Photometabolism of aromatic compounds byRhodopseudomonas palustris

The phototrophic bacteriumRhodopseudomonas palustris has been reported to be versatile in photometabolism of aromatic compounds. However, the kinetics of degradation of aromatic compounds byR. palustris appears not to have been reported in the literature. In this laboratory a photosynthetic bacterium that was identified asRhodopseudomonas palustris(bp) was isolated from a sample collected from a canal where refinery wastewater was discharged. The paper presents the results of the growth of the isolate on different aromatic compounds, their catabolic pathways, and the kinetics of their degradation. The doubling time of the isolate was found to be 23, 22, 17, 27, 20, 24, and 26 h for benzoate,p-hydroxybenzoate, cinnamate,p-coumarate, phenyl valerate, phenyl acetate, and cinnamyl alcohol respectively. It was also found that cinnamate, phenyl valerate, phenyl acetate, and cinnamyl alcohol were converted to benzoate, whilep-coumarate was converted top-hydroxybenzoate. The substrate inhibition constant (Ki) was found to be 1082, 1178, 1362, 1260, 3098, 1347, and 322 mg/L for benzoate,p-hydroxybenzoate, cinnamate,p-coumarate, phenyl valerate, phenyl acetate, and cinnamyl alcohol respectively.     

Biosynthesis of plant-specific phenylpropanoids by construction of an artificial biosynthetic pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biological synthesis of plant secondary metabolites has attracted increasing attention due to their proven or assumed beneficial properties and health-promoting effects. Phenylpropanoids are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. In this study, engineered Escherichia coli containing artificial phenylpropanoid biosynthetic pathways utilizing tyrosine as the initial precursor were established for production of plant-specific metabolites such as ferulic acid, naringenin, and resveratrol. The construction of the artificial pathway utilized tyrosine ammonia lyase and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis, cinnamate/4-coumarate:coenzyme A ligase from Streptomyces coelicolor, caffeic acid O-methyltransferase and chalcone synthase from Arabidopsis thaliana, and stilbene synthase from Arachis hypogaea.     

Improved <Emphasis Type="Italic">p</Emphasis>-hydroxybenzoate production by engineered <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 by using a mixed-substrate feeding strategy

The key precursors for p-hydroxybenzoate production by engineered Pseudomonas putida S12 are phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), for which the pentose phosphate (PP) pathway is an important source. Since PP pathway fluxes are typically low in pseudomonads, E4P and PEP availability is a likely bottleneck for aromatics production which may be alleviated by stimulating PP pathway fluxes via co-feeding of pentoses in addition to glucose or glycerol. As P. putida S12 lacks the natural ability to utilize xylose, the xylose isomerase pathway from E. coli was introduced into the p-hydroxybenzoate producing strain P. putida S12palB2. The initially inefficient xylose utilization was improved by evolutionary selection after which the p-hydroxybenzoate production was evaluated. Even without xylose-co-feeding, p-hydroxybenzoate production was improved in the evolved xylose-utilizing strain, which may indicate an intrinsically elevated PP pathway activity. Xylose co-feeding further improved the p-hydroxybenzoate yield when co-fed with either glucose or glycerol, up to 16.3 Cmol% (0.1 g p-hydroxybenzoate/g substrate). The yield improvements were most pronounced with glycerol, which probably related to the availability of the PEP precursor glyceraldehyde-3-phosphate (GAP). Thus, it was demonstrated that the production of aromatics such as p-hydroxybenzoate can be improved by co-feeding different carbon sources via different and partially artificial pathways. Moreover, this approach opens new perspectives for the efficient production of (fine) chemicals from renewable feedstocks such as lignocellulose that typically has a high content of both glucose and xylose and (crude) glycerol.     

