Unique biosynthesis of dehydroquinic acid?

A search of the genomic sequences of the thermophilic microorganisms Aquifex aeolicus, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii for the first seven enzymes (aroG, B, D, E, K, A, and C ) involved in the shikimic acid biosynthetic pathway reveal two key enzymes are missing. The first enzyme in the pathway, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (aroG) and the second enzyme in the pathway, 5-dehydroquinic acid synthase (aroB) are "missing." The remaining five genes for the shikimate pathway in these organism are present and are similar to the corresponding Escherichia coli genes. The genomic sequences of the thermophiles Pyrococcus abyssi and Thermotoga maritima contain the aroG and aroB genes. Several fungi such as Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pneumocystis carinii f. sp. carinii, and Neurospora crassa contain the gene aroM, a pentafunctional enzyme whose overall activity is equivalent to the combined catalytic activities of proteins expressed by aroB, D, E, K, and A genes. Two of these fungi also lack an aroG gene. A discussion of potential reasons for these missing enzymes is presented.     

Modular engineering of L-tyrosine production in Escherichia coli

Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Site-specific integration and constitutive expression of key genes into Escherichia coli chromosome increases shikimic acid yields

As the key starting material for the chemical synthesis of Oseltamivir, shikimic acid (SA) has captured worldwide attention. Many researchers have tried to improve SA production by metabolic engineering, yet expression plasmids were used generally. In recent years, site-specific integration of key genes into chromosome to increase the yield of metabolites showed considerable advantages. The genes could maintain stably and express constitutively without induction. Herein, crucial genes aroG, aroB, tktA, aroE (encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase, dehydroquinate synthase, transketolase and shikimate dehydrogenase, respectively) of SA pathway and glk, galP (encoding glucokinase and galactose permease) were integrated into the locus of ptsHIcrr (phosphoenolpyruvate: carbohydrate phosphotransferase system operon) in a shikimate kinase genetic defect strain Escherichia coli BW25113 (ΔaroL/aroK, DE3). Furthermore, another key gene ppsA (encoding phosphoenolpyruvate synthase) was integrated into tyrR (encoding Tyr regulator protein). As a result, SA production of the recombinant (SA5/pGBAE) reached to 4.14 g/L in shake flask and 27.41 g/L in a 5-L bioreactor. These data suggested that integration of key genes increased SA yields effectively. This strategy is environmentally friendly for no antibiotic is added, simple to handle without induction, and suitable for industrial production.     

Altered glucose transport and shikimate pathway product yields in E. coli

Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.     

Ultrastructural localisation by protein A-gold immunocytochemistry of 5-enolpyruvylshikimic acid 3-phosphate synthase in a plant cell culture which overproduces the enzyme

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338–16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.Abbreviations EPSP 5-enolpyruvylshikimic acid 3-phosphate     

Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities

The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during a condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. This limitation can be relieved by (i) the recycling of pyruvate from PEP using PEP synthase (Pps) or (ii) use of non-PTS sugars such as xylose. Previous studies have shown the usefulness of overexpressing tktA (encoding transketolase), aroG, and pps (PEP synthase) for DAHP production in an aroB strain unable to utilize DAHP further. In the present study we confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters. In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, as predicted by the stoichiometric analysis. However, this Pps effect is observed only in the presence of both increased AroG and TktA. In xylose mimimal medium, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown here are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.     

Aggregation and separability of the shikimate pathway enzymes in yeasts

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.     

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae

The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141 mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.     

微生物CoQ合成途径及CoQ10生产菌株的分子生物学改造

CoQ10具有呼吸链电子传递者、抗氧化性、调控基因表达等多种生理生化功能,目前不仅用作药物也用作食品添加剂。微生物发酵法是目前生产CoQ10最有效的方法。本文就有关微生物CoQ合成途径及基于CoQ合成途径的CoQ10生产菌株分子生物学改造的策略与研究进展进行了综述和展望。     

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.     

Differential responses of five cherry tomato varieties to water stress: changes on phenolic metabolites and related enzymes

Different tomato cultivars (Solanum lycopersicum L.) with differences in tolerance to drought were subjected to moderate water stress to test the effects on flavonoids and caffeoyl derivatives and related enzymes. Our results indicate that water stress resulted in decreased shikimate pathway (DAHP synthase, shikimate dehydrogenase, phenylalanine ammonium lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase) and phenolic compounds (caffeoylquinic acid derivatives, quercetin and kaempferol) in the cultivars more sensitive to water stress. However, cv. Zarina is more tolerant, and registered a rise in querc-3-rut-pent, kaempferol-3-api-rut, and kaempferol-3-rut under the treatment of water stress. Moreover, this cultivar show increased activities of flavonoid and phenylpropanoid synthesis and decreased in degradation-related enzymes. These results show that moderate water stress can induce shikimate pathway in tolerant cultivar.     

