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PubMed2001

Role of ptsO in carbon-mediated inhibition of the Pu promoter belonging to the pWW0 Pseudomonas putida plasmid.

An investigation was made into the role of the ptsO gene in carbon source inhibition of the Pu promoter belonging to the Pseudomonas putida upper TOL (toluene degradation) operon. ptsO is coexpressed with ptsN, the loss of which is known to render Pu unresponsive to glucose. Both ptsN and ptsO, coding for the phosphoenolpyruvate:sugar phosphotransferase system (PTS) family proteins IIA(Ntr) and NPr, respectively, have been mapped adjacent to the rpoN gene of P. putida. The roles of these two genes in the responses of Pu to glucose were monitored by lacZ reporter technology with a P. putida strain engineered with all regulatory elements in monocopy gene dosage. In cells lacking ptsO, Pu activity seemed to be inhibited even in the absence of glucose. A functional relationship with ptsN was revealed by the phenotype of a double ptsN ptsO mutant that was equivalent to the phenotype of a mutant with a single ptsN disruption. Moreover, phosphorylation of the product of ptsO seemed to be required for C inhibition of Pu, since an H15A change in the NPr sequence that prevents phosphorylation of this conserved amino acid residue did not restore the wild-type phenotype. A genomic search for proteins able to phosphorylate ptsO revealed the presence of two open reading frames, designated ptsP and mtp, with the potential to encode PTS type I enzymes in P. putida. However, neither an insertion in ptsP nor an insertion in mtp resulted in a detectable change in inhibition of Pu by glucose. These results indicate that some PTS proteins have regulatory functions in P. putida that are independent of their recognized role in sugar transport in other bacteria.

Biological Transport, Active↗

PubMed1999

Reconstitution of human thymic implants is limited by human immunodeficiency virus breakthrough during antiretroviral therapy.

Human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu thymic implants depleted of CD4(+) cells can support renewed thymopoiesis derived from both endogenous and exogenous T-cell progenitors after combination antiretroviral therapy. However, successful production of new thymocytes occurs transiently. Possible explanations for the temporary nature of this thymic reconstitution include cessation of the thymic stromal support function, exhaustion of T-cell progenitors, and viral resurgence. Distinguishing between these processes is important for the development of therapeutic strategies aimed at reconstituting the CD4(+) T-cell compartment in HIV-1 infection. Using an HIV-1 strain engineered to express the murine HSA heat-stable antigen surface marker, we explored the relationship between HIV-1 expression and CD4(+) cell resurgence kinetics in HIV-1-depleted SCID-hu implants following drug therapy. Antiviral therapy significantly suppressed HIV-1 expression in double-positive (DP) CD4/CD8 thymocytes, and the eventual secondary decline of DP thymocytes following therapy was associated with renewed viral expression in this cell subset. Thymocytes derived from exogenous T-cell progenitors induced to differentiate in HIV-1-depleted, drug-treated thymic implants also became infected. These results indicate that in this model, suppression of viral replication occurs transiently and that, in spite of drug therapy, virus resurgence contributes to the transient nature of the renewed thymic function.

Animals↗

PubMed2026

The novel transcriptional activator Bhr1 combining NTPase and Zn(II)2Cys6 DNA-binding domains controls (hemi-)cellulase response to mannose-rich substrates in the white-rot fungus Dichomitus squalens.

The regulatory landscape responsible for lignocellulose degradation in white-rot basidiomycete fungi remains largely unexplored. In this study, we characterize a novel transcriptional activator, Bhr1, in the white-rot fungus Dichomitus squalens. Bhr1 exhibits an unusual domain architecture that combines a septin-like P-loop NTPase fold with Zn(II)2Cys6 DNA-binding domains and plays a critical role in activating (hemi-)cellulase enzyme production when D. squalens is exposed to mannose-rich substrates. Using CRISPR/Cas9-mediated gene editing, we generated a bhr1 disruption mutant that displayed distinct phenotypes and enzyme activity profiles on mannose and guar gum compared to the wild type. RNA sequencing data indicate that Bhr1 induces specific (hemi-)cellulase-encoding genes without altering the expression of genes encoding sugar transporters or sugar metabolic enzymes. Phylogenetic analyses show that Bhr1 is basidiomycete specific and largely restricted to saprotrophic and plant-associated Agaricomycetes fungi. Based on the domain architecture of Bhr1 and the effects of its disruption in D. squalens, our findings reveal a lineage-specific regulatory innovation in basidiomycetes that is distinct from those described in ascomycetes. Elucidating the function and evolutionary conservation of Bhr1 advances our understanding of lignocellulose degradation at the molecular level in basidiomycete fungi and may inform studies of their ecological adaptation and the development of biotechnological applications.IMPORTANCEUnderstanding the transcriptional regulatory mechanisms in white-rot fungi, such as Dichomitus squalens, is crucial for advancing our knowledge of lignocellulose degradation. This study identifies D. squalens Bhr1 as a key regulator of (hemi-)cellulase production on mannose-rich substrates and further distinguishes basidiomycete transcription factors involved in plant biomass degradation from their ascomycete counterparts. Our findings highlight the significance of lineage-specific regulators in facilitating adaptive enzyme production for efficient biomass utilization, which is critical to carbon cycling in terrestrial ecosystems. This work establishes a foundation for exploring novel regulatory strategies among wood-degrading fungi, potentially enabling targeted strain engineering in biotechnological applications.