One-pot synthesis of genistein from tyrosine by coincubation of genetically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells

For production of genistein from N-acetylcysteamine-attached p-coumarate (p-coumaroyl-NAC) supplemented to the medium, a chalcone synthase (CHS) gene from Glycyrrhiza echinata, a chalcone isomerase (CHI) gene from Pueraria lobata, and an isoflavone synthase (IFS) gene from G. echinata were placed under the control of the galactose-inducible GAL promoters in pESC vector and were introduced in Saccharomyces cerevisiae. When the recombinant yeast cells (0.5 g wet weight) were used as “enzyme bags” and incubated at 30°C for 48 h in 100 ml of the buffer containing galactose and 1 mM (265 mg/l) p-coumaroyl-NAC, ca. 340 μg genistein/l was produced. Another system consisting of two enzyme bags was also generated for the purpose of production of genistein from tyrosine. One enzyme bag was an Escherichia coli cell containing a phenylalanine ammonia-lyase gene from a yeast, a 4-coumarate/cinnamate:CoA ligase gene from the actinomycete Streptomyces coelicolor A3(2), the CHS gene, and the CHI gene, in addition to the acetyl-CoA carboxylase gene from Corynebacterium glutamicum, all of which were under the control of the isopropyl-β-d-thiogalactopyranoside-inducible T7 promoter, and thus producing (S)-naringenin from tyrosine. The other enzyme bag was a S. cerevisiae cell containing the IFS gene. Coincubation of the E. coli cells (0.5 g wet weight) and S. cerevisiae cells (0.5 g wet weight) at 26°C for 60 h in 20 ml of the buffer containing 3 mM (543 mg/l) tyrosine as the starting substrate yielded ca. 6 mg genistein/l.     

Anaerobic biodegradation ofPara-cresol under three reducing conditions

The anaerobic degradation ofp-cresol was studied with one sediment source under three reducing conditions—denitrifying, sulfidogenic, and methanogenic. Loss ofp-cresol (1 mM) in all the anaerobic systems took initially 3 to 4 weeks. In acclimated culturesp-cresol was degraded in less than a week.p-Cresol was completely metabolized under denitrifying, sulfidogenic, and methanogenic conditions, with formation of nitrogen gas, loss of sulfate, and formation of methane and carbon dioxide, respectively.p-Cresol metabolism proceeded throughp-hydroxybenzal-dehyde andp-hydroxybenzoate under denitrifying and methanogenic conditions. These compounds were rapidly degraded in cultures acclimated top-cresol under all three reducing conditions. These results suggest that the initial pathway ofp-cresol degradation is the same under denitryfying, sulfidogenic, and methanogenic conditions and proceeds via oxidation of the methyl substituent top-hydroxybenzaldehyde andp-hydroxybenzoate. The initial rate ofp-hydroxybenzaldehyde degradation was high in both the unacclimated cultures and in the cultures acclimated top-cresol, suggesting that this step is nonspecific. Benzoate was additionally detected as a metabolite followingp-hydroxybenzoate in the methanogenic cultures, but not in the denitrifying or sulfidogenic cultures. The degradation pathway therefore may diverge afterp-hydroxybenzoate formation depending on which electron acceptor is available.     

Production of alcohol from sugar beet molasses without heat or filter sterilization

Among three esters of p-hydroxybenzoate, n-butyl p-hydroxybenzoate was selected as the best antimicrobial substance. Molasses medium sterilized by this ester was used as a substrate for ethanol production. n-Butyl p-hydroxybenzoate (0.15% w/v) completely inhibited the growth of free yeast cell inoculum, Ca-alginate immobilized yeast inoculum and bacterial contaminants. Immobilization of the yeast cell inoculum in Ca-alginate with castor oil (6% v/v) offered a yeast cell protection against the inhibitory effect of n-butyl p-hydroxybenzoate. The presence of castor oil in this immobilization system did not affect the metabolic activity of the yeast in beads compared to the cells immobilized without castor oil. The yeast cell beads in this system completely utilized up to 25% molasses sugar with an ethanol yield of 10.58%, equal to 83% of its theoretical value. The beads were stable and could be used successfully for seven cycles of batch fermentation. The optimum fermentation temperature using this system was 35°C. Received 21 January 1997/ Accepted in revised form 05 May 1997     

