Degradation of trichloroethylene by recombinant E. coli in continuous culture

Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h^–1.     

Metabolic enhancement due to plasmid maintenance

Summary Plasmid maintenance allows the strain JM109 of Escherichia coli to grow in a minimal defined medium (M9). JM109 carrying no plasmid can hardly grow in M9 whereas JM109 carrying one, two and three plasmids have a clear metabolic advantage over the untransformed strain. In a complex medium like LB (Luria-Bertani Broth) all strains grow well and despite the number of plasmids carried by the host maximum specific growth rates are not severely affected. Our results suggest that the glucose metabolism is an essential factor contributing to this behavior.     

Expression of a deleted variant of human plasminogen in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A recombinant plasmid carrying a modified gene of human plasminogen (mini-plasminogen), lacking four kringle domains and an amino terminal fragment, and containing an additional oligopeptide of six N-terminal histidine residues has been constructed. The plasmid was used for transformation of E. coli JM 109 cells to obtain a strain producing a recombinant modified human plasminogen. The target protein is superexpressed in a form of inclusion bodies and is composed of more than 50% insoluble protein. The renaturated and chromatographically purified protein exhibits amidolytic activity specific for plasminogen proenzyme in a fibrinolytic system.     

Construction of shuttle vectors derived fromAcetobacter xylinum for cellulose-producing bacteriumAcetobacter xylinum

Summary Shuttle vector pUF106 was constructed by ligation ofAcetobacter xylinum plasmid pFF6 toEscherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. pUF106 transformed cellulose-producingA. xylinum ATCC10245 as well asE. coli JM109.     

CLONING OF MELITTIN cDNA FROM HONEYBEE INTO THE HIGH EXPREHION PLASMID OF ESCHERICHIA COLI*

Abstract Total mRNA from venom glands of newly emerged queen bees was reversely transcribed into cDNA and cloned into the EcoRI site of plasmid λgt11; cDNA library for bee venom was thus constructed. PCR technique was used to produce the melittin coding sequence from the cDNA library. A 87 bp product was produced and inserted into the EcoRI and PstI sites of the high level expression vector pBV220. Recombinant plasmid pBM95 was transformed into the competent cells of E.coli JM101. After screening transformants on LB medium with ampicilin, structure of the recombinant plasmid pBM95 from transformants was analyzed and melittin gene in pBM95 was sequenced. The cloned cDNA coding for honey bee melittin was obtained.     

Bioaugmentation of the decolorization rate of acid red GR by genetically engineered microorganism <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109 (pGEX-AZR)

The study showed that the genetically engineered microorganism (GEM) bioaugment successfully the dye wastewater biotreatment systems to enhance acid red GR (ARGR) removal. Escherichia coli JM109 (pGEX-AZR) was the GEM with higher azoreductase activity. The kinetics of the ARGR decolorization by the E. coli JM109 (pGEX-AZR) agreed with Andrews model. The kinetic parameters, r _dye,max, K _s and K _i , were found to be 42.45 mg g⁻¹ h⁻¹, 584.93 mg L⁻¹ and 556.89 mg L⁻¹, respectively. The E. coli JM109 (pGEX-AZR) was tested in anaerobic sequencing batch reactors (AnSBR) in order to enhance the ARGR decolorization. The decolorization rate of ARGR was affected by the amount of E. coli JM109 (pGEX-AZR) inoculation and the best amount of inoculation was 10%. The continuous operations of the four bioreactors with different E. coli JM109 (pGEX-AZR) immobilization supports showed that the E. coli JM109 (pGEX-AZR) could bioaugment decolorization in AnSBRs with suspended and immobilized on macroporous foam carriers. For 42 days continuous operation in the AnSBRs, both the tolerance to ARGR concentration shock and the decolorization rate in these two bioaugmented AnSBRs are higher than those of the other two systems, control system and bioaugmented AnSBRs system with the sodium-alginate immobilized cells, the decolorization rate reached 90%. Changes in microbial community were detected by ribosomal intergenic spacer analysis (RISA) and amplified ribosomal DNA restriction analysis (ARDRA), which revealed that the introduced E. coli JM109 (pGEX-AZR) was persistent in the augmented systems and maintained higher metabolic activity.     