Mannose↗

PubMed2005

Suckling mice were used to detect infectious dengue-2 viruses by intracerebral injection of the full-length RNA transcript.

OBJECTIVE: In this study we established a mouse brain injection system to detect infectious dengue-2 virus produced from the full-length RNA transcripts. METHODS: In vitro transcription was used to synthesize full-length dengue-2 virus RNA from the plasmid pRS424FLDEN2NGC, which was intracerebrally injected into the 6-day-old suckling mice (ICR strains). Engineered dengue-2 viruses were detected in the brain sections using immunohistochemistry staining. RT-PCR followed by restriction endonuclease BstEII digestion was used to confirm the mosquito C6/36 cells cocultured with the mouse brain extract. RESULTS: The mice inoculated with the full-length dengue-2 viral RNA transcript showed paralysis symptoms and died between day 10 and 13 postinjection. The dengue-2 virus-specific antigens (E, Core and NS1) were detected in all the brain and part of the liver sections of the paralyzed mice by immunohistochemistry staining, indicating the existence of dengue-2 virus in these tissues of the suckling mice. The viruses detected in the brains of suckling mice were indeed infectious, which was further confirmed by coculturing mosquito C6/36 cells with the brain extract of the injected mice. CONCLUSIONS: We developed an in vivo approach to detect and produce engineered dengue viruses with infectivity from the full-length plasmid cDNA. This suckling mice system will also aid in screening the infectious viruses that are created by site-directed mutagenesis and is useful for the studies of dengue virus gene function and pathogenesis in the host.

Animals↗

PubMedDate not supplied

Involvement of reactive oxygen species in bacterial killing within epithelial cells.

Several non-phagocytic cells can actively generate the superoxide anion by NAD(P)H oxidases resembling the enzymatic complex typical of phagocytes. Overexpression of periplasmic Cu,ZnSOD rescues invasive E. coli strains from killing within epithelial cells, suggesting that superoxide generation by such cells can oxidatively damage invading bacteria. Pre-treatment of HeLa cells with diphenyl iodonium or 4'-hydroxy-3'-methoxyacetophenone, two inhibitors of NAD(P)H oxidase, significantly enhances intracellular survival of wild type invasive E. coli cells. On the contrary, these inhibitors have no effect on the intracellular survival of an invasive E. coli strain engineered to overexpress Cu,ZnSOD. These results support the hypothesis that superoxide generation by a NAD(P)H oxidase-like complex can limit bacterial survival within epithelial cells and suggest that the role of periplasmic Cu,ZnSOD in bacterial infections is not simply that of conferring protection against the phagocytic oxidative burst.

Enzyme Inhibitors↗

PubMed2002

An Escherichia coli biosensor strain for amplified and high throughput detection of antimicrobial agents.

We report here the construction of a bacterial reporter system for high-throughput screening of antimicrobial agents. The test organism is the Escherichia coli K-12 strain carrying luciferase genes luxC, luxD, luxA, luxB, and luxE from the bioluminescent bacterium Photorhabdus luminescens in a runaway replication plasmid. The replication of the plasmid can be induced, resulting in a change of the plasmid copy number from 1-2/cell to several hundreds per cell within tens of minutes. This increase in plasmid copies is independent of the replication of the host cells. The system will therefore amplify the effects of antibiotics inhibiting bacterial replication machinery, such as fluoroquinolones, and the inhibitory effects can be measured in real time by luminometry. The biosensor was compared with a strain engineered to emit light constitutively, and it was shown to be much more sensitive to various antibiotics than conventional overnight cultivation methods. The approach shows great potential for high-throughput screening of new compounds.

Anti-Bacterial Agents↗

PubMed2003

Amplified detection of transcriptional and translational inhibitors in bioluminescent Escherichia coli K-12.

