, limonene, α-farnesene, squalene, alkanes, butanol, and fatty acids, which can be a substitute for petroleum and fossil fuels in the future, have been elaborated.