Domain structure and interaction within the pentafunctional arom polypeptide.

The AROM locus of Aspergillus nidulans specifies a pentafunctional polypeptide catalysing five consecutive steps leading to the production of 5-enolpyruvylshikimate 3-phosphate in the shikimate pathway. Aided by oligonucleotide-mediated site-directed mutagenesis, the whole AROM locus and various overlapping subfragments from within it have been fused to the powerful hybrid trc promoter in the Escherichia coli plasmid pKK233-2. Expression of these subfragments in appropriate aro mutants of E. coli has (a) allowed the delineation of functional domains within the arom polypeptide, (b) shown that the arom polypeptide falls in two independently folding and functioning regions, the N-terminal half specifying 3-dehydroquinate (DHQ) synthase and EPSP synthase and the C-terminus specifying shikimate kinase, biosynthetic 3-dehydroquinase (DHQase) and shikimate dehydrogenase, and (c) strongly suggested an interaction between the DHQ synthase and EPSP synthase domains to stabilise the EPSP synthase activity. In addition an isoenzyme of biosynthetic DHQase, catabolic DHQase, encoded by the QUTE gene of A. nidulans has been transcribed from the trc promoter and upon isopropyl-thio-beta-D-galactoside induction produces up to 20% of the total soluble cell protein.     

Corynebacterium glutamicum contains 3-deoxy-D-arabino-heptulosonate 7-phosphate synthases that display novel biochemical features

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 2.5.1.54) catalyzes the first step of the shikimate pathway that finally leads to the biosynthesis of aromatic amino acids phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr). In Corynebacterium glutamicum ATCC 13032, two chromosomal genes, NCgl0950 (aroF) and NCgl2098 (aroG), were located that encode two putative DAHP synthases. The deletion of NCgl2098 resulted in the loss of the ability of C. glutamicum RES167 (a restriction-deficient strain derived from C. glutamicum ATCC 13032) to grow in mineral medium; however, the deletion of NCgl0950 did not result in any observable phenotypic alteration. Analysis of DAHP synthase activities in the wild type and mutants of C. glutamicum RES167 indicated that NCgl2098, rather than NCgl0950, was involved in the biosynthesis of aromatic amino acids. Cloning and expression in Escherichia coli showed that both NCgl0950 and NCgl2098 encoded active DAHP synthases. Both the NCgl0950 and NCgl2098 DAHP synthases were purified from recombinant E. coli cells and characterized. The NCgl0950 DAHP synthase was sensitive to feedback inhibition by Tyr and, to a much lesser extent, by Phe and Trp. The NCgl2098 DAHP synthase was slightly sensitive to feedback inhibition by Trp, but not sensitive to Tyr and Phe, findings that were in contrast to the properties of previously known DAHP synthases from C. glutamicum subsp. flavum. Both Co2+ and Mn2+ significantly stimulated the NCgl0950 DAHP synthase's activity, whereas Mn2+ was much more stimulatory than Co2+ to the NCgl2098 DAHP synthase's activity.     

The entry reaction of the plant shikimate pathway is subjected to highly complex metabolite-mediated regulation

The plant shikimate pathway directs bulk carbon flow toward biosynthesis of aromatic amino acids (AAAs, i.e. tyrosine, phenylalanine, and tryptophan) and numerous aromatic phytochemicals. The microbial shikimate pathway is feedback inhibited by AAAs at the first enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DHS). However, AAAs generally do not inhibit DHS activities from plant extracts and how plants regulate the shikimate pathway remains elusive. Here, we characterized recombinant Arabidopsis thaliana DHSs (AthDHSs) and found that tyrosine and tryptophan inhibit AthDHS2, but not AthDHS1 or AthDHS3. Mixing AthDHS2 with AthDHS1 or 3 attenuated its inhibition. The AAA and phenylpropanoid pathway intermediates chorismate and caffeate, respectively, strongly inhibited all AthDHSs, while the arogenate intermediate counteracted the AthDHS1 or 3 inhibition by chorismate. AAAs inhibited DHS activity in young seedlings, where AthDHS2 is highly expressed, but not in mature leaves, where AthDHS1 is predominantly expressed. Arabidopsis dhs1 and dhs3 knockout mutants were hypersensitive to tyrosine and tryptophan, respectively, while dhs2 was resistant to tyrosine-mediated growth inhibition. dhs1 and dhs3 also had reduced anthocyanin accumulation under high light stress. These findings reveal the highly complex regulation of the entry reaction of the plant shikimate pathway and lay the foundation for efforts to control the production of AAAs and diverse aromatic natural products in plants.