The excessive prescription of antimicrobial agents and their use as animal growth promoters lead to the spread of resistance among pathogenic bacteria. Consequently, unnecessary use should be minimized, and new chemicals with novel mechanisms of action are needed. The authors have developed a fast method to measure the activity of antibiotics by means of a genetically engineered strain of Escherichia coli K-12. The system is based on the full-length bacterial luciferase operon coupled to the tetracycline-inducible tetA promoter in the reporter plasmid pTetLux1. Sublethal doses of tetracycline are used to start the luciferase synthesis in cultures that were previously incubated with the antibiotic under investigation. After a variable time frame-from 1 to 4 h, depending on the antimicrobial mode of action-the level of light emission from treated cultures is compared to the level obtained in control cultures. The gap in bioluminescence outlines the antibiotic interference in bacterial metabolism. Throughout this study, freeze-dried sensor cells were used to avoid repeated cultures from day to day. The authors show the results of 10 model antibiotics, representing different molecular structures and mechanisms of action. The results show that no actively dividing cells are needed for sensitive responses, especially when transcriptional and translational inhibitors, directly interfering with the luciferase production, are tested. The assay can be easily automated for high-throughput screening purposes of pharmaceutical industry.

Automation↗

PubMed2006

Gonadectomy-induced adrenocortical neoplasia in the domestic ferret (Mustela putorius furo) and laboratory mouse.

Sex steroid-producing adrenocortical adenomas and carcinomas occur frequently in neutered ferrets, but the molecular events underlying tumor development are not well understood. Prepubertal gonadectomy elicits similar tumors in certain inbred or genetically engineered strains of mice, and these mouse models shed light on tumorigenesis in ferrets. In mice and ferrets, the neoplastic adrenocortical cells, which functionally resemble gonadal steroidogenic cells, arise from progenitors in the subcapsular or juxtamedullary region. Tumorigenesis in mice is influenced by the inherent susceptibility of adrenal tissue to gonadectomy-induced hormonal changes. The chronic elevation in circulating luteinizing hormone that follows ovariectomy or orchiectomy is a prerequisite for neoplastic transformation. Gonadectomy alters the plasma or local concentrations of steroid hormones and other factors that affect adrenocortical tumor development, including inhibins, activins, and Müllerian inhibiting substance. GATA-4 immunoreactivity is a hallmark of neoplastic transformation, and this transcription factor might serve to integrate intracellular signals evoked by different hormones. Synergistic interactions among GATA-4, steroidogenic factor-1, and other transcription factors enhance expression of inhibin-alpha and genes critical for ectopic sex steroid production, such as cytochrome P450 17alpha-hydroxylase/17,20 lyase and aromatase. Cases of human adrenocortical neoplasia have been linked to precocious expression of hormone receptors and to mutations that alter the activity of G-proteins or downstream effectors. Whether such genetic changes contribute to tissue susceptibility to neoplasia in neutered ferrets and mice awaits further study.

Adrenal Cortex Neoplasms↗

PubMed2003

Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum.

AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production.

Brevibacterium↗

PubMed2005

[Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

OBJECTIVE: To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products. METHODS: PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively. RESULTS: Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1. CONCLUSION: An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.

Amino Acid Sequence↗

PubMed2001

Validation of apple cider pasteurization treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes.

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and degrees Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380-94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778. CDC F2833, and CDC H0662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14 degrees Brix was heated under conditions ranging from 60 degrees C for 14 s to 71.1 degrees C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1 degrees C for 14 s. Lower temperatures, or less time at 68.1 degrees C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6 degrees C for 14 s for Salmonella spp. L. monocytogenes survived 68.1 degrees C for 14 s, but survivors died in cider within 24 h at 4 degrees C. Laboratory results were validated with a surrogate E coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1 degrees C for 14 s (Wisconsin recommendations) and at 71.1 degrees C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1 degrees C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.

Adaptation, Physiological↗

PubMed1999

Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy.

A fully human IgG2kappa monoclonal antibody (MAb), E7.6.3, specific to the human epidermal growth factor (EGF) receptor (EGFr) was generated from human antibody-producing XenoMouse strains engineered to be deficient in mouse antibody production and to contain the majority of the human antibody gene repertoire on megabase-sized fragments from the human heavy and kappa light chain loci. The E7.6.3 MAb exhibits high affinity (KD = 5 x 10(-11) M) to the receptor, blocks completely the binding of both EGF and transforming growth factor alpha (TGF-a) to various EGFr-expressing human carcinoma cell lines, and abolishes EGF-dependent cell activation, including EGFr tyrosine phosphorylation, increased extracellular acidification rate, and cell proliferation. The antibody (0.2 mg i.p. twice a week for 3 weeks) prevents completely the formation of human epidermoid carcinoma A431 xenografts in athymic mice. More importantly, the administration of E7.6.3 without concomitant chemotherapy results in complete eradication of established tumors as large as 1.2 cm3. Tumor eradication of A431 xenografts was achieved in nearly all of the mice treated with total E7.6.3 doses as low as 3 mg, administered over the course of 3 weeks, and a total dose of 0.6 mg led to tumor elimination in 65% of the mice. No tumor recurrence was observed for more than 8 months after the last antibody injection, which further indicated complete tumor cell elimination by the antibody. The potency of E7.6.3 in eradicating well-established tumors without concomitant chemotherapy indicates its potential as a monotherapeutic agent for the treatment of multiple EGFr-expressing human solid tumors, including those for which no effective chemotherapy is available. Being a fully human antibody, E7.6.3 is expected to exhibit minimal immunogenicity and a longer half-life as compared with mouse or mouse-derivatized MAbs, thus allowing repeated antibody administration, including in immunocompetent patients. These results suggest E7.6.3 as a good candidate for assessing the full therapeutic potential of anti-EGFr antibody in the therapy of multiple patient populations with EGFr-expressing solid tumors.

