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.

The relevance of enzyme specificity for coenzymes and the presence of 6-phosphogluconate dehydrogenase for polyhydroxyalkanoates production in the metabolism of Pseudomonas sp. LFM046.

Reconstruction of genome-based metabolic model is a useful approach for the assessment of metabolic pathways, genes and proteins involved in the environmental fitness capabilities or pathogenic potential as well as for biotechnological processes development. Pseudomonas sp. LFM046 was selected as a good polyhydroxyalkanoates (PHA) producer from carbohydrates and plant oils. Its complete genome sequence and metabolic model were obtained. Analysis revealed that the gnd gene, encoding 6-phosphogluconate dehydrogenase, is absent in Pseudomonas sp. LFM046 genome. In order to improve the knowledge about LFM046 metabolism, the coenzyme specificities of different enzymes was evaluated. Furthermore, the heterologous expression of gnd genes from Pseudomonas putida KT2440 (NAD+ dependent) and Escherichia coli MG1655 (NADP+ dependent) in LFM046 was carried out and provoke a delay on cell growth and a reduction in PHA yield, respectively. The results indicate that the adjustment in cyclic Entner-Doudoroff pathway may be an interesting strategy for it and other bacteria to simultaneously meet divergent cell needs during cultivation phases of growth and PHA production.

Re-evaluation of cyanophycin synthesis in Corynebacterium glutamicum and incorporation of glutamic acid and lysine into the polymer.

Corynebacterium glutamicum was only examined in the early 2000s as a possible microorganism for the production of the polyamide cyanophycin (multi-L-arginyl-poly-[L-aspartic acid], CGP). CGP is a potential precursor for the synthesis of polyaspartic acid and CGP-derived dipeptides which may be of use in peptide-based clinical diets, as dietary supplements, or in livestock feeds. In the past, C. glutamicum was disregarded for CGP production due to low CGP contents and difficulties in isolating the polymer. However, considering recent advances in CGP research, the capabilities of this organism were revisited. In this study, several cyanophycin synthetases (CphA) as well as expression vectors and cultivation conditions were evaluated. The ability of C. glutamicum to incorporate additional amino acids such as lysine and glutamic acid was also examined. The strains C. glutamicum pVWEx1::cphAΔ1 and C. glutamicum pVWEx1::cphABP1 accumulated up to 14% of their dry weight CGP, including soluble CGP containing more than 40 mol% of the alternative side-chain amino acid lysine. The soluble, lysine-rich form of the polymer was not detected in C. glutamicum in previous studies. Additionally, an incorporation of up to 6 mol% of glutamic acid into the backbone of CGP synthesized by C. glutamicum pVWEx1::cphADh was detected. The strain accumulated up to 17% of its dry weight in soluble CGP. Although glutamic acid had previously been found to replace arginine in the side chain, this is the first time that glutamic acid was found to substitute aspartic acid in the backbone.

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP-transport system.

In this study, we investigated an SBP (DctPAm ) of a tripartite ATP-independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7T . Deletion of dctPAm as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7T , if cultivated on mineral salt medium supplemented with d-glucose, d-galactose, l-arabinose, d-fucose, d-xylose or d-gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctPAm using the broad host vector pBBR1MCS-5::dctPAm . Furthermore, an uptake assay with radiolabeled [14 C(U)]-d-glucose clearly showed that the deletion of dctPAm , dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of KD performing thermal shift assays showed a shift in the melting temperature of DctPAm in the presence of d-gluconic acid (KD 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above-mentioned carbohydrates d-galactonate (KD 10.72 ± 1.4 µM), d-fuconic acid (KD 13.50 ± 1.6 µM) and d-xylonic acid (KD 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.

Assessment of bacterial acyltransferases for an efficient lipid production in metabolically engineered strains of E. coli.

Microbially produced lipids like triacylglycerols or fatty acid ethyl esters are currently of great interest as fuel replacements or other industrially relevant compounds. They can even be produced by non-oleaginous microbes, like Escherichia coli, upon metabolic engineering. However, there is still much room for improvement regarding the yield for a competitive microbial production of lipids or biofuels. We genetically engineered E. coli by expressing fadD, fadR, pgpB, plsB and 'tesA in combination with atfA from Acinetobacter baylyi. A total fatty acid contents of up to 16% (w/w) was obtained on complex media, corresponding to approximately 9% (w/w) triacylglycerols and representing the highest titers of fatty acids and triacylglycerols obtained in E. coli under comparable cultivation conditions, so far. To evaluate further possibilities for an optimization of lipid production, ten promising bacterial wax ester synthase/acyl-Coenzyme A:diacylglycerol acyltransferases were tested and compared. While highest triacylglycerol storage was achieved with AtfA, the mutated variant AtfA-G355I turned out to be most suitable for fatty acid ethyl ester biosynthesis and enabled an accumulation of approx. 500 mg/L without external ethanol supplementation.

Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

Synthesis of a citrulline-rich cyanophycin by use of Pseudomonas putida ATCC 4359.

