Cyanophycin modifications for applications in tissue scaffolding.

Cyanophycin (CGP) is a polypeptide consisting of amino acids-aspartic acid in the backbone and arginine in the side chain. Owing to its resemblance to cell adhesive motifs in the body, it can be considered suitable for use in biomedical applications as a novel component to facilitate cell attachment and tissue regeneration. Although it has vast potential applications, starting with nutrition, through drug delivery and tissue engineering to the production of value-added chemicals and biomaterials, CGP has not been brought to the industry yet. To develop scaffolds using CGP powder produced by bacteria, its properties (e.g., biocompatibility, morphology, biodegradability, and mechanical strength) should be tailored in terms of the requirements of the targeted tissue. Crosslinking commonly stands for a primary modification method for renovating biomaterial features to these extents. Herein, we aimed to crosslink CGP for the first time and present a comparative study of different methods of CGP crosslinking including chemical, physical, and enzymatic methods by utilizing glutaraldehyde (GTA), UV exposure, genipin, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), and monoamine oxidase (MAO). Crosslinking efficacy varied among the samples crosslinked via the different crosslinking methods. All crosslinked CGP were non-cytotoxic to L929 cells, except for the groups with higher GTA concentrations. We conclude that CGP is a promising candidate for scaffolding purposes to be used as part of a composite with other biomaterials to maintain the integrity of scaffolds. The initiative study demonstrated the unknown characteristics of crosslinked CGP, even though its feasibility for biomedical applications should be confirmed by further examinations. KEY POINTS: • Cyanophycin was crosslinked by 5 different methods • Crosslinked cyanophycin is non-cytotoxic to L929 cells • Crosslinked cyanophycin is a promising new material for scaffolding purposes.

A comparative study between two carboxymethylated polysaccharides/protein electrostatic and cross-linked nanogels constructed for caffeic acid and eugenol delivery.

In response to the pressing demand for functional nanomaterials synthesis and applications, two polyelectrolyte complexes (PECs) [electrostatic and cross-linked nanogels (NGs)] loaded individually with caffeic acid (CafA) and eugenol (Eug) demonstrating multifunctionalities were proposed for the first time. Curdlan (Curd) and glucomannan (GM) were carboxymethylated (CMCurd and CMGM) successfully and polymeric ratios of 1:1 and 4:1 (v/v) for chitosan (Cs): CMCurd and lactoferrin (Lf): CMGM were selected for the synthesis of Cs/CMCurd and Lf/CMGM NGs. Due to the use of EDC/NHS, Cs/CMCurd/CafA and Lf/CMGM/Eug NGs possessed very uniform particles sizes of 177 ± 18 and 230 ± 17 nm with marked encapsulation efficiencies (EEs) of 76 ± 4 and 88 ± 3 %, respectively. The formation of a carbonyl-amide linkage in both cross-linked NGs was confirmed by FTIR. It should be noted, the self-assembly was not reliable in retaining enough of the encapsulated compounds. Owing to the excellent physicochemical characteristics of the loaded cross-linked NGs, they were prioritized over the electrostatic ones. Both Cs/CMCurd/CafA and Lf/CMGM/Eug NGs exhibited high colloidal stability over 12 weeks, elevated hemocompatibility, and in vitro serum stability. The generated NGs were also tailored to possess controlled release profiles for CafA and Eug over 72 h. Cs/CMCurd/CafA and Lf/CMGM/Eug NGs had promising antioxidant efficacies and could remarkably inhibit 4 bacterial pathogens at low 2-16 µg/mL concentration of encapsulated NGs compared to their unencapsulated counterparts. Interestingly, the respective NGs could significantly decline the IC50 against colorectal cancer HCT-116 than conventional drugs. Based on these data, it was conferred that the investigated NGs could be promising candidates for functional foods and pharmaceutics.

Novel Biodegradable Nanoparticulate Chain-End Functionalized Polyhydroxybutyrate-Caffeic Acid with Multifunctionalities for Active Food Coatings.