Microbial degradation of components of sewage from phenol production facilities

Processes of aerobic biodegradation of components of phenol production sewage (phenol, acetophenone, dimethylphenylcarbinol, cumene hydroperoxide, α-methylstyrene, benzoate, andp-hydroxybenzoate) by bacterial strains obtained from the collection of the Saratov Institute of Biocatalysis were studied. The metabolic reactions were shown to be oxidative and to have a common catabolic sequence (cumene hydroperoxide-dimethylphenylcarbinol-α-methylstyrene-acetophenone-phenyl acetate-phenol-pyrocatechol-aromatic ring breakage). Benzoate andp-hydroxybenzoate were degraded through the formation of pyrocatechol and protocatechuate, respectively. Metabolic pathways were similar in model mixtures of components and sewage samples.     

Optimization of the solvent-tolerant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> S12 as host for the production of <Emphasis Type="Italic">p</Emphasis>-coumarate from glucose

A Pseudomonas putida S12 strain was constructed that is able to convert glucose to p-coumarate via the central metabolite l-tyrosine. Efficient production was hampered by product degradation, limited cellular l-tyrosine availability, and formation of the by-product cinnamate via l-phenylalanine. The production host was optimized by inactivation of fcs, the gene encoding the first enzyme in the p-coumarate degradation pathway in P. putida, followed by construction of a phenylalanine-auxotrophic mutant. These steps resulted in a P. putida S12 strain that showed dramatically enhanced production characteristics with controlled l-phenylalanine feeding. During fed-batch cultivation, 10 mM (1.7 g l⁻¹) of p-coumarate was produced from glucose with a yield of 3.8 Cmol% and a molar ratio of p-coumarate to cinnamate of 85:1.     

The Origin and Biosynthesis of the Benzenoid Moiety of Ubiquinone (Coenzyme Q) in Arabidopsis

It is not known how plants make the benzenoid ring of ubiquinone, a vital respiratory cofactor. Here, we demonstrate that Arabidopsis thaliana uses for that purpose two separate biosynthetic branches stemming from phenylalanine and tyrosine. Gene network modeling and characterization of T-DNA mutants indicated that acyl-activating enzyme encoded by At4g19010 contributes to the biosynthesis of ubiquinone specifically from phenylalanine. CoA ligase assays verified that At4g19010 prefers para-coumarate, ferulate, and caffeate as substrates. Feeding experiments demonstrated that the at4g19010 knockout cannot use para-coumarate for ubiquinone biosynthesis and that the supply of 4-hydroxybenzoate, the side-chain shortened version of para-coumarate, can bypass this blockage. Furthermore, a trans-cinnamate 4-hydroxylase mutant, which is impaired in the conversion of trans-cinnamate into para-coumarate, displayed similar defects in ubiquinone biosynthesis to that of the at4g19010 knockout. Green fluorescent protein fusion experiments demonstrated that At4g19010 occurs in peroxisomes, resulting in an elaborate biosynthetic architecture where phenylpropanoid intermediates have to be transported from the cytosol to peroxisomes and then to mitochondria where ubiquinone is assembled. Collectively, these results demonstrate that At4g19010 activates the propyl side chain of para-coumarate for its subsequent β-oxidative shortening. Evidence is shown that the peroxisomal ABCD transporter (PXA1) plays a critical role in this branch.     

Heterologous production of flavanones in<Emphasis Type="Italic"> Escherichia coli</Emphasis>: potential for combinatorial biosynthesis of flavonoids in bacteria

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 μg naringenin/l from tyrosine and 750 μg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds. Dedicated to Professor Sir David Hopwood.     

