Regulation of E. coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNA^Phe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Expression of pheA gene using PR-PL tandem promoter of bacteriophage lambda

Summary The genepheA ⁺ coding chorismate niutase P-prephenate dehydratase, one of the regulatory enzymes of phenylalanine biosynthesis, was cloned into the down-stream of P_R-P_L tandem promoter. In this construction, both the native promoter-operator region and the attenuator region ofpheA ⁺ operon were eliminated so as to avoid the repression and attenuation ofpheA ⁺ gene expression. The expression ofpheA ⁺ gene was directed by P_R-P_L tandem promoter of bacteriophage lambda and controlled by a temperature sensitive repressor, cI₈₅₇.It was shown that the expression as well as phenylalanine production was regulated by temperature. Maximum production of phenylalanine, 170 mg/l, was obtained at 40°C. The host strain, MC1065, produced a trace (4 mg/l) of phenylalanine at the same temperature.     

Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli

The bifunctional enzyme chorismate mutase (CM)-prephenate dehydratase (PD), which is encoded by the pheA gene of Escherichia coli, catalyses the two consecutive key steps in phenylalanine biosynthesis. To utilize the enzyme for metabolic engineering of phenylalanine-producing Corynebacterium glutamicum KY10694, the intact gene was cloned on a multicopy vector to yield pEA11. C. glutamicum cells transformed with pEA11 exhibited a more than tenfold increase in CM and PD activities relative to the host cells. Moreover, the level of pheA expression was further elevated a fewfold when cells were starved of phenylalanine, suggesting that the attenuation regulation of pheA expression functions in heterogeneous C. glutanicum. Plasmid pEA11 encoding the wild-type enzyme was mutated to yield pEA22, which specified CM-PD exhibiting almost complete resistance to end-product inhibition. When pEA22 was introduced into KY10694, both the activities of CM and PD were highly maintained throughout the cultivation, thus leading to a 35% increased production (23 g/l) of phenylalanine.     

Hyperproduction of phenylalanine by Escherichia coli: application of a temperature-controllable expression vector carrying the repressor-promoter system of bacteriophage lambda

For the high production of phenylalanine by Escherichia coli, we cloned the pheA^FR and aroF^FR genes (FR = feedback resistant), which encoded chorismate mutase P-prephenate dehydratase and 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase that are feedback inhibition-free as to the endproducts, into a temperature-controllable expression vector composed of the P_R and P_L promoter and a temperature sensitive repressor, cI₈₅₇, of bacteriophage lambda. The plasmid obtained was designated as pSY130-14, and the temperature dependency of expression of the cloned genes and of phenylalanine production was investigated at different temperatures between 30 and 42°C using the strain AT2471 harbouring the plasmid. Above 35°C, the pheA^FR gene and aroF^FR gene expressions, and activities of both enzymes continued to increase up to 42°C. The cell concentration remained constant up to 38.5°C, but started to decrease sharply above 40°C, while the cell concentration of the host strain, AT2471, remained constant at all temperatures tested. The concentration of phenylalanine also depended on the temperature, and the highest production of phenylalanine, 18.6 g l⁻¹, was obtained from glucose at 38.5°C in a 2.5 1 reactor.     

Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF ^wt or pheA ^fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF ^wt and pheA ^fbr improved the titer of l-Phe to 4.46 ± 0.06 g l^?1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA ^fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l^?1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.     

Genetic aspects of aromatic amino acid biosynthesis in Lactococcus lactic

Polymerase chain reaction (PCR) primers designed from a multiple alignment of predicted amino acid sequences from bacterial aroA genes were used to amplify a fragment of Lactococcus lactis DNA. An 8 kb fragment was then cloned from a lambda library and the DNA sequence of a 4.4 kb region determined. This region was found to contain the genes tyrA, aroA, aroK, and pheA, which are involved in aromatic amino acid biosynthesis and folate metabolism. TyrA has been shown to be secreted and AroK also has a signal sequence, suggesting that these proteins have a secondary function, possibly in the transport of amino acids. The aroA gene from L. lactis has been shown to complement an E. coli mutant strain deficient in this gene. The arrangement of genes involved in aromatic amino acid biosynthesis in L. lactis appears to differ from that in other organisms.     