Animals↗

PubMed2000

Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1.

Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH.

3T3 Cells↗

PubMed2001

[Cloning and expression of the genes of glutathione synthetases].

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.

Cloning, Molecular↗

PubMed2001

[Studies on synthesis of glutathione by E. coli BL21 (pTrc-gsh) coupled with Saccharomyces cerevisiae].

During synthesis of GSH by the engineered strain E. coli BL21(pTrc-gsh) coupled with Saccharomyces cerevisiae producing ATP from adenosin, the inconsistency of two systems in the concentration of phosphate buffer was solved by decreasing concentration to 250 mmol/L. The conditions under 250 mmol/L phosphate buffer were optimized and the yield of GSH was 1.6 g/L, which was higher than that of summation by two systems under the same conditions respectively. Addition of glycine later after glutamate and cysteine weakened the inhibition of GSH to GSHI. It made the yield of GSH reach to 2.13 g/L which was 30.7% higher than the control.

Adenosine↗

PubMed2002

[Gene cloning and expression of CTLA-4 in Streptomyces lividans].

Gene cloning and expression of CTLA-4 was performed in S. lividans with two new types of different signal peptides--vsi and gpp to investigate the secretory expression efficiency of CTLA-4. The hsCTLA-4 gene was fused to the vsi sequences and then inserted into the shuttle vector-pUWL-219. At the same time, the hsCTLA-4 was inserted into the downstream of gpp signal peptide in the plasmid pLNSP. Then the recombinant plasmids were transformed into S. lividans TK24 respectively. The two engineering strains were named as S. lividans [pUWL219-VC] and S. lividans [pLNSP/CTLA-4]. The result of SDS-PAGE and Western blotting show that the recombinant strain S. lividans [pUWL219-VC] and S. lividans [pLNSP/CTLA-4] can express CTLA-4 with about MW 13,000 which is similar to those reported earlier and have immunoactivity. It is the first time that CTLA-4 expression in S. lividans is reported.

Abatacept↗

PubMed2000

[The selection of fluoroacetate-resistant mutant from E. coli MMR204 and its influence on the expression of heterologous GL-7ACA acylase].

In the cultivation of gene engineered strain of Escherichia coli on glucose medium, excretion and accumulation of acetic acid inhibit not only cell growth but also the the expression of heterologous protein. It is obvious that the desirable host strain maintaining acetate at a low level is one of the approaches to increase the production of recombinant protein. The present article deals with the selection of mutants of E. coli DP19, DP8, which grow on the medium containing pyruvate as the sole carbon source in the presence of 50 mmol/L fluoroacetic acid. It is shown that mutant DP19 is defective in its phosphotransacetylase(PTA) activity and accumulates less acetate in the medium, while DP8 is defective in acetate kinase (ACK) and accumulates similar level of acetate comparing with its parent. Using pta- mutant E. coli DP19 as host, the expression of GL-7ACA acylase gene on the recombinant plasmid pMR24 is improved, and the yield of enzyme activity in flask fermentation is about twice as much as its parent.

Acetate Kinase↗

PubMed2002

[Cloning and expression of Vitreoscilla hemoglobin in Streptomyces aureofaciens].

Vitreoscilla hemoglobin Gene was cloned in Streptomyces aureofaciens through E. coli-Streptomyces shuttle plasmids constructed by both pIJ699-pUC19(vhb) and pIJ702-pBR322(vhb). Under low dissolved oxygen conditions, expression of hemoglobin enhanced CTC yield of engineering strain more about 40%-60% that that of control by improving oxygen transmission in cells. Hemoglobin expression by Promoter of tetracycline resistance gene was more effective for oxygen transmission than that by its native promoter regulated by dissolved oxygen concentrations in environments of locally low concentrations of dissolved oxygen.

Bacterial Proteins↗