An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of TnΩP_BAD to place the highly regulable, arabinose inducible P_BAD promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P_BAD promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.     

The Escherichia coli rhamnose promoter rhaP BAD is in Pseudomonas putida KT2440 independent of Crp–cAMP activation

We developed an expression vector system based on the broad host range plasmid pBBR1MCS2 with the Escherichia coli rhamnose-inducible expression system for applications in Pseudomonas. For validation and comparison to E. coli, enhanced green fluorescent protein (eGFP) was used as a reporter. For further characterization, we also constructed plasmids containing different modifications of the rhaP _BAD promoter. Induction experiments after the successful transfer of these plasmids into Pseudomonas putida KT2440 wild-type and different knockout strains revealed significant differences. In Pseudomonas, we observed no catabolite repression of the rhaP _BAD promoter, and in contrast to E. coli, the binding of cyclic adenosine monophosphate (cAMP) receptor protein (Crp)–cAMP to this promoter is not necessary for induction as shown by deletion of the Crp binding site. The crp ⁻ mutant of P. putida KT2440 lacked eGFP expression, but this is likely due to problems in rhamnose uptake, since this defect was complemented by the insertion of the l-rhamnose-specific transporter rhaT into its genome via transposon mutagenesis. Other global regulators like Crc, PtsN, and CyoB had no or minor effects on rhamnose-induced eGFP expression. Therefore, this expression system may also be generally useful for Pseudomonas and other γ-proteobacteria.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

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.     

Construction and characterization of a recombinant whole-cell biocatalyst of Escherichia coli expressing styrene monooxygenase under the control of arabinose promoter

A recombinant Escherichia coli (pBAB1) containing styrene monooxygenase (SMO) was developed for the conversion of styrene to enantiopure (S)-styrene oxide that is an important chiral building block in organic synthesis. The styAB genes encoding SMO was cloned into a multicopy plasmid under the tightly regulated promoter of bacterial l-arabinose operon which is inducible by l-arabinose. The recombinant showed that expression level of StyA protein and whole-cell SMO activities were varied depending on the concentration of the inducer l-arabinose. The maximum SMO activity was 108 U/g cdw when the cells were induced with 0.2% l-arabinose. SDS-PAGE and Western blot analyses indicated that whole-cell SMO activity was strongly correlated with the expression level of StyA protein. Organic-aqueous two-phase experiment could yield 50 mM enantiopure (S)-styrene oxide in organic phase in 18 h, but the recombinant SMO activity was unstable during the reaction. The expression of styAB under the control of l-arabinose promoter was significantly repressed in the presence of glucose.     

Bioconversion of L-tyrosine to L-DOPA by a novel bacterium Bacillus sp. JPJ

l-DOPA is an amino acid derivative and most potent drug used against Parkinson’s disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of l-DOPA from l-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of l-DOPA from l-tyrosine in buffer (pH 8) containing 1 mg ml⁻¹ cell mass incubated at 40°C for 60 min. The combination of CuSO₄ and l-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml⁻¹, respectively. The activated charcoal 2 mg ml⁻¹ was essential for maximum bioconversion of l-tyrosine to l-DOPA and the crude tyrosinase activity was 2.7 U mg⁻¹ of tyrosinase. Kinetic studies showed significant values of Y _p/s (0.994), Q _s (0.500) and q _s (0.994) after optimization of the process. The production of l-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC–MS. This is the first report on rapid and efficient production of l-DOPA from l-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems.     

Optimal Production of Poly-γ-glutamic Acid by Metabolically Engineered Escherichia coli

Metabolically-engineered Escherichia coli strains were developed by cloning poly-γ-glutamic acid (γ-PGA) biosynthesis genes, consisting of pgsB, pgsC and pgsA, from Bacillus subtilis The metabolic and regulatory pathways of γ-PGA biosynthesis in E. coli were analyzed by DNA microarray. The inducible trc promoter and a constitutive promoter (P_HCE) derived from the d-amino acid aminotransferase (D-AAT) gene of Geobacillus toebii were employed. The constitutive HCE promoter was more efficient than inducible trc promoter for the expression of γ-PGA biosynthesis genes. DNA microarray analysis showed that the expression levels of several NtrC family genes, glnA, glnK, glnG, yhdX, yhdY, yhdZ, amtB, nac, argT and cbl were up-regulated and sucA, B, C, D genes were down-regulated. When (NH₄)₂SO₄ was added at 40 g/l into the feeding solution, the final γ-PGA concentration reached 3.7 g/l in the fed-batch culture of recombinant E. coli/pCOpgs.     