Derepression of Uridine Diphosphate-Glucose Pyrophosphorylase (galU) in capR(lon), capS, and capT Mutants and Studies on the galU Repressor

Mutation of the capR(lon), capS, or capT genes in Escherichia coli K-12 causes overproduction of capsular polysaccharide leading to a mucoid phenotype. Several of the enzymes involved in capsular polysaccharide synthesis are derepressed in cap mutants. Previously it was shown that uridine diphosphate-glucose (UDPG) pyrophosphorylase, an enzyme involved in the synthesis of three of the nucleotide sugar precursors of the capsule, is derepressed in capR mutants. The control of galU, the gene which codes for UDPG pyrophosphorylase, is described in this study. In addition, it has been found that the enzyme is also derepressed in capS and capT mutants. The effect of galU gene dosage in cap mutants and the wild-type strain (all lysogenic for 80) was studied by infecting them with the purified transducing phage 80dgalU. The level of UDPG pyrophosphorylase increased in proportion to the number of galU copies added. The rate of enzyme synthesis in the mutants was about sixfold higher than in the wild type per galU gene added for multiplicities of infection from one to twenty. Thus, all the galU copies added to the wild-type lysogen were repressed. We obtain greater than 20 galU copies per cell by infecting the nonlysogenic strain which allows multiplication of 80dgalU. With some number of galU copies greater than 20, the rate of UDPG pyrophosphorylase synthesis in the wild type approaches the mutant rate of synthesis. The results suggest that there may indeed be a galU repressor pool in the cell which can be completely titrated. This pool must be composed of more than 20 galU repressor molecules. Since the capR, capS, and capT gene products or combinations thereof are known to control other widely separated operons of the cell besides the galU gene, it is postulated that the galU repressor may be capable of binding other operators. This would account for the relatively large pool of galU repressors per cell.     

Molecular cloning and sequencing of the phenol hydroxylase gene from Pseudomonas putida BH

A genomic library of the phenol-degrading bacterium Pseudomonas putida BH was constructed in the broad host range cosmid pVK100 and introduced into Escherichia coli HB101. One of the recombinant cosmids recovered from catechol- and/or 2-hydroxymuconic semialdehyde-accumulating clones, pS10–45, had a 19.6-kb insert fragment which allowed P. putida KT2440 to grow on phenol as a sole carbon and energy source. Subcloning and expression studies indicated that the phenol hydroxylase gene cluster (pheA) is located on a 6.1-kb SacI fragment. The results of DNA sequencing of the SacI fragment revealed that the pheA gene cluster encodes a multicomponent phenol hydroxylase.     

Shared Metabolic Pathways in a Coevolved Insect-Bacterial Symbiosis

The symbiotic bacterium Buchnera aphidicola lacks key genes in the biosynthesis of five essential amino acids (EAAs), and yet its animal hosts (aphids) depend on the symbiosis for the synthesis of these EAAs (isoleucine, leucine, methionine, phenylalanine, and valine). We tested the hypothesis, derived from genome annotation, that the missing Buchnera reactions are mediated by host enzymes, with the exchange of metabolic intermediates between the partners. The specialized host cells bearing Buchnera were separated into a Buchnera fraction and a Buchnera-free host cell fraction (HF). Addition of HF to isolated Buchnera preparations significantly increased the production of leucine and phenylalanine, and recombinant enzymes mediating the final reactions in branched-chain amino acid and phenylalanine synthesis rescued the production of these EAAs by Buchnera preparations without HF. The likely precursors for the missing proximal reactions in isoleucine and methionine synthesis were identified, and they differed from predictions based on genome annotations: synthesis of 2-oxobutanoate, the aphid-derived precursor of isoleucine synthesis, was stimulated by homoserine and not threonine via threonine dehydratase, and production of the homocysteine precursor of methionine was driven by cystathionine, not cysteine, via reversal of the transsulfuration pathway. The evolution of shared metabolic pathways in this symbiosis can be attributed to host compensation for genomic deterioration in the symbiont, involving changes in host gene expression networks to recruit specific enzymes to the host cell.     

Phenylalanine and tyrosine biosynthesis in Escherichia coli K-12: mutants derepressed for 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (phe), 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthetase (tyr), chorismate mutase T-prephenate dehydrogenase, and transaminase A

Mutant strains of Escherichia coli have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthetase (phe) is derepressed, in addition to those enzymes of tyrosine biosynthesis previously shown to be controlled by the gene tyrR. The major enzyme of the terminal pathway of phenylalanine biosynthesis chorismate mutase-prephenate dehydratase is not derepressed in these strains. Genetic analysis of the mutants shows that the mutation or mutations causing derepression map close to previously reported tyrR mutations. A study of one of the mutations has shown it to be recessive to the wild-type allele in a diploid strain. It is proposed that the tyrR gene product is involved in the regulation of the synthesis of DAHP synthetase (phe) as well as the synthesis of DAHP synthetase (tyr), chorismate mutase-prephenate dehydrogenase, and transaminase A.     