Overproduction ofd-Aminoacylase fromAlcaligenes xylosoxydanssubsp.xylosoxydansA-6 inEscherichia coliand Its Purification

We constructed the high-expression plasmid forD-aminoacylase fromAlcaligenes xylosoxydanssubsp.xylosoxydansA-6. The appropriate Shine–Dalgarno sequence (AAGGAG) was introduced to the eight bases upstream of start codon (ATG) ofD-aminoacylase structural gene by site-directed mutagenesis, and then the 1.75-kb DNA fragment including the open reading frame was inserted into the downstream of the tac promoter of plasmid vector pKK223-3. The resultant plasmid, which was named pKNSD2, showed a highD-aminoacylase activity inEscherichia coliJM109 cells transformed with it. The enzyme was purified to homogeneity in only two steps with a final yield of 24% (sp act, 2023 U/mg).     

Plasmid size can affect the ability of Escherichia coli to produce high-quality plasmids

Large molecular weight plasmids are often used in gene therapy and DNA vaccines. To investigate the effect of plasmid size on the performance of Escherichia coli host strains during plasmid preparation, we employed E. coli JM109 and TOP10 cells to prepare four plasmids ranging from 4.7 to 16.8?kb in size. Each plasmid was extracted from JM109 and TOP10 cells using an alkaline lysis mini-preparation method. However, when commercial kits were used to extract the same plasmids from JM109 cells, the large molecular weight plasmids substantially degraded, compared with their smaller counterparts. No degradation was observed when the four plasmids were extracted from E. coli TOP10 cells using the same commercial kit. We conclude, therefore, that the performance of E. coli in high quality plasmid preparations can be affected by plasmid size.     

Microbial transformation of benzene to <Emphasis Type="Italic">cis</Emphasis>-3,5-cyclohexadien-1,2-diols by recombinant bacteria harboring toluene dioxygenase gene <Emphasis Type="Italic">tod</Emphasis>

Toluene dioxygenase (TDO) catalyzes asymmetric cis-dihydroxylation of aromatic compounds. To achieve high efficient biotransformation of benzene to benzene cis-diols, Pseudomonas putida KT2442, Pseudomonas stutzeri 1317, and Aeromonas hydrophila 4AK4 were used as hosts to express TDO gene tod. Plasmid pSPM01, a derivative of broad-host plasmid pBBR1MCS-2 harboring tod from plasmid pKST11, was constructed and introduced into the above three strains. Their abilities to catalyze the biotransformation of benzene to benzene cis-diols, namely, cis-3,5-cyclohexadien-1,2-diols abbreviated as DHCD, were examined. In shake-flask cultivation under optimized culture media and growth condition, benzene cis-diols production by recombinant P. putida KT2442 (pSPM01), P. stutzeri 1317 (pSPM01), and A. hydrophila 4AK4 (pSPM01) were 2.68, 2.13, and 1.17 g/l, respectively. In comparison, Escherichia coli JM109 (pSPM01) and E. coli JM109 (pKST11) produced 0.45 and 0.53 g/l of DHCD, respectively. When biotransformation was run in a 6-l fermenter, DHCD production in P. putida KT2442 (pSPM01) was approximately 60 g/l; this is the highest DHCD production yield reported so far.     

Production of l-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P_LP_R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.     

Extension of Chronological Lifespan by ScEcl1 Depends on Mitochondria in Saccharomyces cerevisiae

We constructed two plasmids that have a strong tac promoter and a structural gene for tryptophanase of Enterohacter aerogenes SM-18 (pKT901EA) or Escherichia coli K-12 (pKT951EC). The tryptophanase activity of E. coli JM109 transformed with pKT90lEA (JM109/pKT901EA) was inducible with isopropyl-β-D-thiogalactopyranoside, and 3.6 times higher than that of E. aerogenes SM-18. Cells of JM109/pKT901EA induced for tryptophanase synthesized L-tryptophan from indole, ammonia, and pyruvate more efficiently than E. aerogenes SM-18. Although JM109/pKT951EC expressed a similar level of tryptophanase activity to that of JM109/pKT901EA, the synthesis of L-tryptophan by the cells of JM109/pKT951EC did not proceed well compared with JM109/pKT901EA. Tryptophanases from E. aerogenes and E. coli K-12 were purified, and their properties were investigated. The purified E. aerogenes tryptophanase showed higher stability against heat inactivation than E. coli tryptophanase.     

Prokaryotic expression,purification and functional characterization of human FHL3

Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42°C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.     

