Coupling dairy wastewaters for nutritional balancing and water recycling: sustainable heterologous 2-phenylethanol production by engineered cyanobacteria.

Microalgae biotechnology is hampered by the high production costs and the massive usage of water during large-volume cultivations. These drawbacks can be softened by the production of high-value compounds and by adopting metabolic engineering strategies to improve their performances and productivity. Today, the most sustainable approach is the exploitation of industrial wastewaters for microalgae cultivation, which couples valuable biomass production with water resource recovery. Among the food processing sectors, the dairy industry generates the largest volume of wastewaters through the manufacturing process. These effluents are typically rich in dissolved organic matter and nutrients, which make it a challenging and expensive waste stream for companies to manage. Nevertheless, these rich wastewaters represent an appealing resource for microalgal biotechnology. In this study, we propose a sustainable approach for high-value compound production from dairy wastewaters through cyanobacteria. This strategy is based on a metabolically engineered strain of the model cyanobacterium Synechococcus elongatus PCC 7942 (already published elsewhere) for 2-phenylethanol (2-PE). 2-PE is a high-value aromatic compound that is widely employed as a fragrance in the food and cosmetics industry thanks to its pleasant floral scent. First, we qualitatively assessed the impact of four dairy effluents on cyanobacterial growth to identify the most promising substrates. Both tank-washing water and the liquid effluent of exhausted sludge resulted as suitable nutrient sources. Thus, we created an ideal buffer system by combining the two wastewaters while simultaneously providing balanced nutrition and completely avoiding the need for fresh water. The combination of 75% liquid effluent of exhausted sludge and 25% tank-washing water with a fine-tuning ammonium supplementation yielded 180 mg L-1 of 2-PE and a biomass concentration of 0.6 gDW L-1 within 10 days. The mixture of 90% exhausted sludge and 10% washing water produced the highest yield of 2-PE (205 mg L-1) and biomass accumulation (0.7 gDW L-1), although in 16 days. Through these treatments, the phosphates were completely consumed, and nitrogen was removed in a range of 74%-77%. Overall, our approach significantly valorized water recycling and the exploitation of valuable wastewaters to circularly produce marketable compounds via microalgae biotechnology, laying a promising groundwork for subsequent implementation and scale-up.

Combining metabolite doping and metabolic engineering to improve 2-phenylethanol production by engineered cyanobacteria.

2-Phenylethanol (2-PE) is a rose-scented aromatic compound, with broad application in cosmetic, pharmaceutical, food and beverage industries. Many plants naturally synthesize 2-PE via Shikimate Pathway, but its extraction is expensive and low-yielding. Consequently, most 2-PE derives from chemical synthesis, which employs petroleum as feedstock and generates unwanted by products and health issues. The need for "green" processes and the increasing public demand for natural products are pushing biotechnological production systems as promising alternatives. So far, several microorganisms have been investigated and engineered for 2-PE biosynthesis, but a few studies have focused on autotrophic microorganisms. Among them, the prokaryotic cyanobacteria can represent ideal microbial factories thanks to their ability to photosynthetically convert CO2 into valuable compounds, their minimal nutritional requirements, high photosynthetic rate and the availability of genetic and bioinformatics tools. An engineered strain of Synechococcus elongatus PCC 7942 for 2-PE production, i.e., p120, was previously published elsewhere. The strain p120 expresses four heterologous genes for the complete 2-PE synthesis pathway. Here, we developed a combined approach of metabolite doping and metabolic engineering to improve the 2-PE production kinetics of the Synechococcus elongatus PCC 7942 p120 strain. Firstly, the growth and 2-PE productivity performances of the p120 recombinant strain were analyzed to highlight potential metabolic constraints. By implementing a BG11 medium doped with L-phenylalanine, we covered the metabolic burden to which the p120 strain is strongly subjected, when the 2-PE pathway expression is induced. Additionally, we further boosted the carbon flow into the Shikimate Pathway by overexpressing the native Shikimate Kinase in the Synechococcus elongatus PCC 7942 p120 strain (i.e., 2PE_aroK). The combination of these different approaches led to a 2-PE yield of 300 mg/gDW and a maximum 2-PE titer of 285 mg/L, 2.4-fold higher than that reported in literature for the p120 recombinant strain and, to our knowledge, the highest recorded for photosynthetic microorganisms, in photoautotrophic growth condition. Finally, this work provides the basis for further optimization of the process aimed at increasing 2-PE productivity and concentration, and could offer new insights about the use of cyanobacteria as appealing microbial cell factories for the synthesis of aromatic compounds.

Computational Analysis of Dynamic Light Exposure of Unicellular Algal Cells in a Flat-Panel Photobioreactor to Support Light-Induced CO2 Bioprocess Development.

