Versatile Applications of Cyanobacteria in Biotechnology.

Cyanobacteria are blue-green Gram-negative and photosynthetic bacteria which are seen as one of the most morphologically numerous groups of prokaryotes. Because of their ability to fix gaseous nitrogen and carbon dioxide to organic materials, they are known to play important roles in the universal nutrient cycle. Cyanobacteria has emerged as one of the promising resources to combat the issues of global warming, disease outbreaks, nutrition insecurity, energy crises as well as persistent daily human population increases. Cyanobacteria possess significant levels of macro and micronutrient substances which facilitate the versatile popularity to be utilized as human food and protein supplements in many countries such as Asia. Cyanobacteria has been employed as a complementary dietary constituent of feed for poultry and as vitamin and protein supplement in aquatic lives. They are effectively used to deal with numerous tasks in various fields of biotechnology, such as agricultural (including aquaculture), industrial (food and dairy products), environmental (pollution control), biofuel (bioenergy) and pharmaceutical biotechnology (such as antimicrobial, anti-inflammatory, immunosuppressant, anticoagulant and antitumor); recently, the growing interest of applying them as biocatalysts has been observed as well. Cyanobacteria are known to generate a numerous variety of bioactive compounds. However, the versatile potential applications of cyanobacteria in biotechnology could be their significant growth rate and survival in severe environmental conditions due to their distinct and unique metabolic pathways as well as active defensive mechanisms. In this review, we elaborated on the versatile cyanobacteria applications in different areas of biotechnology. We also emphasized the factors that could impede the implementation to cyanobacteria applications in biotechnology and the execution of strategies to enhance their effective applications.

Phosphonates enantiomers receiving with fungal enzymatic systems.

BACKGROUND: Phosphonates derivatives are in the area of interests because of their unique chemical-physical features. These compounds manifest variety of biological interactions within the sensitive living cells, including impact on particular enzymes activities. Biological "cause and effect" interactions are based upon the specific matching between the structures and/or compounds and this is usually the result of proper optical configurations of particular chiral moieties. Presented research is targeted to the phosphonates with the heteroatom incorporated in their side functionalities. Such molecules are described as possible substrates of bioconversion for the first time lately and this field is not fully explored. RESULTS: Presented research is targeted to the synthesis of pure hetero-phosphonates enantiomers. The catalytic activity of yeasts and moulds were tested towards two substrates: the thienyl and imidazole phosphonates to resolve their racemic mixtures. Biotransformations conditions differed depending on the outcome, what included changing of following parameters: type of cultivation media, bioprocess duration (24-72 h), additional biocatalyst pre-treatment (24-48 h starvation step triggering the secondary metabolism). (S)-1-amino-1-(3-thienyl)methylphosphonate was produced with the assistance of R. mucilaginosa or A. niger (e.e. up to 98% and yield up to 100%), starting from the 3 mM of substrate racemic mixture. Bioconversion of racemic mixture of 3 mM of (1-amino-1-(4-imidazole)methylphosphonic acid) resulted in the synthesis of S-isomer (up to 95% of e.e.; 100% of yield) with assistance of R. mucilaginosa. 24 h biotransformation was conducted with biomass preincubated under 48-hour starvation conditions. Such stereoselective resolution of the racemic mixtures of substrates undergoes under kinetic control with the conversion of one from the enantiomers. CONCLUSIONS: Composition of the culturing media and pre-incubation in conditions of nutrient deficiency were significant factors influencing the results of kinetic resolution of racemic mixtures of phosphonic substrates and influencing the economic side of the biocatalysis e.g. by determining the duration of whole biocatalytic process.

(S)-Thienyl and (R)-Pirydyl phosphonate Derivatives Synthesized by Stereoselective Resolution of Their Racemic Mixtures With Rhodotorula mucilaginosa (DSM 70403) - Scaling Approaches.