Biocontrol of bacterial soft rot of calla lily by elicitor HarpinXoo and N-acyl homoserine lactonase (AttM)

Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum is a serious plant disease in Zantedeschia spp. (also called calla lily). In this study, two independent genes (a N-acyl homoserine lactonase gene attM from Agrobacterium tumefaciens and a hypersensitive response and pathogenicity gene hrf1 from Xanthomonas oryzae pv. oryzae), transcribed by a strong and constitutive Escherichia coli promoter P _lpp , respectively, were cloned into plasmid pUC19, and was transformed into E. coli, creating strain JM109/pPHA. The result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) assay showed that both genes (hrf1 and attM) were successfully expressed in one plasmid system in strain JM109/pPHA. The expressed Harpin_Xoo (Hrf1) and AttM protein had the ability of inducing hypersensitive response (HR) in nonhost tobacco and degrading the N-acyl homoserine lactones (AHLs) produced by P. carotovorum subsp. carotovorum, respectively, whereas Harpin_Xoo and AttM protein did not seem to interfere with the normal growth of this pathogen. In planta, strain JM109/pPHA could significantly reduce the soft rot disease severity on dormant tubers (control efficiency: 92.8%) or potted plants (control efficiency: 92.4%) of calla lily. We have first demonstrated the both biocontrol effects of Harpin_Xoo and AttM proteins (also described as Quorum interference) on the bacterial soft rot disease of calla lily, caused by P. carotovorum subsp. carotovorum. This work provided a potential way to control this serious plant disease.     

Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors

Cultivation conditions for the extracellular production of a hybrid β-glucanase from Bacillus were established by using Escherichiacoli JM109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid β-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of β-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of β-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of β-glucanase [E. coli JM109(pLF3)] was tested on the fermenter scale. Received: 1 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

Inhibition Site of Cupric Ions on the Growth of Thiobacillus ferrooxidans on Sulfur-salts Medium

The cDNA sequence coding for tuna growth hormone (tGH) was placed under the control of the repressible acid phosphatase (PHO5) promoter of a yeast, Saccharomyces cerevisiae, in an expression plasmid, pAM82. The yeast cells transformed with the plasmid synthesized tGH only when the cDNA was attached to the vector through a synthetic oligonucleotide linker having a similar sequence to the 5′-flanking region of the PHO5 structural region. The amount of tGH produced in yeast cells accounted for more than 3% of the total cellular protein and the product was immunologically identified as tGH by Western blotting using polyclonal antibodies specific to tGH.     

Correlation between Two Glycine tRNAs and Corresponding Glycyl tRNA Synthetases

A gene encoding a stereo-specific secondary alcohol dehydrogenase (CpSADH) that catalyzed the oxidation of (S)-1,3-BDO to 4-hydroxy-2-butanone was cloned from Candida parapsilosis. This CpSADH-gene consisted of 1,009 nucleotides coding for a protein with M _r 35,964. A recombinant Escherichia coli JM109 strain harboring the expression plasmid, pKK-CPA1, produced (R)-1,3-BDO (93.5% ee, 94.7% yield) from the racemate without any additive to regenerate NAD⁺ from NADH.     

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

cDNA for mRNA of tuna growth hormone (GH) was cloned by screening a cDNA library constructed from tuna pituitary gland poly(A)⁺ RNA. The nucleotide sequence of cDNA (911 bases) revealed an open reading frame of 615 nucleotides, including a sequence (51 bases) for a possible secretory protein leader peptide. Noncoding regions were found in the nucleotide sequences up- (5′-terminal: 65 bases) and down- (3′-terminal: 231 bases) stream of the open reading frame. An amino-acid sequence deduced from the nucleotide sequence of the cDNA was identical with that determined in the purified tuna GH. Tuna GH was composed of 187 amino acids, and had a calculated molecular weight of 21275. Amino-acid sequencing showed that there was one possible N-glycosylation site at Asn (Asn-Cys-Thr). Tuna GH showed amino-acid sequence homologies with chum salmon (67%), yellow tail (90%) and with human (32%) growth hormones.     

Influence of site-directed mutagenesis on protein assembly and solubility of tadpole H-chain ferritin

In order to understand the influence of ferroxidase center on the protein assembly and solubility of tadpole ferritin, three mutant plasmids, pTH58K, pTH61G, and pTHKG were constructed with the aid of site-directed mutagenesis and mutant proteins were produced inEscherichia coli. Mutant ferritin H-subunits produced by the cells carrying plasmids pTH58K and pTHKG were active soluble proteins, whereas the mutant obtained from the plasmid pTH61G was soluble only under osmotic stress in the presence of sorbitol and betaine. Especially, the cells carrying pTH61G together with the plasmid pGroESL harboring the molecular chaperone genes produced soluble ferritin. The mutant ferritin H-subunits were all assembled into ferritin-like holoproteins. These mutant ferritins were capable of forming stable iron cores, which means the mutants are able to accumulate iron with such modified ferroxidase sites. Further functional analysis was also made on the individual amino acid residues of ferroxidase center.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.