The bioactivities of polyhydroxyalkanoates have been curtailed owing to the lack of bioactive functional groups in their backbones. In this regard, polyhydroxybutyrate (PHB) produced from new locally isolated Bacillus nealsonii ICRI16 was chemically modified for enhancing its functionality, stability as well as solubility. First, PHB was transformed to PHB-diethanolamine (PHB-DEA) by transamination. Subsequently, for the first time, the chain ends of the polymer were substituted by caffeic acid molecules (CafA), generating novel PHB-DEA-CafA. The chemical structure of such a polymer was confirmed by Fourier-transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance (1H NMR). The modified polyester demonstrated improved thermal behavior compared to PHB-DEA as was shown by thermogravimetric analysis, derivative thermogravimetry, and differential scanning calorimetry analyses. Interestingly, 65% of PHB-DEA-CafA was biodegraded in a clay soil environment after 60 days at 25 °C, while 50% of PHB was degraded within the same period. On another avenue, PHB-DEA-CafA nanoparticles (NPs) were successfully prepared with an impressive mean particle size of 223 ± 0.12 nm and high colloidal stability. The nanoparticulate polyester had powerful antioxidant capacity with an IC50 of 32.2 mg/mL, which was the result of CafA loading in the polymer chain. More importantly, the NPs had a considerable effect on the bacterial behavior of four food pathogens, inhibiting 98 ± 0.12% of Listeria monocytogenes DSM 19094 after 48 h of exposure. Finally, the raw polish sausage coated with NPs had a significantly lower bacterial count of 2.11 ± 0.21 log cfu/g in comparison to other groups. When all these positive features are recognized, the polyester described herein could be considered as a good candidate for commercial active food coatings.

Medium-Chain-Length Fatty Acid Catabolism in Cupriavidus necator H16: Transcriptome Sequencing Reveals Differences from Long-Chain-Length Fatty Acid ß-Oxidation and Involvement of Several Homologous Genes.

The number of genes encoding ß-oxidation enzymes in Cupriavidus necator H16 (synonym, Ralstonia eutropha H16) is high, but only the operons A0459-A0464 and A1526-A1531, each encoding four genes for ß-oxidation enzymes, were expressed during growth with long-chain-length fatty acids (LCFAs). However, we observed that C. necator ΔA0459-A0464 ΔA1526-A1531 and C. necator H16 showed the same growth behavior during growth with decanoic acid and shorter FAs. The negative effect of the deletion of these two operons increased with an increasing chain length of the utilized FAs. Transcriptome sequencing (RNA-Seq) revealed the expression profiles of genes involved in the catabolism of medium-chain-length fatty acids (MCFAs) in C. necator H16. Operon A0459-A0464 was expressed only during growth with nonanoic acid, whereas operon A1526-A1531 was highly expressed during growth with octanoic and nonanoic acid. The gene clusters B1187-B1192 and B0751-B0759 showed a log2 fold change in expression of up to 4.29 and 4.02, respectively, during growth with octanoic acid and up to 8.82 and 5.50, respectively, with nonanoic acid compared to sodium gluconate-grown cells. Several acyl-CoA ligases catalyze the activation of MCFAs with coenzyme A (CoA), but fadD3 (A3288), involved in activation of LCFAs, was not detected. The expression profiles of C. necator strain ΔA0459-A0464 ΔA1526-A1531 showed that the growth with nonanoic acid resulted in the expression of further ß-oxidation enzyme-encoding genes. Additional insights into the transport of FAs in C. necator H16 revealed the complexity and putative involvement of the DegV-like protein encoded by A0463 in the transport of odd-chain-length FAs and of siderophore biosynthesis in the transport mechanism. IMPORTANCE Although Cupriavidus necator H16 has been used in several studies to produce polyhydroxyalkanoates from various lipids, the fatty acid metabolism is poorly understood. The ß-oxidation of long-chain-length FAs has been investigated, but the tremendous number of homologous genes encoding ß-oxidation enzymes hides the potential for variances in the expressed genes for catabolism of shorter FAs. The catabolism of medium-chain-length FAs and connected pathways has not been investigated yet. As more sustainable substrates such as lipids and the production of fatty acids and fatty acid derivates become more critical with the dependency on fossil-based substances, understanding the complex metabolism in this highly diverse workhorse for biotechnology, C. necator, is inevitable. For further metabolic engineering and construction of production strains, we investigated the metabolism during growth on medium-chain-length FAs by RNA-Seq.