Rhodotorula mucilaginosa was successfully applied as a biocatalyst for the enantioselective resolution of the racemic mixtures of heteroatom phosphonates derivatives, resulting in receiving the following enantiomers: (S)-1-amino-1(2-thienyl)methylphosphonic acid (Product 1) and (R)-1-amino-1-(3'pirydyl) methylphosphonic acid (Product 2). Biological synthesis of both products is reported for the first time. Pure (S)-1-amino-1-(2-thienyl)methylphosphonic acid (Product 1) was isolated with a conversion degree of 50% after 24 h of biotransformation was conducted on a laboratory scale under moderate conditions (1.55 mM of substrate 1, 100 mL of distilled water, 135 rpm, 25°C; Method A). The scale was enlarged to semi-preparative one, using a simplified flow-reactor (Method C; 3.10 mM of substrate 1) and immobilized biocatalyst. The product was isolated with a conversion degree of 50% just after 4 h of biotransformation. Amino-1-(3'pirydyl)methylphosphonic acid (Substrate 2) was converted according to novel procedure, by the immobilized biocatalyst - Rhodotorula mucilaginosa. The process was carried out under moderate conditions (3.19 mM - substrate 2 solution; Method C1) with the application of a simplified flow reactor system, packed with the yeasts biomass entrapped in 4% agar-agar solution. Pure (R)-amino-1-(3'pirydyl)methylphosphonic (50% of conversion degree) was received within only 48 h.

Biogenic synthesis of silica nanoparticles from corn cobs husks. Dependence of the productivity on the method of raw material processing.

Corn processing generates thousands of tons of cob husks, which still contains many valuable elements. To make the most of these wastes, they are applied as substrates for biotransformation's procedures. This approach allowed converting or releasing, the elements deposited in the plant material and obtaining valuable products. Thus bioconversion of corn cob husks (CCH) using a fungus of the Fusarium culmorum genus resulted in obtaining silica nanoparticles of defined size and morphology. SEM analysis excluded their presence on the surface of the substrate. FTIR confirmed the presence of siloxane bonds and O-Si-O bonds in post-biotransformation fluid. Using the Heteropoly Blue Method, it was checked that the highest concentration of silica during 16-day biotransformation falls on the 7th day of the process, in which both the substrate sterilization and the process of the biocatalyst starvation were of key importance. Using the STEM and EDX analysis, it was proved that the obtained nanoparticles with a spherical form are structured and their dimensions are ~40 and ~70 nm. ICP-OES proved that the overall process efficiency was 47%. Such nanoparticles can be successfully used in the medical industry.

Reductive capabilities of different cyanobacterial strains towards acetophenone as a model substrate - Prospect of applications for chiral building blocks synthesis.

Bioreductive capabilities of four morphologically different strains of cyanobacteria have been assessed in this work. Arthrospira maxima, Leptolyngbya foveolarum, Nodularia sphaerocarpa and Synechococcus bigranulatus were applied as catalysts for the reduction of acetophenone to the corresponding chiral phenylethyl alcohol. The process was modified regarding substrate concentration, duration of pre-cultivation period, duration of biotransformation, light regime and glucose addition to the culture media. Obtained results clearly showed that cyanobacteria were active towards acetophenone what resulted in the substrate reduction to (S)-1-phenylethanol with high enantiomeric excess. The reaction efficiency increased with the biotransformation time, but the higher concentration of substrate limited the process yield. Also, all tested strains performed reaction with the highest efficacy under continuous light regime. The most active strains - N. sphaerocarpa and S. bigranulatus carried out the conversion of 1 mM acetophenone with high efficiency of respectively 97.6% and 96.2% after 13 days of biotransformation. A. maxima reached 45.8% of conversion after 13 days of biotransformation whereas L. foveolarum did not exceed 20%. The enantiomeric excesses were respectively 98.8%- A. maxima, 91.7%- L. foveolarum, 72.6%- S. bigranulatus and N. sphaerocarpa 16.2%.

First biological conversion of chiral heterophosphonate derivative - Scaling and paths of conversion discussion.

Presented work describes the first approach for the biocatalytic resolution of racemic mixtures of heterophosphonate derivative. Penicillium funiculosum and Rhodotorula mucilaginosa were successfully applied for the biological conversion of racemic mixture of 1-amino-1-(3'-pyridyl)methylphosphonic acid 3. Both microorganisms carried out the kinetically driven process leading to conversion of one from the substrate enantiomers, leaving the second one unreacted. Application of R. mucilaginosa allowed obtaining pure enantiomer of the substrate (yield 100%, e.e 100% - unreacted isomer) after 24 h of biotransformation of 3 in the laboratory scale process (Method E), applying biocatalyst pre-treatment step - 24 h of starvation. In case of other biocatalyst, application of whole cells of P. funiculosum in laboratory scale process, also resulted in conversion of the racemic mixture of substrate 3via oxidative deamination into ketone derivative, which was then bioreduced (second step of the process) into 1-hydroxy-1-(3'-pyridyl)methylphosphonic acid 4. This time two products were isolated: unreacted substrate and hydroxy compound 4. Conversion degree ranged from 30% (standard procedure, method A) to even 70% (with extra addition of sodium pyruvate - method B2). However, in this case, bioconversion was not enantioselective - products: amino- and hydroxyderivative were obtained as racemic mixtures. Both biocatalysts were also tested towards the scaling so other biocatalytic procedures were introduced - with immobilized fungal mycelium. In case of Rhodotorula mucilaginosa this approach failed (data not shown) but Penicillium funiculosum turned out to be active and also selective. Thus, application of this biocatalyst in the half-preparative scale, continuous-flow bioprocess (Method C2) resulted in the obtaining of pure S-3 (100% e.e.) isomer with the 100% of conversion degree, without any side products. Recorded NMR spectra allowed confirming the reaction progress and its selectivity and also postulating possible mechanism of conversion.

Nanosilica synthesis mediated by Aspergillus parasiticus strain.

Rice husks (RHs) are plant waste materials abundant in phytoliths silica bodies. These were used as starting material for fungal-mediated biotransformation leading to the synthesis of a high-value added product. A strain of Aspergillus parasiticus was capable of transforming the amorphous silica conglomerates into structured nanoparticles (NPs) in the process of RHs biotransformation. Silica NPs were produced extracellularly and their size ranged from 3 to 400 nm depending on the biotransformation conditions and the post-biotransformation supernatant processing. To characterize the NP's structure and dimension, SEM, STEM, EDX and FTIR technics were applied. These demonstrated and confirmed that pyramid (400 nm), cubical (85 nm) and spherical (3 nm and 24 ± 8 nm) forms of silica NPs were obtained.

Fungal synthesis of chiral phosphonic synthetic platform - Scope and limitations of the method.

Chiral hydroxyphosphonates due to their wide range of biological properties are industrially important chemicals. Chemical synthesis of their optical isomers is expensive, time consuming and not friendly to the environment, so biotransformations are under consideration. Among others, these compounds act as enzymes inhibitors. This makes the bioconversions of phosphonates, especially scaling experiments, hard to perform. Biocatalysis is one of the methods that can be applied in synthesis of optically pure compounds. To increase the efficiency of the process with whole cell biocatalysts, it is essential to ensure optimal reaction conditions that minimize cellular stress and can enhance the metabolic activity of cells. The present investigation focuses on the scaling up of the kinetic resolution of racemic mixture of 2-butyryloxy-2-(ethoxy-P-phenylphosphinyl)acetic acid, applying free and immobilized form of the fungal biocatalysts and two operation systems: shake flask and recirculated fixed-bed batch reactor. Protocols of effective mycelium immobilization on polyurethane foams were set for T. purpurogenus IAFB 2512, F. oxysporum, P. commune. The best results of biotransformation were obtained with the immobilized P. commune in the column recirculated fixed-bed batch reactor. The conversion reaches 56% (maximal for the kinetic process) and the enantiomeric enrichment of the isomers mixture ranges between 82 and 93% (93% for ester of RP,R conformation). All biocatalysts exhibit SP-preference toward tested compound, what is essential because of importance of the phosphorus atom chirality for its biological activity.

Reductive activity of free and immobilized cells of cyanobacteria toward oxophosphonates-comparative study.