Biosynthesis and accumulation of rosmarinic acid in suspension cultures ofColeus blumei

This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH cinnamic acid 4-hydroxylase - 4CL 4-coumarate:CoA ligase - HPPR hydroxyphenylpyruvate reductase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - PAL phenylalanine ammonia-lyase - RAS rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase) - TAT tyrosine aminotransferase     

Formation of syringyl-rich lignins in maize as influenced by feruloylated xylans and <Emphasis Type="Italic">p</Emphasis>-coumaroylated monolignols

Abstract Grass cell walls are atypical because their xylans are acylated with ferulate and lignins are acylated with p-coumarate. To probe the role and interactions of these p-hydroxycinnamates during lignification, feruloylated primary cell walls isolated from maize cell suspensions were lignified with coniferyl and sinapyl alcohols and with varying levels of p-coumarate esters. Ferulate xylan esters enhanced the formation of wall-bound syringyl lignin more than methyl p-coumarate, however, maximal concentrations of syringyl lignin were only one-third that of guaiacyl lignin. Including sinapyl p-coumarate, the presumed precursor of p-coumaroylated lignins, with monolignols unexpectedly accelerated peroxidase inactivation, interfered with ferulate copolymerization into lignin, and had minimal or adverse effects on cell wall lignification. Free phenolic groups of p-coumarate esters in isolated maize lignin and pith cell walls did not undergo oxidative coupling with each other or with added monolignols. Thus, the extensive formation of syringyl-rich lignins and the functional role of extensive lignin acylation by p-coumarate in grasses remains a mystery.     

Monoclonal antibodies to p-coumarate

p-Coumaric acid is one of the predominant phenolic acids acylating the cell walls of grasses; p-coumarates are mainly esterified by lignins and arabinoxylans. Here we describe the production and characterisation of two monoclonal antibodies against p-coumarates.The 5-O-pCou-Ara(1 → 4)Xyl was chemically synthesized and conjugated to a carrier protein. Two interesting antibodies were obtained, hereinafter named INRA-COU1 and INRA-COU2. The specificity of these monoclonal antibodies has been evaluated using competitive-inhibition assays with different oligosaccharides and phenolic compounds. INRA-COU1, recognized free p-coumaric acid or p-coumarate esters. INRA-COU1 did not react with any of the other hydroxycinnamic acids and related compounds found in plants. INRA-COU2, only recognizes esterified p-coumarate. These antibodies were used to study the localization of p-coumarates in the cell walls of grasses. Immunocytochemical analyses indicated noticeable amounts of p-coumarate in the cell walls of the aleurone layer of wheat grain, in the epiderm of cereal straw, and in the exoderm of wheat root.The use of these antibodies will contribute to a better understanding of the organisation and developmental dynamics of cell walls in Graminaceae.     

Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase

A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters). Received: 14 July 2000 / Accepted: 8 August 2000     

Role of protein kinases of human red cell membrane in deformability and aggregation changes

The proteomic analysis has shown that the red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation alterations. The exposure of red blood cells (RBCs) to some chemical compounds has led to a change in the RBC microrheological properties. When forskolin (10 μM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator were added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p<0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p<0.01). The red cell aggregation (RBCA) was significantly decreased under these conditions (p<0.01). Markedly less changes of deformability were found after RBC incubation with protein kinase stimulator C (PKC)—phorbol 12-myristate 13-acetate (PMA). This drug reduced the red cell aggregation only slightly. The red cell tyrosine phosphotase activity was changed by N-vanadat and a significant RBCD rise and RBCA lowering were obtained. The similar effect was found when the cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor—lavendustin eliminated the above mentioned effects.     

