Characterizing isoprene production in cyanobacteria - Insights into the effects of light, temperature, and isoprene on Synechocystis sp. PCC 6803.

Engineering cyanobacteria for the production of isoprene and other terpenoids has gained increasing attention in the field of biotechnology. Several studies have addressed optimization of isoprene synthesis in cyanobacteria via enzyme and pathway engineering. However, only little attention has been paid to the optimization of cultivation conditions. In this study, an isoprene-producing strain of Synechocystis sp. PCC 6803 and two control strains were grown under a variety of cultivation conditions. Isoprene production, as quantified by modified membrane inlet mass spectrometer (MIMS) and interpreted using Flux Balance Analysis (FBA), increased under violet light and at elevated temperature. Increase of thermotolerance in the isoprene producer was attributed to the physical presence of isoprene, similar to plants. The results demonstrate a beneficial effect of isoprene on cell survival at higher temperatures. This increased thermotolerance opens new possibilities for sustainable bio-production of isoprene and other products.

Mapping competitive pathways to terpenoid biosynthesis in Synechocystis sp. PCC 6803 using an antisense RNA synthetic tool.

BACKGROUND: Synechocystis sp. PCC 6803 utilizes pyruvate and glyceraldehyde 3-phosphate via the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of terpenoids. Considering the deep connection of the MEP pathway to the central carbon metabolism, and the low carbon partitioning towards terpenoid biosynthesis, significant changes in the metabolic network are required to increase cyanobacterial production of terpenoids. RESULTS: We used the Hfq-MicC antisense RNA regulatory tool, under control of the nickel-inducible PnrsB promoter, to target 12 different genes involved in terpenoid biosynthesis, central carbon metabolism, amino acid biosynthesis and ATP production, and evaluated the changes in the performance of an isoprene-producing cyanobacterial strain. Six candidate targets showed a positive effect on isoprene production: three genes involved in terpenoid biosynthesis (crtE, chlP and thiG), two involved in amino acid biosynthesis (ilvG and ccmA) and one involved in sugar catabolism (gpi). The same strategy was applied to interfere with different parts of the terpenoid biosynthetic pathway in a bisabolene-producing strain. Increased bisabolene production was observed not only when interfering with chlorophyll a biosynthesis, but also with carotenogenesis. CONCLUSIONS: We demonstrated that the Hfq-MicC synthetic tool can be used to evaluate the effects of gene knockdown on heterologous terpenoid production, despite the need for further optimization of the technique. Possible targets for future engineering of Synechocystis aiming at improved terpenoid microbial production were identified.

Metabolic engineering of Synechocystis sp. PCC 6803 for improved bisabolene production.

Terpenoids are a wide class of organic compounds with industrial relevance. The natural ability of cyanobacteria to produce terpenoids via the methylerythritol 4-phosphate (MEP) pathway makes these organisms appealing candidates for the generation of light-driven cell factories for green chemistry. Here we address the improvement of the production of (E)-α-bisabolene, a valuable biofuel feedstock, in Synechocystis sp. PCC 6803 via sequential heterologous expression of bottleneck enzymes of the native pathway. Expression of the bisabolene synthase is sufficient to complete the biosynthetic pathway of bisabolene. Expression of a farnesyl-pyrophosphate synthase from Escherichia coli did not influence production of bisabolene, while enhancement of the MEP pathway via additional overexpression of 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and IPP/DMAPP isomerase (IDI) significantly increased production per cell. However, in the absence of a carbon sink, the overexpression of DXS and IDI leads to significant growth impairment. The final engineered strain reached a volumetric titre of 9 â€‹mg â€‹L-1 culture of bisabolene after growing for 12 days. When the cultures were grown in a high cell density (HCD) system, we observed an increase in the volumetric titres by one order of magnitude for all producing-strains. The strain with improved MEP pathway presented an increase twice as much as the remaining engineered strains, yielding more than 180 â€‹mg â€‹L-1 culture after 10 days of cultivation. Furthermore, the overexpression of these two MEP enzymes prevented the previously reported decrease in the bisabolene specific titres when grown in HCD conditions, where primary metabolism is usually favoured. We conclude that fine-tuning of the cyanobacterial terpenoid pathway is crucial for the generation of microbial platforms for terpenoid production on industrial-scale.

Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes.

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol-∆sps, ∆ggpS, and ∆sps∆ggpS-were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis' native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803.

Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. Operating in a two-step semi-batch mode over a period of eight days, volumetric yields of (E)-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L-1. Likewise, yields of the sesquiterpene alcohols (-)-patchoulol and (-)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L-1 and 96.3 ± 2.2 mg * L-1, respectively. While specific productivity was compromised particularly for (E)-α-bisabolene in the HDC system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for (E)-α-bisabolene than for the hydroxylated terpenoids. Together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria.