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.     

Two tandem promoters to increase gene expression in Lactococcus lactis

Two plasmids, pPAH and pAH, containing a staphylokinase variant gene (sakXH) under the control of two tandem promoters (P₃₂-P_lacA) or promoter P_lacA alone were constructed and introduced into Lactococcus lactis MG5267. The expression of sakXH in the strain MG5267(pPAH) was approximately twice as high as that in the strain MG5267(pAH), according to the formation of fibrinolytic halos on fibrinolytic plates detected at the same conditions, indicating that the two tandem promoters were stronger than one alone. Difference between the expressions of sakXH under the inducible and non-inducible conditions suggested that P_lacA retained its feature as an inducible promoter when fused to promoter P₃₂.     

A Single mri Slice Does Not Accurately Predict Visceral and Subcutaneous Adipose Tissue Changes During Weight Loss

Earlier cross‐sectional studies found that a single magnetic resonance imaging (MRI) slice predicts total visceral and subcutaneous adipose tissue (VAT and SAT) volumes well. We sought to investigate the accuracy of trunk single slice imaging in estimating changes of total VAT and SAT volume in 123 overweight and obese subjects who were enrolled in a 24‐week CB‐1R inverse agonist clinical trial (weight change, ?7.7 ± 5.3 kg; SAT change, ?5.4 ± 4.9 l, VAT change, ?0.8 ± 1.0 l). VAT and SAT volumes at baseline and 24 weeks were derived from whole‐body MRI images. The VAT area 5–10 cm above L₄—L₅ (A_+5–10) (R² = 0.59–0.70, P < 0.001) best predicted changes in VAT volume but the strength of these correlations was significantly lower than those at baseline (R² = 0.85–0.90, P < 0.001). Furthermore, the L₄—L₅ slice poorly predicted VAT volume changes (R² = 0.24–0.29, P < 0.001). Studies will require 44–69% more subjects if (A_+5–10) is used and 243–320% more subjects if the L₄—L₅ slice is used for equivalent power of multislice total volume measurements of VAT changes. Similarly, single slice imaging predicts SAT loss less well than cross‐sectional SAT (R² = 0.31–0.49 vs. R² = 0.52–0.68, P < 0.05). Results were the same when examined in men and women separately. A single MRI slice 5–10 cm above L₄—L₅ is more powerful than the traditionally used L₄—L₅ slice in detecting VAT changes, but in general single slice imaging poorly predicts VAT and SAT changes during weight loss. For certain study designs, multislice imaging may be more cost‐effective than single slice imaging in detecting changes for VAT and SAT.     

Efficient expression of a promoter-controlled gene: Tandem promoters of lambda PR and PL functional in enteric bacteria

We describe an expression vector that functions in enteric bacteria. The vector contains the coliphage λ promoters P_R and P_L and entire P_R and P_L operators in tandem upstream from the multiple cloning sites containing the kanamycin-resistant gene. The vector also specifies a ribosome binding site and a thermolabile repressor, cI₈₅₇, and the P_RM promoter. These promoters as well as lacUV5 and trp promoters were inserted into the EcoRI site of pKO-1 plasmid so that they drove the expression of a reporter gene, galactokinase (galK). The P_RP_L promoter showed the highest efficiency of galK expression in the Escherichia coli strain K12ΔH1Δtrp; it was strong in Klebsiella aerogenes, and weak in Serratia marcescens and Citrobacter freundii.     

Expression of cDNA fragment encoding sperm membrane peptide inE. coli

A secretory high-level expression cloning vector designated as pSBC-20 was constructed by inserting a DNA fragment encoding the signal peptide of ompA protein into pBV 220 vector. Any foreign DNA fragment can be inserted into the polylinker cloning sites located after the secretion signal sequence. The cloned foreign gene is under the control of the P _R -P _L promoter while the expression of the gene is regulated by the cI-gene product. The products are secreted into the periplasmic space of bacteria or into the medium. A recombinant plasmid (pRSD-220) was constructed by inserting the 210 bp from RSD-2, a cDNA encoding a peptide fragment of human sperm protein, into the EcoRI site of pSBC-20. TheE. coli cells transformed with pRSD-220 were propagated at 30 °C, then incubated at 42 °C for several hrs. The cloned gene product was secreted into the culture medium at a high rate. The yield was about 60 mg of gene product per liter of cultured medium.     

Identification of a novel sugar-H+ symport protein, FucP, for transport of L-fucose into Escherichia coli

l -Fucose (6-deoxy-l -galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli is mediated by An l -fucose-H⁺ symport activity, in order to determine the nature of a putative transporter encoded by the E. coli fucP gene and Identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda O_l P_l promoters, induction of the T7 promoter resulted in the expression of [¹⁴C]-l -fucose uptake activity and the concomitant expression of a [³⁵S]-Met-labelled 32 kDa protein at levels too tow for detection by staining with Coomassie briiiiant blue or for protein sequencing, induction of the lambda O_l P_l promoter caused the appearance of l -fucose-H₊ symport activity and of a Coomassie brilliant blue-stained 32 kDa membrane protein expressed at high levels sufficient for identification as FucP by N-terminal protein sequencing. The FucP protein is, therefore, a sugar-H₊ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.     

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.     

Activation of the expression of the microcin C51 operon upon glucose starvation of cells at the exponential growth phase

It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the ^S subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon ( P_mcc-lac ), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P_mcc-lac expression upon severe glucose starvation occurred in rpoS ⁺ and rpoS ^– strains. In cells carrying the rpoD800 mutation that renders the ⁷⁰ subunit of RNA polymerase temperature-sensitive, an activation of P_mcc-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors —nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol—did not activate P_mcc-lac expression in cells at the exponential growth phase.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 48–52.Original Russian Text Copyright © 2005 by Veselovskii, Metlitskaya, Lipasova, Bass, Khmel.     

Anaerobic synthesis of succinic acid by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strains with activated NAD<Superscript>+</Superscript>-reducing pyruvate dehydrogenase complex

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD⁺-reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acid from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids through deletion of the genes ackA, pta, poxB, and ldhA (SGM0.1) in E. coli MG1655 strain and by additional introduction of the Bacillus subtilis pyruvate carboxylase (SGM0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 P_L-aceEF-lpdA and SGM0.1 P_L-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P_L-aceEF-lpdA and SGM0.1 P_L-aceEF-lpdA [pPYC] exceeded the corresponding yields of control strains by 2 and 33% in the absence and by 9 and 26% in the presence in media of HCO₃⁻ ion. It is concluded that an increase in the succinic acid production by strain SGM0.1 P_L-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive branch of the tricarboxylic acid cycle, is caused by activation of the glyoxylate shunt.     

Utilization of promoter regions of coliphage lambda in Bacillus subtilis

Summary In vivo studies with galactokinase monitoring system demonstrated that the coliphage lambda P_RP_L promoter regions could be utilized in B. subtilis. These promoter regions were preferentially utilized during the stationary growth phase of B. subtilis. However, these promoter regions were not controlled by the cI₈₅₇ gene at permissive or non-permissive temperature in B. subtilis, although the P _RM promoter was utilized in B. subtilis. S1-nuclease mapping suggests that B. subtilis RNA polymerase recognizes specific sequences in P _R promoter region that is not utilized in E. coli.     

Growth,energy and nitrogen budgets and efficiencies of the growing larvae of Megachile pacifica (Panzer) (Hymenoptera: Megachilidae)

Summary The larvae of Megachile pacifica, the leafcutter bee, develop within a cell constructed of pieces of leaf by the adult female which also provides a plug of nectar and pollen, the sole source of nutrient for the larvae. In this study, eggs (1 per cell) hatched in 2–3 days and larvae were fully grown after a further 9 days at 28° C.The mean larval dry weight (dw) when fully grown (P _L ) was 18.89 mg. Larvae ate 45.99 mg (dw) (C) of their food leaving 0.90 mg (dw) unconsumed. The faeces (FU) weighed an average of 5.62 mg (dw) and their cocoon weighed 2.68 mg (dw) (P _E ). The components of their energy budget (C=(P _L +P _E )+R+FU) were C=1079.4 J, P _L =569.1 J, P _E =62.25 J, R (metabolic energy measured with a Gilson respirometer)=218.3 J and FU=134.4 J. R calculated by difference was 313.6 J.Full grown larvae contained 1.17 mg of nitrogen (N). They egested 0.19 mg N and incorporated 0.16 mg N in the cocoons. They ingested 1.51 mg N (measured) — the calculated consumption of nitrogen (by difference) was 1.52 mg.The ecological efficiencies showed that these larvae are among the most efficient invertebrate converters of energy and nitrogen yet recorded. The assimilation efficiencies (A·C ^-1 ) were 87.5% (energy), 87.2% (N), the net ecological efficiencies (P·A ^-1 ) were 66.8% (energy) and 100% (N) and the gross ecological efficiencies (P·C ^-1 ) were 58.5% (energy) and 87.6% (N). the production to respiration ratio (x100) was 201.5% using calculated R.     