Influences of cultural medium component on the production of poly(γ-glutamic acid) byBacillus sp. RKY3

In this study, the cultural medium used for the efficient production of γ-PGA with a newly isolatedBacillus sp. RKY3 was optimized. It was necessary to supplement the culture medium withl-glutamic acid and an additional carbon source in order to induce the effective production of γ-PGA. The amount of γ-PGA increased with the addition ofl-glutamic acid to the medium. The addition of 90 g/Ll-glutamic acid to the medium resulted in the maximal yield of γ-PGA (83.2 g/L). The optimum nitrogen source was determined to be peptone, but corn steep liquor, a cheap nutrient, was also found to be effective for γ-PGA production. Both the γ-PGA production and cell growth increased rapidly with the addition of small amounts of K₂HPO₄ and MgSO₄·7H₂O.Bacillus sp. RKY3 appears to require Mg²⁺, rather than Mn²⁺, for γ-PGA production, which is distinct from the production protocols associated with other, previously reported bacteria.Bacillus sp. RKY3 may also have contributed some minor γ-PGA depolymerase activity, resulting in the reduction of the molecular weight of the produced γ-PGA at the end of fermentation.     

Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.     

Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P _lac , P _tac , or P _BAD derived from Escherichia coli, or promoter regions of phaC1 (P _phaC ) or phaP1 (P _phaP ) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P _lac , P _tac , P _phaC , and P _phaP mediated constitutive gene expression, among which P _tac was the strongest promoter. lacI-P _tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P _BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P _phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P _phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.     

<Emphasis Type="SmallCaps">l</Emphasis>-Asparaginase Production by Moderate Halophilic Bacteria Isolated from Maharloo Salt Lake

l-Asparaginase is an anti-neoplastic drug used in lymphoblastic leukemia chemotherapy. Nowadays, this enzyme derived from bacterial sources, mostly l-asparaginase II from Escherichia coli and in lesser amount l-asparaginase of Erwinia sp. has medical utilization. The long-term usage of these agents leads to allergic reactions and new asparaginase with new immunological characteristics is required. Halophilic bacteria might contain l-asparaginase with novel immunological properties that can be used in hypersensitive patients. In this experiment, we have screened moderate Halophilic bacteria for l-asparaginase production ability and showed that Halophilic bacteria produce intra- and extracellular l-asparaginase. Bacillus sp. BCCS 034 was found to produce the highest l-asparaginase (1.64 IU/ml supernatant) extracellularly.     

l-Stereoselective amino acid amidase with broad substrate specificity from Brevundimonas diminuta: characterization of a new member of the leucine aminopeptidase family

Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaA_Bd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal activity at 50°C and pH 7.5. LaaA_Bd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was largely restored by the addition of Co²⁺ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co²⁺, suggesting that LaaA_Bd is a Co²⁺-dependent enzyme. LaaA_Bd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaA_Bd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence of seven open-reading frames (ORFs), one of which (laaA _Bd ) encodes the amidase. LaaA_Bd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA _Bd gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaA_Bd in cell-free extracts of the E. coli transformant was 25.9 units mg⁻¹ with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720.     

Positive and negative regulation of expression of the l-sorbose (sor) operon by SorC in Klebsiella pneumoniae

Summary In Klebsiella pneumoniae the gene products involved in the degradation of the ketose l-sorbose are encoded in the sor operon. It comprises, besides structural genes for uptake and catabolism, a promoter-proximal gene sorC, encoding a protein SorC of M_r 40 kDa, for which no enzymatic function has been detected. All sor genes are coordinately expressed and inducible by l-sorbose. Polar insertions and frameshift mutations in sorC cause a pleiotropic negative effect on the expression of all other sor genes. This defect is complemented in trans by the wild-type sorC ⁺ allele for frameshift mutations, but not for polar insertions. A single promoter for all sor genes, for which SorC is the activator, thus seems to be located in front of sorC. The repressor activity of SorC was demonstrated by complementation of constitutive sorC alleles with a sorC ⁺ allele leading to inducible expression of all sor genes, including sorC, which, as visualized by the use of a series of lacZ fusions, thus autoregulates its expression, both as an activator and a repressor.     