Synthesis of cyanophycin (multi-L-arginyl-poly-L-aspartic acid, CGP) in recombinant organisms is an important option to obtain sufficiently large amounts of this polymer with a designed composition for use as putative precursors for biodegradable technically interesting chemicals. Therefore, derivates of CGP, harbouring a wider range of constituents, are of particular interest. As shown previously, cyanophycin synthetases with wide substrate ranges incorporate other amino acids than arginine. Therefore, using an organism, which produces the required supplement by itself, was the next logical step. Former studies showed that Pseudomonas putida strain ATCC 4359 is able to produce large amounts of L-citrulline from L-arginine. By expressing the cyanophycin synthetase of Synechocystis sp. PCC 6308, synthesis of CGP was observed in P. putida ATCC 4359. Using an optimised medium for cultivation, the strain was able to synthesise insoluble CGP amounting up to 14.7 ± 0.7% (w/w) and soluble CGP amounting up to 28.7 ± 0.8% (w/w) of the cell dry matter, resulting in a total CGP content of the cells of 43.4% (w/w). HPLC analysis of the soluble CGP showed that it was composed of 50.4 ± 1.3 mol % aspartic acid, 32.7 ± 2.8 mol % arginine, 8.7 ± 1.6 mol % citrulline and 8.3 ± 0.4 mol % lysine, whereas the insoluble CGP contained less than 1 mol % of citrulline. Using a mineral salt medium with 1.25 or 2% (w/v) sodium succinate, respectively, plus 23.7 mM L-arginine, the cells synthesised insoluble CGP amounting up to 25% to 29% of the CDM with only a very low citrulline content.

Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production.

The numerous physiological functions of the nonessential amino acid L-aspartate, the semi-essential amino acid L-arginine, and the essential amino acid L-lysine, made them attractive for a wide range of nutritional and/or therapeutic applications. Furthermore, the administration of these amino acids as mixtures or as dipeptides for higher bioavailability is scientifically approved, and various commercial products of these forms are already available on the market. Although the industrial production of dipeptides is, with few exceptions, in an early stage, several strategies have been established and are compared in this review. Additionally, the recent developments in the technical production of aspartate-arginine and aspartate-lysine dipeptides from the biopolymer cyanophycin produced in microorganisms are discussed. Cyanophycin-derived dipeptides are produced exclusively by biotechnological procedures, probably possess higher bioavailability and may be used as better alternatives to the widely applied amino acid mixtures. Thus, the pivotal advantages and the potential applications of these dipeptides as well as of their constituting amino acids in nutrition and therapy are also discussed. Special emphasis is dedicated to arginine due to its numerous physiological roles in many cardiovascular, genitourinary, gastrointestinal, and immune disorders.

Occurrence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons.

Petroleum (or crude oil) is a complex mixture of hydrocarbons. Annually, millions of tons of crude petroleum oil enter the marine environment from either natural or anthropogenic sources. Hydrocarbon-degrading bacteria (HDB) are able to assimilate and metabolize hydrocarbons present in petroleum. Crude oil pollution constitutes a temporary condition of carbon excess coupled to a limited availability of nitrogen that prompts marine oil-degrading bacteria to accumulate storage compounds. Storage lipid compounds such as polyhydroxyalkanoates (PHAs), triacylglycerols (TAGs), or wax esters (WEs) constitute the main accumulated lipophilic substances by bacteria under such unbalanced growth conditions. The importance of these compounds as end-products or precursors to produce interesting biotechnologically relevant chemicals has already been recognized. In this review, we analyze the occurrence and accumulation of lipid storage in marine hydrocarbonoclastic bacteria. We further discuss briefly the production and export of lipophilic compounds by bacteria belonging to the Alcanivorax genus, which became a model strain of an unusual group of obligate hydrocarbonoclastic bacteria (OHCB) and discuss the possibility to produce neutral lipids using A. borkumensis SK2.

Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production.

Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures. For these purposes, new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals. An important group of these chemicals are those that comprise N-functionalities. Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals. This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylassetrade mark or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals.

Microdiesel: Escherichia coli engineered for fuel production.

Biodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l(-1) and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.

Engineering the genotype of Acinetobacter sp. strain ADP1 to enhance biosynthesis of cyanophycin.

To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. DeltaastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.

Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants.

Polyhydroxybutyrate (PHB) is a member of a class of thermoelastic polymers called polyhydroxyalkanoates that serve many bacteria as intracellular storage molecules for carbon and energy. Transgenic plants provide a potential means of producing this polymer cost-effectively. To date, however, few reports of the successful production of this polymer have been published, with the exception of work with transgenic Arabidopsis. Using a variety of chimeric constructs, we have determined that the constitutive, chloroplast-localized expression of one of the genes involved in PHB production-the beta-ketothiolase (phbA) gene-is detrimental to the efficient production of transgenic PHB. The alternate use of either inducible or somatically activated promoters allowed the construction of transgenic PHB-producing potato (Solanum tuberosum) and tobacco (Nicotiana tabacum) plants, although the amount of PHB formed was still rather low. Taking advantage of an inducible promoter, the maximal amount of PHB produced in transgenic potato was 0.09 mg g(-1) dry weight. In transgenic tobacco using a somatically activated promoter, up to 3.2 mg g(-1) dry weight was accumulated. In Arabidopsis, the formation of high levels of PHB had previously been shown to be accompanied by severe negative effects on growth and development of the plant. Phasins are proteins known from PHB-producing bacteria speculated to serve as protectants against the highly hydrophobic surface of the PHB granules in the bacterial intracellular milieu. Co-expression of the phasin gene in parallel with the PHB synthesis genes, however, did not lead to reduced symptom development.