Improved cadaverine production from mutant Klebsiella oxytoca lysine decarboxylase

Cadaverine has the potential to become an important platform chemical for the production of nylon. Previously, a system that overexpresses the Klebsiella oxytoca lysine decarboxylase in Escherichia coli was engineered. The system was optimized by codon optimization, and tuning the expression level of the gene by testing various promoters and inducer concentrations. Here, we further improved the system by optimizing the sequence located in the region of the ribosome‐binding site in order to enhance translation efficiency. We also identified mutant lysine decarboxylase enzymes that demonstrated enhanced cadaverine‐production ability. Together, these modifications increased cadaverine production in the system by 50%, and the system has a yield of 80% from lysine‐HCl under the conditions we tested. This is the first time that a system to produce cadaverine using the lysine decarboxylase from K. oxytoca performed at a level that is competitive with the traditional systems using the E. coli lysine decarboxylases in both lab‐scale and batch fermentation conditions.     

Catabolism of putrescine and spermidine in embryogenic and non-embryogenic callus lines of Picea abies

The oxidation of putrescine and spermidine were studied in embryogenic and nonembryogenic cell cultures of Picea abies (L.) Karst., with [1,4-¹⁴C]-putrescine and [1,4-¹⁴C]-spermidine as substrates. Activities of putrescine and spermidine oxidation varied at every developmental stage in both cultures. Putrescine was oxidized ca 5 times as fast both in embryogenic and non-embryogenic tissue as spermidine. Diamine and especially polyamine oxidase activity increased markedly in both tissues towards the end of the culturing. In maturing embryos and in ageing non-embryogenic cultures, enzyme activities were lower than in non-differentiated embryogenic calli. Aminoguanidine (1 m M ) inhibited di- and polyamine oxidation in non-embryogenic tissue by >60% and >30%, respectively. The pH optimum for putrescine oxidation was 8.0, but in non-embryogenic tissue spermidine was degraded even more actively at pH 5.0. [¹⁴C]-Spermidine was catabolized to [¹⁴C]-putrescine. Pyrroline dehydrogenase activity was observed in non-embryogenic spruce tissue cultures.     

The role of L-arginine in toxic liver failure: interrelation of arginase,polyamine catabolic enzymes and nitric oxide synthase

Summary. The existing interrelation in metabolic pathways of L-arginine to polyamines, nitric oxide (NO) and urea synthesis could be affected in sepsis, inflammation, intoxication and other conditions. The role of polyamines and NO in the toxic effect of mercury chloride on rat liver function was studied. Administration of mercury chloride for 24 h led to significantly elevated plasma activities of Alanine transaminase (ALT) and Aspartate transaminase (AST). Malondyaldehyde (MDA) levels were unaffected (p > 0.05) and arginase activity was significantly decreased (p < 0.05) while nitrate/nitrite production was significantly elevated (p < 0.001) in liver tissue. Polyamine oxidase (PAO) and diamine oxidase (DAO) activities, enzymes involved in catabolism of polyamines, were decreased. L-arginine supplementation to intoxicated rats potentiated the effect of mercury chloride on NO production and it was ineffective on arginase activity. Results obtained in this study show that mercury chloride-induced toxicity leads to abnormally high levels of ALT and AST that may indicate liver damage with the involvement of polyamine catabolic enzymes and NO.     

Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses

Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H₂O₂, one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.     

Spatial and functional correlation between diamine-oxidase and peroxidase activities and their dependence upon de-etiolation and wounding in chick-pea stems

The activities of diamine oxidase (DAO, EC 1.4.3.6) and peroxidase (POD, EC 1.11.1.7) were determined along the stems of light-grown Cicer arietinum L. (chick-pea) seedlings. Enzyme activities were evaluated in the soluble, lightly bound (salt extraction) and tightly bound (Driselase digestion) wall fractions, and in residual fractions obtained from the different internodes. Apparent tissue distributions of both enzymes and lignin depositions were visualised by means of histochemical and immunohistochemical techniques. A close relationship was found between DAO and POD activities in the soluble and wall fractions along the stem. The biochemical activities of both enzymes decreased from the base to the apex of the stem in parallel with the distribution pattern of lignifying tissues in this organ. A similar activity gradient was found for each enzyme along the epidermis of the whole organ. Moreover, deetiolation elicited a rise in the activities of both enzymes in this tissue. Wounding chick-pea stems induced parallel increases in DAO and POD activities in the soluble and wall fractions. In-situ histochemical detection of both enzymes demonstrated the parallel occurrence of the DAO/POD system and lignosuberised depositions in the cell walls adjacent to the wound site. The patterns of POD isoforms resulting from the wound-healing process were determined by means of starch-gel electrophoresis. In addition to changes in relative intensity of enzyme bands in soluble and wall fractions, a new POD isoform, possibly related to the wounding response, appeared in the soluble fraction. This isoform was shown to be lightly bound to cell walls as it could be detected in the extracellular fluids obtained from wound-healed seedlings. On the basis of the above-mentioned results, a strict spatial and functional correlation can be inferred between DAO and POD in chick-pea, and probably in other Leguminosae species, in accordance with previous evidence indicating an integrated role for these enzymes in the apoplast.Abbreviations DAO diamine oxidase - POD peroxidase - Syr syringaldazine The authors wish to thank Professor M.M. Altamura (Università La Sapienza, Rome, Italy) for helpful discussion. We also thank C. Castellacci for technical assistance and M.I. Augeri for helping in figure preparation. This research was supported by the Ministero della Pubblica Istruzione, Italy.     