Sustainable curdlan biosynthesis by Rahnella variigena ICRI91 via alkaline hydrolysis of Musa sapientum peels and its edible, active and modified hydrogel for Quercetin controlled release.

Despite the high demand for curdlan (Curd), its industrial implementation has not reached a mature stage due to the high cost of simple sugar feed stocks. Herein, Musa sapientum peels hydrolysate (MPH) was proposed for the first time as a sustainable medium for Curd generation and as an ameliorated functional biomaterial for quercetin (Quer) sustained release. In this study, banana peels have been hydrolysed by 3 % NaOH catalyst/ 60 °C, yielding high concentration of glucose 20.5 ± 0.04 and 24.3 ± 0.11 g/L and reducing sugar amount, respectively. Meanwhile, a novel local Rahnella variigena ICRI91 strain was isolated from soil, that was useful for Curd production and identified by 16S rRNA analysis. Furthermore, three-batch fermentation models were carried out using MPH for obtaining a sufficient yield of Curd. R. variigena ICRI91 accumulated a satisfactory Curd concentration; 10.3 ± 0.25 g/L; using 60 g/L MPH. On the other hand, the strain produced an impressive Curd yield; 21.5 ± 0.13 g/L with an attained productivity of 0.179 ± 0.01 g/L/h and a sugar consumption of 68 ± 0.25 % as the MPH content increased to 100 g/L. For the first time, Curd hydrogel was modified by different amount of Xylitol (Xyl), reaching good mechanical performance; 3.1 MPa and 75 % for tensile strength (TS) and elongation at break (EB), respectively. Curd/Xyl (3/5) hydrogel was then integrated with nanometer-sized quercetin nanocrystals (Quer NCs, 83 ± 0.12 nm) with high colloidal stability of -23 ± 0.05 mV. The interconnected H- bonding between Xyl and Curd was confirmed by FTIR and SEM. The generated biomaterial was tailored to exhibit a sustained Quer release over 72 h. It also has improved antibacterial efficacy against four bacterial pathogens compared to that of a free drug. In recognition of these merits, an edible polymeric nanomaterial has been proposed for the functional food and biomedicine sectors.

The Hyperproduction of Polyhydroxybutyrate Using Bacillus mycoides ICRI89 through Enzymatic Hydrolysis of Affordable Cardboard.

Bioplastics are contemplated as remarkable substitutes for conventional plastics to accommodate green technological advancements. However, their industrial production has not been fully implemented owing to the cost of carbon resources. From another perspective, valorizing different paper mill wastes has become a prominent research topic. These materials may serve as an affording sustainable feedstock for bioplastic production. Adjustment of cardboard waste hydrolysate as suitable fermentation media for production of bacterial polyhydroxyalkanoates (PHAs) has been investigated. Cardboard samples were defibered and dried before enzymatic hydrolysis. The enzymatic degradation of commercial cellulase was monitored over 15 days. Interestingly, 18.2 ± 0.2 g/L glucose yield was obtained from 50 g cardboard samples using a 1.5% (v/v) enzyme concentration. The samples exhibited maximum weight loss values of 69-73%. Meanwhile, five soil samples were collected from local sites in Lodz, Poland. A total of 31 bacterial isolates were screened and cultured on Nile blue plates. Analysis of the 16S rRNA gene sequence of the most potent producer revealed 100% similarity to Bacillus mycoides. Cardboard hydrolysates whole medium, modified MSM with cardboard hydrolysate and nitrogen depleted MSM with cardboard hydrolysate were utilized for PHA production, followed by PHA productivity and cell dry weight (CDW) estimation compared to glucose as a standard carbon source. An impressive PHA accumulation of 56% CDW was attained when the waste hydrolysate was used as a carbon source. FTIR and NMR analysis of the isolated PHA indicated that functional groups of the polymer were related to PHB (polyhydroxybutyrate). Thermal analysis demonstrates that PHB and PHB-CB (PHB produced from cardboard hydrolysate) have degradation temperatures of 380 and 369 °C, respectively, which reflect the high thermal stability and heat resistance compared to the same properties for a standard polymer. This is the first demonstration of full saccharification of corrugated cardboard paper waste for high-level production of PHA. In addition, the attained PHB productivity is one of the highest levels achieved from a real lignocellulosic waste.

Unveiling steps of the TDP degradation pathway in Variovorax paradoxus TBEA6.

Since the role of biobased plastics increases every year, the search for alternatives to petrol-based polymers is very important. Variovorax paradoxus TBEA6 is able to grow with 3,3'-thiodipropionic acid (TDP) as sole source for carbon and energy. TDP can be used as a precursor substrate for the synthesis of polythioesters (PTE). To increase the feasibility of PTE synthesis, a good understanding of the degradation pathway of TDP in V. paradoxus TBEA6 is essential. Therefore, two putative 3-hydroxyisobutyryl-CoA hydrolases (VPARA_03110 & VPARA_05510) and two putative 3-hydroxypropionate dehydrogenases (VPARA_41140 & VPARA_54550) were investigated in this study. The deletion mutant V. paradoxus ∆VPARA_05510 showed a TDP-negative phenotype during growth experiments. The ability to grow with TDP as sole carbon source was successfully restored by complementation. Supernatant analysis revealed that the deletion mutant did not metabolize TDP or 3MP anymore. A specific enzyme activity up to 0.032 U/mg for the purified 3-hydroxyisobutyryl-CoA hydrolase VPARA_05510 was determined. A shift in the proteins (VPARA_54550) melting temperature of 6 °C with 2000 µM 3HP in comparison to protein without ligand was observed during thermal shift assays with the putative 3-hydroxypropionate dehydrogenase.

Naturally-Sourced Antibacterial Polymeric Nanomaterials with Special Reference to Modified Polymer Variants.

Despite the recent advancements in treating bacterial infections, antibiotic resistance (AR) is still an emerging issue. However, polymeric nanocarriers have offered unconventional solutions owing to their capability of exposing more functional groups, high encapsulation efficiency (EE) and having sustained delivery. Natural polymeric nanomaterials (NMs) are contemplated one of the most powerful strategies in drug delivery (DD) in terms of their safety, biodegradability with almost no side effects. Every nanostructure is tailored to enhance the system functionality. For example, cost-effective copper NPs could be generated in situ in cellulose sheets, demonstrating powerful antibacterial prospects for food safety sector. Dendrimers also have the capacity for peptide encapsulation, protecting them from proteolytic digestion for prolonged half life span. On the other hand, the demerits of naturally sourced polymers still stand against their capacities in DD. Hence, Post-synthetic modification of natural polymers could play a provital role in yielding new hybrids while retaining their biodegradability, which could be suitable for building novel super structures for DD platforms. This is the first review presenting the contribution of natural polymers in the fabrication of eight polymeric NMs including particulate nanodelivery and nanofabrics with antibacterial and antibiofilm prospects, referring to modified polymer derivatives to explore their full potential for obtaining sustainable DD products.

Theoretical Studies of Cyanophycin Dipeptides as Inhibitors of Tyrosinases.

The three-dimensional structure of tyrosinase has been crystallized from many species but not from Homo sapiens. Tyrosinase is a key enzyme in melanin biosynthesis, being an important target for melanoma and skin-whitening cosmetics. Several studies employed the structure of tyrosinase from Agaricus bisporus as a model enzyme. Recently, 98% of human genome proteins were elucidated by AlphaFold. Herein, the AlphaFold structure of human tyrosinase and the previous model were compared. Moreover, tyrosinase-related proteins 1 and 2 were included, along with inhibition studies employing kojic and cinnamic acids. Peptides are widely studied for their inhibitory activity of skin-related enzymes. Cyanophycin is an amino acid polymer produced by cyanobacteria and is built of aspartic acid and arginine; arginine can be also replaced by other amino acids. A new set of cyanophycin-derived dipeptides was evaluated as potential inhibitors. Aspartate-glutamate showed the strongest interaction and was chosen as a leading compound for future studies.

The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.

Insights into the Degradation of Medium-Chain-Length Dicarboxylic Acids in Cupriavidus necator H16 Reveal ß-Oxidation Differences between Dicarboxylic Acids and Fatty Acids.

Many homologous genes encoding ß-oxidation enzymes have been found in the genome of Cupriavidus necator H16 (synonym Ralstonia eutropha H16). By proteome analysis, the degradation of adipic acid was investigated and showed differences from the degradation of hexanoic acid. During ß-oxidation of adipic acid, activation with coenzyme A (CoA) is catalyzed by the two-subunit acyl-CoA ligase encoded by B0198 and B0199. The operon is completed by B0200 encoding a thiolase catalyzing the cleavage of acetyl-CoA at the end of the ß-oxidation cycle. C. necator ΔB0198-B0200 strain showed improved growth on adipic acid. Potential substitutes are B1239 for B0198-B0199 and A0170 as well as A1445 for B0200. A deletion mutant without all three thiolases showed diminished growth. The deletion of detected acyl-CoA dehydrogenase encoded by B2555 has an altered phenotype grown with sebacic acid but not adipic acid. With hexanoic acid, acyl-CoA dehydrogenase encoded by B0087 was detected on two-dimensional (2D) gels. Both enzymes are active with adipoyl-CoA and hexanoyl-CoA as substrates, but specific activity indicates a higher activity of B2555 with adipoyl-CoA. 2D gels, growth experiments, and enzyme assays suggest the specific expression of B2555 for the degradation of dicarboxylic acids. In C. necator H16, the degradation of carboxylic acids potentially changes with an increasing chain length. Two operons involved in growth with long-chain fatty acids seem to be replaced during growth on medium-chain carboxylic acids. Only two deletion mutants showed diminished growth. Replacement of deleted genes with one of the numerous homologous is likely. IMPORTANCE The biotechnologically interesting bacterium Cupriavidus necator H16 has been thoroughly investigated. Fifteen years ago, it was sequenced entirely and annotated (A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian, et al., Nat Biotechnol 24:1257-1262, 2006, https://doi.org/10.1038/nbt1244). Nevertheless, the degradation of monocarboxylic fatty acids and dicarboxylic acids has not been elucidated completely. C. necator is used to produce value-added products from affordable substrates. One of our investigations' primary targets is the biotechnological production of organic acids with different and specific chain lengths. The versatile metabolism of carboxylic acids recommends C. necator H16 as a candidate for producing value-added organic products. Therefore, the metabolism of these compounds is of interest, and, for different applications in industry, understanding such central metabolic pathways is crucial.

Cyanophycin Modifications-Widening the Application Potential.

A circular bioeconomy approach is essential to slowing down the fearsome ongoing climate change. Replacing polymers derived from fossil fuels with biodegradable biobased polymers is one crucial part of this strategy. Cyanophycin is a polymer consisting of amino acids produced by cyanobacteria with many potential applications. It consists mainly of aspartic acid and arginine, however, its composition may be changed at the production stage depending on the conditions of the polymerization reaction, as well as the characteristics of the enzyme cyanophycin synthetase, which is the key enzyme of catalysis. Cyanophycin synthetases from many sources were expressed heterologously in bacteria, yeast and plants aiming at high yields of the polymer or at introducing different amino acids into the structure. Furthermore, cyanophycin can be modified at the post-production level by chemical and enzymatic methods. In addition, cyanophycin can be combined with other compounds to yield hybrid materials. Although cyanophycin is an attractive polymer for industry, its usage as a sole material remains so far limited. Finding new variants of cyanophycin may bring this polymer closer to real-world applications. This short review summarizes all modifications of cyanophycin and its variants that have been reported within the literature until now, additionally addressing their potential applications.

Enzymatic and Chemical Approaches for Post-Polymerization Modifications of Diene Rubbers: Current state and Perspectives.