The outer membrane TolC is involved in cysteine tolerance and overproduction in <Emphasis Type="Italic">Escherichia coli</Emphasis>

l-Cysteine is an important amino acid in terms of its industrial applications. We previously found marked production of l-cysteine directly from glucose in recombinant Escherichia coli cells by the combination of enhancing biosynthetic activity and weakening the degradation pathway. Further improvements in l-cysteine production are expected to use the amino acid efflux system. Here, we identified a novel gene involved in l-cysteine export using a systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection). Among the 3,985 nonessential gene mutants, tolC-disrupted cells showed hypersensitivity to l-cysteine relative to wild-type cells. Gene expression analysis revealed that the tolC gene encoding the outer membrane channel is essential for l-cysteine tolerance in E. coli cells. However, l-cysteine tolerance is not mediated by TolC-dependent drug efflux systems such as AcrA and AcrB. It also appears that other outer membrane porins including OmpA and OmpF do not participate in TolC-dependent l-cysteine tolerance. When a low-copy-number plasmid carrying the tolC gene was introduced into E. coli cells with enhanced biosynthesis, weakened degradation, and improved export of l-cysteine, the transformants exhibited more l-cysteine tolerance and production than cells carrying the vector only. We concluded that TolC plays an important role in l-cysteine tolerance probably due to its export ability and that TolC overexpression is effective for l-cysteine production in E. coli. Natthawut Wiriyathanawudhiwong and Iwao Ohtsu contributed equally to this work.     

Production characteristics of interferon-α using an l-arabinose promoter system in a high-cell-density culture

 Using high-cell-density culture of Escherichia coli under the control of an l-arabinose promoter (P_araB), several factors affecting the production of recombinant protein and the formation of inclusion bodies were studied. The inducer, l-arabinose, showed a maximal induction level above 10.7 mM in the final concentration. The concentration of inducer also affected the partition of interferon-α (IFN-α) into the soluble form and inclusion bodies. Induction kinetics of the rate of accumulation of IFN-α on the P_araB promoter showed a slower rate than those of other promoter systems, for example T7, lac or tac. These innate characteristics of P_araB enabled cells to grow continuously in spite of the metabolic burden induced by the expression of foreign protein. The duration time of induction could control the expression of both soluble and insoluble protein. The ratio of yeast extract to glycerol (N/C ratio) in feeding media significantly affected both the production level of recombinant protein and inclusion body formation. The reason for decreasing specific bioactivity during induction can be explained by the increased proportion of inclusion bodies in the total expressed IFN-α. Received: 21 May 1999 / Received last revision: 16 August 1999 / Accepted: 2 September 1999     

The properties of l-fucose binding agglutinin associated with the cell wall of Rhizoctonia solani

The adherence of Escherichia coli B cells to cell wall associated-agglutinin of the soil borne plant pathogen Rhizoctonia solani, was inhibited by l-fucose, l-galactose, trypsin, SDS, cycloheximide and Na₂-EDTA. The coiling of the biocontrol agent Trichoderma harzianum around Rhizoctonia hyphae was prevented by SDS, cycloheximide, Na₂-EDTA and methyl--l-fucoside — an inhibitor of Rhizoctonia agglutinin not metabolized by both fungi. The possible role of the agglutinin in Trichoderma-Rhizoctonia interaction is discussed.     

Metabolic pathways and biotechnological production of l-cysteine

l-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are commercially produced by fermentation, cysteine is mainly produced by protein hydrolysis. However, synthetic or biotechnological products have been preferred in the market. Biotechnological processes for cysteine production, both enzymatic and fermentative processes, are discussed. Enzymatic process, the asymmetric hydrolysis of dl-2-amino-Δ²-thiazoline-4-carboxylic acid to l-cysteine, has been developed and industrialized. The l-cysteine biosynthetic pathways of Escherichia coli and Corynebacterium glutamicum, which are used in many amino acid production processes, are also described. These two bacteria have basically same l-cysteine biosynthetic pathways. l-Cysteine-degrading enzymes and l-cysteine-exporting proteins both in E. coli and C. glutamicum are also described. In conclusion, for the effective fermentative production of l-cysteine directly from glucose, the combination of enhancing biosynthetic activity, weakening the degradation pathway, and exploiting the export system seems to be effective.