Intramolecular and intermolecular C-H activation at a cationic Pt center

Syntheses and C-H bond activation reactions of the novel electrophilic Pt^II complexes [(tmeda)Pt(CH₃)(OEt₂)][BAr₁], [(tmeda)Pt(CH₃)(THF)][BAr_f], and [(tmeda)Pt(CH₃)(NC₅F₅)][BAr_f] are described {[BAr_f]⁻ = [(3,5-C₆H₃(CF₃)₂)₄B]⁻} (tmeda is N,N,N′,N′-tetramethylethylenediamine), [(tmeda)Pt(CH₃)(OEt₂)][BAr_f] and [(tmeda)Pt(CH₃)(THF)][BAr_f] are unstable at room temperature, yielding methane and the Fischer carbene Pt^II hydrides, [(tmeda)Pt(=C(CH₃)(OCH₂CH₃))(H)][BAr_f] and . The methane liberated from [(tmeda)Pt(CH₃)(OEt₂-d₁₀)][BAr_f] consists of an isotopomeric mixture, (CH₄, CH₃D, CH₂D₂ and CHD₃), indicating a multiple H/D exchange reaction following the C-D activation and prior to methane loss. [(tmeda)Pt(CH₃)(THF-d₈)][BAr] liberates CH₄ and CH₃D. Methane-¹³C, cyclohexane, toluene, and benzene react with [(tmeda)Pt(CH₃)(NC₅F₅)][BAr_f] to yield methane and new organoplatinum complexes. Deuterated alkanes and arenes react with [(tmeda)Pt(CH₃)(NC₅F₅] [BAr_f] to give a mixture of methane isotopomers. The relevance of these results to the oxidation of alkanes by aqueous platinum complexes is discussed.     

Androgenic sensitivity of polyamine-oxidizing enzyme activities in female rat tissues

Summary. Effects of testosterone (10 μg/100 g body weight) on polyamine-oxidizing enzyme activities in female rat uterus, liver and kidney were demonstrated. Testosterone-treated rats exhibited 2.07 fold (p < 0.002) higher uterine polyamine oxidase (PAO) activity and 1.93 fold (p < 0.02) higher diamine oxidase (DAO) activity, as compared to the controls. In the liver, testosterone caused an elevation in PAO (1.39 fold, p < 0.05), but not in DAO activity, whereas in kidney the hormone stimulated DAO (1.30 fold, p < 0.05), but not PAO activity. The effects observed suggest a possible role for testosterone in the modulation of polyamine levels in the female organs studied and especially in uterus. Received May 12, 1999, Accepted December 16, 1999     

Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy

Bone morphogenetic proteins (BMPs) are members of the TGF-β (transforming growth factor-beta) superfamily, of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus expansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4 gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective of the present study was to detect polymorphism in intronic, exonic and 3′ un-translated regions of BMP4 gene in Indian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning > 40%), Osmanabadi, Sangamneri (Twinning 20–30%), Sirohi and Ganjam (Twinning < 10%)) differing in prolificacy and geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) was used to design primers for the amplification of a targeted region followed by direct DNA sequencing to identify the genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic region and the 3′ flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutation resulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534 locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predominant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhrana breed with a genotype frequency of 0.02. A microsatellite was identified in the 3′ flanking region, only 20 nucleotides downstream from the termination site of the coding region, as a short sequence with more than nineteen continuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of a non-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is the first report of a mutation in the coding region of the caprine BMP4 gene. But whether the reproduction trait of goat is associated with the BMP4 polymorphism, needs to be further defined by association studies in more populations so as to delineate an effect on it.     

Effect of spermidine and its metabolites on the activity of pea seedlings diamine oxidase and the problems of biosensing of biogenic amines with this enzyme

Spermidine is one of the several biogenic amines, produced during the microbial decarboxylation of proteins. Individual biogenic amines in the formed mixtures are frequently analyzed with oxygen sensor based biosensors, as their content serves as a good biomarker for the determination of food quality. In these biosensors, diamine oxidase from pea seedlings (PSAO), catalyzing the oxidation of various biogenic amines by dissolved oxygen is commonly used for the bio-recognition of amines. However, in the presence of spermidine and/or its metabolite 1,3-diaminopropane, the activity of PSAO and the sensitivity of PSAO-based biosensors decrease due to inhibition. The inhibition constant of soluble spermidine, acting as an inhibiting substrate toward PSAO, was found to be (40 ± 15) mM in freshly prepared solution and (0.28 ± 0.05) mM in solution, incubated 30 days at room temperature. The inhibition constant of 1,3-diaminopropane, acting as a competitive inhibitor, was (0.43 ± 0.12) mM as determined through the oxidation reaction of cadaverine. The metabolic half-life of soluble spermidine was 7 days at room temperature and 186 days at 4 °C. The kinetic measurements were carried out with an oxygen sensor; the composition of the solution of degraded spermidine was analyzed with MS.     

Metabolic engineering for the production of dicarboxylic acids and diamines

Microbial production of chemicals and materials from renewable carbon sources is becoming increasingly important to help establish sustainable chemical industry. In this paper, we review current status of metabolic engineering for the bio-based production of linear and saturated dicarboxylic acids and diamines, important platform chemicals used in various industrial applications, especially as monomers for polymer synthesis. Strategies for the bio-based production of various dicarboxylic acids having different carbon numbers including malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (C13), tetradecanedioic acid (C14), and pentadecanedioic acid (C15) are reviewed. Also, strategies for the bio-based production of diamines of different carbon numbers including 1,3-diaminopropane (C3), putrescine (1,4-diaminobutane; C4), cadaverine (1,5-diaminopentane; C5), 1,6-diaminohexane (C6), 1,8-diaminoctane (C8), 1,10-diaminodecane (C10), 1,12-diaminododecane (C12), and 1,14-diaminotetradecane (C14) are revisited. Finally, future challenges are discussed towards more efficient production and commercialization of bio-based dicarboxylic acids and diamines.