Diene rubbers are polymeric materials which present elastic properties and have double bonds in the macromolecular backbone after the polymerization process. Post-polymerization modifications of rubbers can be conducted by enzymatic or chemical methods. Enzymes are environmentally friendly catalysts and with the increasing demand for rubber waste management, biodegradation and biomodifications have become hot topics of research. Some rubbers are renewable materials and are a source of organic molecules, and biodegradation can be conducted to obtain either oligomers or monomers. On the other hand, chemical modifications of rubbers by click-chemistry are important strategies for the creation and combination of new materials. In a way to expand the scope of uses to other non-traditional applications, several and effective modifications can be conducted with diene rubbers. Two groups of efficient tools, enzymatic, and chemical modifications in diene rubbers, are summarized in this review. By analyzing stereochemical and reactivity aspects, the authors also point to some applications perspectives for biodegradation products and to rational modifications of diene rubbers by combining both methodologies.

Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes.

Chitin is one of the most abundant biopolymers. Due to its recalcitrant nature and insolubility in accessible solvents, it is often considered waste and not a bioresource. The products of chitin modification such as chitosan and chitooligosaccharides are highly sought, but their preparation is a challenging process, typically performed with thermochemical methods that lack specificities and generate hazardous waste. Enzymatic treatment is a promising alternative to these methods, but the preparation of multiple biocatalysts is costly. In this manuscript, we biochemically characterised chitin deacetylases of Mucor circinelloides IBT-83 and utilised one of them for the construction of the first eukaryotic, polycistronic expression system employing self-processing 2A sequences. The three chitin-processing enzymes; chitin deacetylase of M. circinelloides IBT-83, chitinase from Thermomyces lanuginosus, and chitosanase from Aspergillus fumigatus were expressed under the control of the same promoter in methylotrophic yeast Pichia pastoris and characterised for their synergistic action towards their respective substrates.

Incorporation of alternative amino acids into cyanophycin by different cyanophycin synthetases heterologously expressed in Corynebacterium glutamicum.

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a biopolymer that could be used in various fields, for example, as a potential precursor for the synthesis of polyaspartic acid or for the production of CGP-derived dipeptides. To extend the applications of this polymer, it is therefore of interest to synthesize CGP with different compositions. A recent re-evaluation of the CGP synthesis in C. glutamicum has shown that C. glutamicum is a potentially interesting microorganism for CGP synthesis with a high content of alternative amino acids. This study shows that the amount of alternative amino acids can be increased by using mutants of C. glutamicum with altered amino acid biosynthesis. With the DM1729 mutant, the lysine content in the polymer could be increased up to 33.5 mol%. Furthermore, an ornithine content of up to 12.6 mol% was achieved with ORN2(Pgdh4). How much water-soluble or insoluble CGP is synthesized is strongly related to the used cyanophycin synthetase. CphADh synthesizes soluble CGP exclusively. However, soluble CGP could also be isolated from cells expressing CphA6308Δ1 or CphA6308Δ1_C595S in addition to insoluble CGP in all examined strains. The point mutation in CphA6308Δ1_C595S partially resulted in a higher lysine content. In addition, the CGP content could be increased to 36% of the cell dry weight under optimizing growth conditions in C. glutamicum ATCC13032. All known alternative major amino acids for CGP synthesis (lysine, ornithine, citrulline, and glutamic acid) could be incorporated into CGP in C. glutamicum.

3,3'-Thiodipropionic acid (TDP), a possible precursor for the synthesis of polythioesters: identification of TDP transport proteins in Variovorax paradoxus TBEA6.

3,3'-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

In vitro studies on the degradation of common rubber waste material with the latex clearing protein (Lcp1VH2) of Gordonia polyisoprenivorans VH2.

The enzymatic degradation of the rubber polymer poly(cis-1,4-isoprene), e.g. by the latex clearing protein Lcp1VH2 of Gordonia polyisoprenivorans VH2 has been demonstrated with latex milk or pure isoprene-rubber particles, recently. Unfortunately, carbon black filled vulcanized rubber (CFVR) making the biggest part of worldwide rubber wastes, contains several harmful additives making microbial and enzymatic rubber degradation challenging. However, this study demonstrates the successful enzymatic cleavage of industrially produced CFVR. The formation of the cleavage products, oligo(cis-1,4-isoprenoids), from incubating CFVR particles with Lcp1VH2 was detected by HPLC-MS. Various organic solvents were tested to remove harmful or inhibiting additives like antioxidants to enhance product formation. The pretreatment of CFVR particles, especially with chloroform or cyclohexane, significantly improved the degradation. It was also demonstrated that reducing the particles size and thus increasing the enzymatically accessible surface area of the particles led to a strong acceleration of the degradation process. Furthermore, ATR-IR analyses showed that Lcp1VH2 led to the functionalization of the rubber particle surface with carbonyl groups by cleaving isoprene chains, still linked to the particle. Both, the oligo(cis-1,4-isoprenoids) as well as the functionalized rubber particles, are potentially important products, which can be reused as fine chemicals or as additives in rubber production. The present study, showing the enzymatic degradation of common CFVR for the first time, takes an important step towards a new way of rubber waste disposal and indicates the economic feasibility of an efficient and environmentally friendly recycling process by using the rubber oxygenase Lcp1VH2.