Study on russet-related enzymatic activity and gene expression in ‘Shine Muscat’ grape treated with GA3 and CPPU

Physiological (metabolite analysis) and molecular (gene expression) approaches were used to understand the mechanism underlying russet formation in response to the application of GA₃ and CPPU (Forchlorfenuron) in a Japanese table grape cultivar ‘Shine Muscat’. Several different concentrations of GA₃ and GA₃?+?CPPU [25?mg?L^?1 GA₃ (A), 25?mg?L^?1 GA₃?+?5?mg?L^?1 CPPU (B), 25?mg?L^?1 GA₃?+?10?mg?L^?1 CPPU (C), and 25?mg?L^?1 GA₃?+?15?mg?L^?1 CPPU (D)] were applied to grape berry clusters at two weeks after flowering (WAF). No russet was observed on the berries treated with the ‘C’ combination. Lower levels of phenylalanine ammonia-lyase (PAL) activity was observed in the treated samples, relative to the untreated material. Reduced peroxide (POD) activity was also observed in response to different treatments, while the expression of Peroxidase 17 and Phenylalanine ammonia-lyase G1 genes mirrored lignin content. Increased activity of 4-coenzyme A ligase (4CL) may contribute to decreasing the level of russet and help to improve grape berry quality.     

Production of tyrosine from sucrose or glucose achieved by rapid genetic changes to phenylalanine-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> strains

Escherichia coli K12 strains producing l-phenylalanine were converted to l-tyrosine-producing strains using a novel genetic method for gene replacement. We deleted a region of the E. coli K12 chromosome including the pheA gene encoding chorismate mutase/prephenate dehydratase, its leader peptide (pheL), and its promoter using a new polymerase chain reaction-based method that does not leave a chromosomal scar. For high level expression of tyrA, encoding chorismate mutase/prephenate dehydrogenase, its native promoter was replaced with the strong trc promoter. The linked ΔpheLA and Ptrc-tyrA::Kan^R genetic modifications were moved into l-phenylalanine producing strains by generalized transduction to convert l-phenylalanine-producing strains to l-tyrosine-producing strains. Moreover, introduction of a plasmid carrying genes responsible for sucrose degradation into these strains enabled l-tyrosine-production from sucrose.     

High-affinity binding ofl-glutamate to chick retinal membranes

Binding ofl-[³H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P₁), or terminals from the inner plexiform layer (P₂) was studied. Na⁺-dependent and Na⁺-independent binding to these membranes was demonstrated. Na⁺-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (K _B=0.55 M) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [³H]Glutamate binding to P₁ and P₂ fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [³H]glutamate to physiologically relevant receptors in the chick retina.     

Implications of an anomalous intracellular electrical response in bullfrog corneal epithelium

Summary The ionic dependencies of the transepithelial and intracellular electrical parameters were measured in the isolated frog cornea. In NaCl Ringer's the intracellular potential differenceV _sc measured under short-circuit conditions depolarized by nearly the same amount after either increasing the stromal-side KCl concentration from 2.5 to 25mm or exposure to 2mm BaCl₂ (K⁺ channel blocker). With Ba²⁺ the depolarization of theV _sc by 25mm K⁺ was reduced to one-quarter of the control change. If the Cl-permselective apical membrane resistanceR _o remained unchanged, the relative basolateral membrane resistanceR _i, which includes the lateral intercellular space, increased at the most by less than twofold after Ba²⁺. These effects in conjunction with the depolarization of theV _sc by 62 mV after increasing the stromal-side K⁺ from 2.5 to 100mm in Cl-free Ringer's as well as the increase of the apparent ratio of membrane resistances (a=R _o/R_i) from 13 to 32 are all indicative of an appreciable basolateral membrane K⁺ conductance. This ratio decreased significantly after exposure to either 25mm K⁺ or Ba²⁺. The decline ofR _o/R_i with 25mm K⁺ appears to be anomalous since this decrease is not consistent with just an increase of basolateral membrane conductance by 25mm K⁺, but rather perhaps a larger decrease ofR _o thanR _iAlso an increase of lateral space resistance may offset the effect of decreasingR _i with 25mm K⁺. In contrast,R _o/R_i did transiently increase during voltage clamping of the apical membrane potential differenceV _o and exposure to 25mm K⁺ on the stromal side. This increase and subsequent decrease ofR _o/R_i supports the idea that increases in stromal K⁺ concentration may produce secondary membrane resistance changes. These effects onR _o/R_i show that the presence of asymmetric ionic conductance properties in the apical and basolateral membranes can limit the interpretative value of this parameter. The complete substitution of Na⁺ withn-methyl-glucamine in Cl-free Ringer's on the stromal side hyperpolarized theV _sc by 6 mV whereas 10^–4 m ouabain depolarized theV _sc by 7 mV. Thus the basolateral membrane contains K⁺, Na⁺ and perhaps Cl^– pathways in parallel with the Na/K pump component.     