A kinetic and structural comparison of chorismate mutase/prephenate dehydratase from mutant strains of Escherichia coli K12 defective in the PheA gene

Three classes of mutant strains of Escherichia coli K12 defective in pheA, the gene coding for chorismate mutase/prephenate dehydratase, have been isolated: (1) those lacking prephenate dehydratase activity, (2) those lacking chorismate mutase activity, and (3) those lacking both activities. Chorismate mutase/prephenate dehydratase from the second class of mutants was less sensitive to inhibition by phenylalanine than wild-type enzyme and, along with the defective enzyme from the third class of mutants, could not be purified by affinity chromatography on Sepharosyl-phenylalanine. Pure chorismate mutase/prephenate dehydratase protein was prepared from two strains belonging to the first class. The chorismate mutase activity of these enzymes is kinetically similar to that of the wild-type enzyme except for a two- to threefold increase in both the K_a for chorismate and the K_is for inhibition by prephenate. In both cases only one change in the tryptic fingerprint was detected, resulting from a substitution of the threonine residue in the peptide Gln·Asn·Phe·Thr·Arg. This suggests that this residue is catalytically or structurally essential for the dehydratase activity.     

Continuous synthesis of partially derepressed aspartate transcarbamylase during the division cycle of Escherichia coli B-r

The pattern of synthesis of aspartate transcarbamylase during the division cycle of Escherichia coli B/r was determined for several steady-state levels of partially derepressed synthesis of the enzyme. The pattern of synthesis was measured by repressing ATCase synthesis with uracil in populations growing on the surfaces of membrane filters and measuring enzyme activity as a function of time in newborn cells eluted from the populations. The variations in levels of steady-state partial derepression of ATCase synthesis were achieved by including 6-aza-uracil in the growth medium or by using a mutant possessing an increased level of partially derepressed ATCase synthesis compared to the parental strain. The results indicated that the enzyme was synthesized continuously during the division cycle at all levels of partial derepression. The observed absence of step-wise ATCase synthesis during the division cycle, which had been reported previously, was not due to a limitation of the method of analysis since a step-wise pattern was observed when it was expected, i.e. after ATCase synthesis was pulse-derepressed in an exponentially growing population. The possibility that previous reports of periodic synthesis of partially derepressed ATCase in E. coli (Kuempel et al., 1965; Goodwin, 1969a) were manifestations of a disturbance in cellular metabolism caused by the techniques used for synchronization is discussed.     

Genetic Studies of Leucine Biosynthesis in Bacillus subtilis

The mutations in a series of leucine auxotrophs isolated after treatment with nitrosoguanidine, ultraviolet light, and ICR-191 have been mapped between ilvC and pheA on the Bacillus subtilis chromosome. A fine structure map of the region was constructed by transformation. Analysis of several strains by assaying levels of their leucine bioysnthetic enzymes has shown that the region encodes three enzymes. The order of the genes with respect to the biosynthetic steps catalyzed by the gene products is 1–3–2.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

The effect of an ilvA Mu phage insertion on ilv gene expression in Escherichia coli K-12

Physiological analysis of an E. coli K-12 strain carrying a Mu phage integrated into the ilvA structural gene shows that there is a polar affect on ilvD gene expression, whereas, the ilvE gene maintains a normal multivalent regulation response. It was also demonstrated that the ilvC and ilvB genes can be derepressed and repressed in response to ilv multivalent control. These experiments demonstrate that the ilvE structural gene can be regulated independently from the ilvA and ilvD structural genes and that the ilvC structural gene does not require the complete ilvA gene product (threonine deaminase) for its induction.     

Expression of glnB and a glnB-Like Gene (glnK) in a Ribulose Bisphosphate Carboxylase/Oxygenase-Deficient Mutant of Rhodobacter sphaeroides

In a ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant of Rhodobacter sphaeroides, strain 16PHC, nitrogenase activity was derepressed in the presence of ammonia under photoheterotrophic growth conditions. Previous studies also showed that reintroduction of a functional RubisCO and Calvin-Benson-Bassham (CBB) pathway suppressed the deregulation of nitrogenase synthesis in this strain. In this study, the derepression of nitrogenase synthesis in the presence of ammonia in strain 16PHC was further explored by using a glnB::lacZ fusion, since the product of the glnB gene is known to have a negative effect on ammonia-regulated nif control. It was found that glnB expression was repressed in strain 16PHC under photoheterotrophic growth conditions with either ammonia or glutamate as the nitrogen source; glutamine synthetase (GS) levels were also affected in this strain. However, when cells regained a functional CBB pathway by trans complementation of the deleted genes, wild-type levels of GS and glnB expression were restored. Furthermore, a glnB-like gene, glnK, was isolated from this organism, and its expression was found to be under tight nitrogen control in the wild type. Surprisingly, glnK expression was found to be derepressed in strain 16PHC under photoheterotrophic conditions in the presence of ammonia.