Crystal structure of the sugar acid-binding protein CxaP from a TRAP transporter in Advenella mimigardefordensis strain DPN7T.

Recently, CxaP, a sugar acid substrate binding protein (SBP) from Advenella mimigardefordensis strain DPN7T , was identified as part of a novel sugar uptake strategy. In the present study, the protein was successfully crystallized. Although several SBP structures of tripartite ATP-independent periplasmic transporters have already been solved, this is the first structure of an SBP accepting multiple sugar acid ligands. Protein crystals were obtained with bound d-xylonic acid, d-fuconic acid d-galactonic and d-gluconic acid, respectively. The protein shows the typical structure of an SBP of a tripartite ATP-independent periplasmic transporter consisting of two domains linked by a hinge and spanned by a long α-helix. By analysis of the structure, the substrate binding site of the protein was identified. The carboxylic group of the sugar acids interacts with Arg175, whereas the coordination of the hydroxylic groups at positions C2 and C3 is most probably realized by Arg154 and Asn151. Furthermore, it was observed that 2-keto-3-deoxy-d-gluconic acid is bound in protein crystals that were crystallized without the addition of any ligand, indicating that this molecule is prebound to the protein and is displaced by the other ligands if they are available. DATABASE: Structural data of CxaP complexes are available in the worldwide Protein Data Bank (https://www.rcsb.org) under the accession codes 7BBR (2-keto-3-deoxy-d-gluconic acid), 7BCR (d-galactonic acid), 7BCN (d-xylonic acid), 7BCO (d-fuconic acid) and 7BCP (d-gluconic acid).

Post-Synthetic Enzymatic and Chemical Modifications for Novel Sustainable Polyesters.

Because of their biodegradability, compostability, compatibility and flexible structures, biodegradable polymers such as polyhydroxyalkanoates (PHA) are an important class of biopolymers with various industrial and biological uses. PHAs are thermoplastic polyesters with a limited processability due to their low heat resistance. Furthermore, due to their high crystallinity, some PHAs are stiff and brittle. These features result sometimes in very poor mechanical characteristics with low extension at break values which limit the application range of some natural PHAs. Several in vivo approaches for PHA copolymer modifications range from polymer production to enhance PHA-based material performance after synthesis. The methods for enzymatic and chemical polymer modifications are aiming at modifying the structures of the polyesters and thereby their characteristics while retaining the biodegradability. This survey illustrates the efficient use of enzymes and chemicals in post-synthetic PHA modifications, offering insights on these green techniques for modifying and improving polymer performance. Important studies in this sector will be reviewed, as well as chances and obstacles for their stability and hyper-production.

What Has Been Trending in the Research of Polyhydroxyalkanoates? A Systematic Review.

Over the past decades, enormous progress has been achieved with regard to research on environmentally friendly polymers. One of the most prominent families of such biopolymers are bacterially synthesized polyhydroxyalkanoates (PHAs) that have been known since the 1920s. However, only as recent as the 1990s have extensive studies sprung out exponentially in this matter. Since then, different areas of exploration of these intriguing materials have been uncovered. However, no systematic review of undertaken efforts has been conducted so far. Therefore, we have performed an unbiased search of up-to-date literature to reveal trending topics in the research of PHAs over the past three decades by data mining of 2,227 publications. This allowed us to identify eight past and current trends in this area. Our study provides a comprehensive review of these trends and speculates where PHA research is heading.