Cyanobacterial cell factories trace a vibrant pathway to climate change neutrality and sustainable development owing to their ability to turn carbon dioxide-rich waste into a broad portfolio of renewable compounds, which are deemed valuable in green chemistry cross-sectorial applications. Cell factory design requires to define the optimal operational and cultivation conditions. The paramount parameter in biomass cultivation in photobioreactors is the light intensity since it impacts cellular physiology and productivity. Our modeling framework provides a basis for the predictive control of light-limited, light-saturated, and light-inhibited growth of the Synechocystis sp. PCC 6803 model organism in a flat-panel photobioreactor. The model here presented couples computational fluid dynamics, light transmission, kinetic modeling, and the reconstruction of single cell trajectories in differently irradiated areas of the photobioreactor to relate key physiological parameters to the multi-faceted processes occurring in the cultivation environment. Furthermore, our analysis highlights the need for properly constraining the model with decisive qualitative and quantitative data related to light calibration and light measurements both at the inlet and outlet of the photobioreactor in order to boost the accuracy and extrapolation capabilities of the model.

Response of the thylakoid proteome of Synechocystis sp. PCC 6803 to photohinibitory intensities of orange-red light.

Photoautotrophic growth of Synechocystis sp. PCC 6803 in a flat-panel photobioreactor, run in turbidostat mode under increasing intensities of orange-red light (636 nm), showed a maximal growth rate (0.12 h-1) at 300 µmolphotons m-2 s-1, whereas first signs of photoinhibition were detected above 800 µmolphotons m-2 s-1. To investigate the dynamic modulation of the thylakoid proteome in response to photoinhibitory light intensities, quantitative proteomics analyses by SWATH mass spectrometry were performed by comparing thylakoid membranes extracted from Synechocystis grown under low-intensity illumination (i.e. 50 µmolphotons m-2 s-1) with samples isolated from cells subjected to photoinhibitory light regimes (800, 950 and 1460 µmolphotons m-2 s-1). We identified and quantified 126 proteins with altered abundance in all three photoinhibitory illumination regimes. These data reveal the strategies by which Synechocystis responds to photoinibitory growth irradiances of orange-red light. The accumulation of core proteins of Photosystem II and reduction of oxygen-evolving-complex subunits in photoinhibited cells revealed a different turnover and repair rates of the integral and extrinsic Photosystem II subunits with variation of light intensity. Furthermore, Synechocystis displayed a differentiated response to photoinhibitory regimes also regarding Photosystem I: the amount of PsaD, PsaE, PsaJ and PsaM subunits decreased, while there was an increased abundance of the PsaA, PsaB, Psak2 and PsaL proteins. Photoinhibition with 636 nm light also elicited an increased capacity for cyclic electron transport, a lowering of the amount of phycobilisomes and an increase of the orange carotenoid protein content, all presumably as a photoprotective mechanism against the generation of reactive oxygen species.

Analysis of the light intensity dependence of the growth of Synechocystis and of the light distribution in a photobioreactor energized by 635 nm light.

Synechocystis gathered momentum in modelling studies and biotechnological applications owing to multiple factors like fast growth, ability to fix carbon dioxide into valuable products, and the relative ease of genetic manipulation. Synechocystis physiology and metabolism, and consequently, the productivity of Synechocystis-based photobioreactors (PBRs), are heavily light modulated. Here, we set up a turbidostat-controlled lab-scale cultivation system in order to study the influence of varying orange-red light intensities on Synechocystis growth characteristics and photosynthetic activity. Synechocystis growth and photosynthetic activity were found to raise as supplied light intensity increased up to 500 µmol photons m-2 s-1 and to enter the photoinhibition state only at 800 µmol photons m-2 s-1. Interestingly, reverting the light to a non-photo-inhibiting intensity unveiled Synechocystis to be able to promptly recover. Furthermore, our characterization displayed a clear correlation between variations in growth rate and cell size, extending a phenomenon previously observed in other cyanobacteria. Further, we applied a modelling approach to simulate the effects produced by varying the incident light intensity on its local distribution within the PBR vessel. Our model simulations suggested that the photosynthetic activity of Synechocystis could be enhanced by finely regulating the intensity of the light incident on the PBR in order to prevent cells from experiencing light-induced stress and induce their exploitation of areas of different local light intensity formed in the vessel. In the latter case, the heterogeneous distribution of the local light intensity would allow Synechocystis for an optimized usage of light.

Atypical effect of temperature tuning on the insertion of the catalytic iron-sulfur center in a recombinant [FeFe]-hydrogenase.

The expression of recombinant [FeFe]-hydrogenases is an important step for the production of large amount of these enzymes for their exploitation in biotechnology and for the characterization of the protein-metal cofactor interactions. The correct assembly of the organometallic catalytic site, named H-cluster, requires a dedicated set of maturases that must be coexpressed in the microbial hosts or used for in vitro assembly of the active enzymes. In this work, the effect of the post-induction temperature on the recombinant expression of CaHydA [FeFe]-hydrogenase in E. coli is investigated. The results show a peculiar behavior: the enzyme expression is maximum at lower temperatures (20°C), while the specific activity of the purified CaHydA is higher at higher temperature (30°C), as a consequence of improved protein folding and active site incorporation.