This report, based on the previous studies, compares the reductive activity of different modes of following photobiocatalysts (on laboratory and preparative scale): Arthrospira maxima, Nostoc cf. muscorum and Nodularia sphaerocarpa, toward diethyl esters of 2-oxopropylphosphonate (1), 2-oxo-2-phenylethylphosphonate (2), and 2-oxobutylphosphonate (3). It was confirmed that immobilization in alginate matrix do not affect the activity and viability of the biocatalysts. Corresponding (S)-hydroxyphosphonates (1a-3a) were obtained with similar efficiency compared to the free-cell mode with the yield and of the optical purity e.e respectively (e.g., N. sphaerocarpa experiments): (1) yield: 21 %, e.e. 84 %; (2) yield 97 %, e.e. 97; (3) yield 21 %, e.e. 89 %. Scaling up the processes for the best biocatalyst, N. sphaerocarpa, indicated that the use of free-living cells of cyanobacteria is more effective (640 mg of substrate 2, 44 % of yield, 91 % of e.e.), compared to the column bioreactor packed with immobilized cells of this photobiocatalyst (384 mg of substrate 2, 38 % of yield, 86 % of e.e). In the case of free and immobilized cells of N. cf. muscorum, agitation of the medium was the crucial activity mediator. Shaking culture of free cells of N. cf. muscorum converted the diethyl 2-oxo-2-phenylethylphosphonate (2) with the yield of 43 % (99 % of e.e.) compared to 18 % (99 % of e.e., stationary culture). Immobilized cells of this cyanobacterium were also more active toward (2) under shaking conditions (28 % of yield, 99 % of e.e.) than free ones without agitation.

Enantio convergent biotransformation of O,O-dimethyl-4-oxoazetidin-2-ylphosphonate using fungal cells of Penicillium minioluteum and purified enzymes.

This report presents the bioconversion of O,O-dimethyl-4-oxoazetidin-2-ylphosphonate 1 performed in two ways: with the enzymatic system of P. minioluteum and with the application of purified enzymes: penicillinase and two proteases of different origin. Recorded NMR spectra allowed confirming the reaction progress and also postulating possible mechanism of conversion. The path of bioconversion was defined as enantio convergent process for both modes of applied biocatalysts. This means that kinetically driven resolution of racemic mixture of the substrate leads to the one enantiomer of the product. The bioconversion started from ester bond hydrolysis (equally in both enantiomers) with the conversion degree from 30% (whole-cell) to 35% (isolated enzymes) and with the production of optically pure monoester (compound 2; 100% of e.e). For whole-cell bioprocess it was the initiative step for the enantioselective amide bond hydrolysis, what resulted in synthesis of desired product 3-amino-3-phosphonopropanoic acid 4. However, the most effective enzymatic hydrolysis of ester bond performed with penicillinase from Enterobacter cloacae led only to the monoester product 2.

Lipases and whole cell biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid and its ester.

A wide spectrum of commercially available lipases and microbial whole cells catalysts were tested for biotransformations of 2-hydroxy-2-(ethoxyphenylphosphinyl)acetic acid 1 and its butyryl ester. The best results were achieved for biocatalytic hydrolysis of ester: 2-butyryloxy-2-(ethoxyphenylphosphinyl)acetic acid 2 performed by lipase from Candida cylindracea, what gave optically active products with 85% enantiomeric excess, 50% conversion degree and enantioselectivity 32.9 for one pair of enantiomers. Also enzymatic systems of Penicillium minioluteum and Fusarium oxysporum were able to hydrolyze tested compound with high enantiomeric excess (68-93% ee), enantioselectivity (44 for one pair of enantiomers) and conversion degree about 50-55%. Enzymatic acylation of hydroxyphosphinate was successful in case when porcine pancreas lipase was used. After 4days of biotransformation the conversion reaches 45% but the enantiomeric enrichment of the isomers mixture do not exceed 43%. Obtained chiral compounds are valuable derivatizing agents for spectroscopic (NMR) evaluation of enantiomeric excess for particular compounds (e.g. amino acids).

Fungal platform for direct chiral phosphonic building blocks production. Closer look on conversion pathway.

The application of Rhodospirillum toruloides strain allowed resolving the chemically synthesized racemic mixtures of following chiral aminophosphonic acids: 1-aminoethylphosphonic acid (1), 1-amino-1-iso-propyl-1-phosphonic acid (2), 1-amino-1-phenylmethylphosphonic acid (4) and 1-amino-2-phenylethylphosphonic acid (3). The applied protocols resulted in obtaining pure (R)-1-aminoethylphosphonic acid (100 % of e.e.) and enantiomerically enriched mixtures of other phosphonates (73 % e.e. of (S)-1-amino-1-phenylmethylphosphonic acid, 51 % e.e. of (R)-1-amino-2-phenylethylphosphonic acid and 40 % e.e. of (S)-1-amino-2-methylpropylphosphonic acid). Products are valuable chiral building blocks and serve as aminophosphonic acids platform for further applications. Performed experiments allowed to define the path of xenobiotics bioconversion.