The improvement of riboflavin production in <Emphasis Type="Italic">Ashbya gossypii</Emphasis> via disparity mutagenesis and DNA microarray analysis

We generated a high riboflavin-producing mutant strain of Ashbya gossypii by disparity mutagenesis using mutation of DNA polymerase δ in the lagging strand, resulting in loss of DNA repair function by the polymerase. Among 1,353 colonies generated in the first screen, 26 mutants produced more than 3 g/L of riboflavin. By the second screen and single-colony isolation, nine strains that produced more than 5.2 g/L of riboflavin were selected as high riboflavin-producing strains. These mutants were resistant to oxalic acid and hydrogen peroxide as antimetabolites. One strain (W122032) produced 13.7 g/L of riboflavin in a 3-L fermentor using an optimized medium. This represents a ninefold improvement on the production of the wild-type strain. Proteomic analysis revealed that ADE1, RIB1, and RIB5 proteins were expressed at twofold higher levels in this strain than in the wild type. DNA microarray analysis showed that purine and riboflavin biosynthetic pathways were upregulated, while pathways related to carbon source assimilation, energy generation, and glycolysis were downregulated. Genes in the riboflavin biosynthetic pathway were significantly overexpressed during both riboflavin production and stationary phases, for example, RIB1 and RIB3 were expressed at greater than sixfold higher levels in this strain compared to the wild type. These results indicate that the improved riboflavin production in this strain is related to a shift in carbon flux from β-oxidation to the riboflavin biosynthetic pathway.     

Functional male sterility in tomato (Lycopersicon esculentum Mill.) and its application in hybrid seed production

Advantages and disadvantages in using functional male sterility (positional sterile — ps, positional sterile 2 — ps 2, and excerted stigma — ex) in tomato hybrid seed production and attempts to elaborate systems for their more efficacious use in breeding were discussed in this review. It was concluded that the application of one of these types of sterility, (ps 2) in practice, although in a limited number of countries, showed the functional male sterility in tomato was a potential not to be underestimated in developing approaches that aimed at reducting the time and cost associated with hybrid seed production.     

Structure of the O-antigen and characterization of the O-antigen gene cluster of Escherichia coli O108 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic (8-epilegionaminic) acid

On mild acid degradation of the lipopolysaccharide of Escherichia coli O108, the O-polysaccharide was isolated and studied by sugar analysis and one- and two-dimensional ¹H- and ¹³C-NMR spectroscopy. The polysaccharide was found to contain an unusual higher sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-d-galacto-non-2-ulosonic acid (di-N-acetyl-8-epilegionaminic acid, 8eLeg5Ac7Ac). The following structure of the tetrasaccharide repeating unit of the polysac-charide was established: →4)-α-8eLegp5Ac7Ac-(2→6)-α-D-Galp-(1→3)-α-L-FucpNAc-(1→3)-α-D-GlcpNAc-(1→. Functions of the E. coli O108 antigen biosynthetic genes, including seven putative genes for synthesis of 8eLeg5Ac7Ac, were assigned by sequencing the O-antigen gene cluster along with comparison with gene databases and known biosynthetic pathways for related nonulosonic acids.     

The Occurrence of Ferredoxin-sulfite Reductase in Barley Roots

A simple method was established for determining 10 preservatives, butylhydroxyanisole and dibutylhydroxytoluene in food. Steam distillation was carried out, and the distillate was trapped with dichloromethane and distilled water. After acidification and addition of sodium chloride, food additives were extracted from aqueous phase with dichloromethane. The food additives were analyzed with a gas Chromatograph equipped with a dual column system of 10% FFAP and 5% DEGS + 1% H₃PO₄. Column temperature was increased from 140 to 210°C at the rate of 3°C/min, Fluorene was used as an internal standard.

Ethyl p-hydroxybenzoate and isopropyl p-hydroxybenzoate were not separated with the 10% FFAP column, but other food additives were simultaneously determined with this column. With the 5% DEGS + 1% H₃PO₄ column, isobutyl p-hydroxybenzoate and propyl p-hydroxybenzoate were not separated, but the others were simultaneously determined.

Added recovery tests were carried out on about 38 foods.