Intracellular nitrite accumulation: The cause of growth inhibition of Microcystis aeruginosa exposure to high nitrite level

The aim of the present study is to test the role of intracellular nitrite in external nitrite suppressing algal growth. We examined the growth of Microcystis aeruginosa at different nitrite levels under high nitrate conditions and without nitrate conditions. There were higher intracellular nitrite and lower P_m^chla, R_d ^chla, α^chl, maximum cell density and specific growth rate in high nitrate group than nitrate absence group at 5 mg NO₂^?‐N L^?1. At 10 and 15 mg NO₂^?‐N L^?1, P_m^chla, R_d ^chla, α^chl, maximum cell densities and specific growth rates in the high nitrate group became higher than those of the nitrate absence group, while a lower intracellular nitrite in the high nitrate group than nitrate absence group was observed. In addition, the intracellular nitrite and the growth of M. aeruginosa in the high nitrate group did not change from 5 to 10 mg NO₂^?‐N L^?1. In the nitrite uptake experiment, with nitrite concentration increasing from 5 to 15 mg NO₂^?‐N L^?1, maximum nitrite uptake rate of alga increased, and half‐saturation constant of alga decreased. These results indicate that external nitrite inhibited algal growth through stimulating intracellular nitrite rise, which resulted from overexpression of nitrite transporter.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, J_M and K_m of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, J_M, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the K_m for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the K_m for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high K_m and is not additive with the Na⁺-dependent flux.     

Characteristics of the vacuolar membrane ofNitella

Summary Taking advantage of vacuolar perfusion, concentrations of K⁺, Cl^–, and H⁺ in the vacuole ofNitella pulchella were changed in a wide range. Both the potential difference (E _vo ) and specific resistance (R _vo ) between the vacuole and the external medium were scarcely affected by K⁺ in the vacuole, while they responded sensitively to K⁺ in the external medium. E _vo also responded to Cl^– in both internal (vacuolar) and external medium. However, the sign of the response was opposite to that expected from the constant field assumption.R _vo was almost independent of Cl^–-concentrations of both internal and external medium.The response ofE _vo to internal pH was similar to that of external pH. Between pH's 4 and 8,E _vo changed by about 10 mV for one unit change of both external and internal pH.E _vo responded very sensitively to internal pH in the strongly acid region (30–60 mV at pH 3–4) irrespective of the concentration of KCl in the vacuole. In the alkaline region, however,E _vo responded to vacuolar pH only when the KCl concentration in the vacuole was low (0.1 mM).R _vo increased significantly when the vacuolar pH was lowered to 4 or 3.Increase in tonicity of the vacuolar medium to twice normal caused no significant change in bothE _vo andR _vo , while it raised the threshold for excitation.Even when the chemical potential gradient between the internal and external medium was made zero by replacing the cell sap for the same solution used for the external medium, a significant amount ofE _vo was observed. The short-circuit current which was first outward decreased to zero or changed its direction with time. Light did not affect the current. These facts show that the possibility for the contribution of an ion pump toE _vo can be excluded.The results were discussed under the assumption that responses ofE _vo andR _vo to either internal or external ions reflect the passive property of either tonoplast or plasmalemma.     

Construction of Secretory Expression System Suitable to Express Glucagon Under the Control of PL Promoter

It was reported that P_L promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His¹] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD₆₀₀ unit of cells in shake flask. The yield of [Des-His¹] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.Received: 26 September 2002 / Accepted: 24 October 2002     

Role of calcium and calmodulin antagonist in photosynthesis and salinity tolerance inChlorella vulgaris

To cast light upon the role of Ca¹⁺ and calmodulin on photosynthetic rate (P_n), dark respiration (R_D) and amino acid and protein contents in salinity stressed and non-stressedChlorella cultures, the Ca²⁺ chelator EGTA [ethylene glycol-bis-(2-aminoethyl ether)-N,N- tetraacetate] and the calmodulin antagonist TFP (trifluperazine) were used. TFP markedly inhibited P_N while EGTA exerted a slight, if any, effect on P_N. NaCl tolerance, on the other side, was markedly abolished by TFP that inhibited P_N and lowered rate of proline accumulation. Calmodulin might be involved in osmoregulation and salt tolerance ofChlorella. R_D, however, was markedly enhanced by EGTA and Ca²⁺-free medium and hence the Ca²⁺ deprivation increased stress severity exerted by NaCl. Combinations of Na⁺ and Ca²⁺ enhanced P_N, decreased R_D and proline content in comparison with an osmotically equivalent reference culture containing only NaCl. Addition of Ca²⁺ to TFP treated cultures failed to reactivate calmodulin for proline synthesis. However, when Ca²⁺ was added to EGTA-treated cultures, only relatively reduced proline contents were recorded.