2-Aminoethylphosphonate utilization by the cold-adapted Geomyces pannorum P11 strain.

Cold-adapted strain of Geomyces pannorum P11 was found to mineralize of phosphorus-carbon bond-containing compound--2-aminoethylphosphonic acid (2-AEP, ciliatine). The biodegradation process proceeded in the phosphate-independent manner. Ciliatine-metabolizing enzymes' activity was detectable in cell-free extracts prepared from psychrophilic G. pannorum pregrown on 4 mM 2-AEP. Phosphonoacetaldehyde hydrolase (phosphonatase) activity in a partially purified extract was demonstrated at 10 °C.

Chiral phosphinate degradation by the fusarium species: scope and limitation of the process.

Biodegradable capacities of fungal strains of Fusarium oxysporum (DSMZ 2018) and Fusarium culmorum (DSMZ 1094) were tested towards racemic mixture of chiral 2-hydroxy-2-(ethoxyphenylphosphinyl) acetic acid-a compound with two stereogenic centres. The effectiveness of decomposition was dependent on external factors such as temperature and time of the process. Optimal conditions of complete mineralization were established. Both Fusarium species were able to biodegrade every isomer of tested compound at 30°C, but F. culmorum required 10 days and F. oxysporum 11 days to accomplish the process, which was continuously monitored using the (31)P NMR technique.

Comparative study of fungal cell disruption--scope and limitations of the methods.

Simple and effective protocols of cell wall disruption were elaborated for tested fungal strains: Penicillium citrinum, Aspergillus fumigatus, Rhodotorula gracilis. Several techniques of cell wall disintegration were studied, including ultrasound disintegration, homogenization in bead mill, application of chemicals of various types, and osmotic shock. The release of proteins from fungal cells and the activity of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, in the crude extracts were assayed to determine and compare the efficacy of each method. The presented studies allowed adjusting the particular method to a particular strain. The mechanical methods of disintegration appeared to be the most effective for the disintegration of yeast, R. gracilis, and filamentous fungi, A. fumigatus and P. citrinum. Ultrasonication and bead milling led to obtaining fungal cell-free extracts containing high concentrations of soluble proteins and active glucose-6-phosphate dehydrogenase systems.

Biocatalytic resolution of enantiomeric mixtures of 1-aminoethanephosphonic acid.

Several fungal strains, namely Bauveria bassiana, Cuninghamella echinulata, Aspergillus fumigatus, Penicillium crustosum and Cladosporium herbarum, were used as biocatalysts to resolve racemic mixtures of 1-aminoethanephosphonic acid using L/D amino acid oxidase activity. The course of reaction was analyzed by 31P-NMR in the presence of cyclodextrin used as chiral discriminating agent. The best result (42% e.e of R-isomer) was obtained with a strain of Cuninghamella echinulata.

Application of the Beauveria bassiana strain for the enantioselective oxidation of the diethyl 1-hydroxy-1-phenylmethanephosphonate.

Biotransformation of diethyl 1-hydroxy-1-phenylmethanephosphonate using fungi Beauveria bassiana allowed resolving the racemic mixture of the substrate and due to the biocatalyst and reaction conditions modifications, leading to desired optical isomer.

A simple and green procedure for the microbial effective synthesis of 1-phenylethyl alcohol in both enantiomeric forms.

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40-80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker's yeast.

Application of fungi as biocatalysts for the reduction of diethyl 1-oxoalkylphosphonates in anhydrous hexane.

Five different species of microorganisms, namely, Rhodotorula rubra, Rhodotorula glutinis, Cladosporium sp., Verticillium sp., and baker's yeast, turned out to be useful biocatalysts for enantioselective reduction of a variety of diethyl 1-oxoalkylphosphonates. To suppress substrate decomposition, bioreductions were carried out under anhydrous conditions, using lyophilized cells immobilized on Celite R 630. The influence of reaction conditions such as biotransformation time and chemical additives on the yield of the reaction is discussed.