Characterization of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes and mutants revealed highly complex metabolite-mediated feedback regulation of the plant shikimate pathway.     

The roles of the shikimate pathway genes,aroA and aroB,in virulence,growth and UV tolerance of Burkholderia glumae strain 411gr‐6

Burkholderia glumae is the major causal agent of bacterial panicle blight of rice, which is a growing disease problem for rice growers worldwide. In our previous study, some B. glumae strains showed pigmentation phenotypes producing at least two (yellow–green and purple) pigment compounds in casein–peptone–glucose agar medium. The B. glumae strains LSUPB114 and LSUPB116 are pigment‐deficient mutant derivatives of the virulent and pigment‐proficient strain 411gr‐6, having mini‐Tn5gus insertions in aroA encoding 3‐phosphoshikimate 1‐carboxyvinyltransferase and aroB encoding 3‐dehydroquinate synthase, respectively. Both enzymes are known to be involved in the shikimate pathway, which leads to the synthesis of aromatic amino acids. Here, we demonstrate that aroA and aroB are required for normal virulence in rice and onion, growth in M9 minimal medium and tolerance to UV light, but are dispensable for the production of the phytotoxin toxoflavin. These results suggest that the shikimate pathway is involved in bacterial pathogenesis by B. glumae without a significant role in the production of toxoflavin, a major virulence factor of this pathogen.     

Pulse experiments as a prerequisite for the quantification of in vivo enzyme kinetics in aromatic amino acid pathway of Escherichia coli

Glucose pulse experiments were performed to elucidate their effects on the carbon flux into the aromatic amino acid pathway in different Escherichia coli strains. Using a 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP, aroB(-))-producing strain, a fed-batch fermentation strategy specialized for glucose pulse experiments was developed and further applied for 3-dehydroshikimate (DHS, aroE(-))- and shikimate 3-phosphate (S3P, aroA(-))-producing E. coli strains. The strains overexpress a feedback-resistant DAHP synthase and additional enzymes to prevent rate-limiting steps in the aromatic amino acid pathway. Changes of carbon flux into the aromatic amino acid pathway were determined via extracellular metabolite accumulations using (1)H NMR and HPLC measurements. As an important result, a close relationship between pulse intensity and aromatic metabolite formation rates was identified. The more downstream an aromatic pathway intermediate was located, the stronger the glucose pulse intensity had to be in order to detect significant changes in product formation. However, with the experimental conditions chosen, changes after pulse were detected even for shikimate 3-phosphate, the most downstream accumulating metabolite of this experimental series. Hence glucose pulse experiments are assumed to be a promising tool even for the analysis of final pathway products such as, for example, L-phenylalanine.     

Overproduction of, and interaction within, bifunctional domains from the amino- and carboxy-termini of the pentafunctional AROM protein of Aspergillus nidulans

The pentafunctional AROM protein in Aspergillus nidulans and other fungi catalyses five consecutive enzymatic steps leading to the production of 5-enolpyruvylshikimate 3-phosphate (EPSP) in the shikimate pathway. The AROM protein has five separate enzymatic domains that have previously been shown to display a range of abilities to fold and function in isolation as monofunctional enzymes. In this communication, we report (1) the stable overproduction of a bifunctional protein containing the 3-dehydroquinate (DHQ) synthase and EPSP synthase activities in Escherichia coli to around 10% of the total cell protein; (2) that both the DHQ synthase and EPSP synthase activities in the over-produced fragment are enzymatically active as judged by their ability to complement aroA and aroB mutants of E. coli; (3) that the EPSP synthase domain is only enzymatically active when covalently attached to the DHQ synthase domain (the cis arrangement). When DHQ synthase and EPSP synthase are produced concomitantly by transcribing sequences encoding the individual domains from separate plasmids in the same bacterial cell (the trans arrangement) no overproduction or enzyme activity can be detected for the EPSP synthase domain; (4) the EPSP synthase domain can be stably overproduced as a fusion protein with glutathione S-transferase (GST), however the EPSP synthase in this instance is enzymatically inactive; (5) a protein containing an enzymatically inactive DHQ synthase domain in the cis arrangement with EPSP synthase domain is stably overproduced with enzymatically active EPSP synthase; (6) the two C-terminal domains of the AROM protein specifying the 3-dehydroquinase and shikimate dehydrogenase domains can be overproduced in A. nidulans using a specially constructed expression vector. This same bi-domain fragment however is not produced in E. coli when identical coding sequences are transcribed from a prokaryotic expression vector. These data support the view that multifunctional/multidomain proteins do not solely consist of independent units covalently linked together, but rather that certain individual domains interact to varying degrees to stabilise enzyme activity.