Fed-batch cultivation of an oxygen-dependent inducible promoter system, the nar promoter in Escherichia coli with an inactivated nar operon

The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific beta-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific beta-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific beta-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture.     

Characterization of an oxygen-dependent inducible promoter,the Escherichia coli nar promoter,in gram-negative host strains

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.     

Development and characterization of an oxygen-dependent inducible promoter system, the modified nar promoter in a mutant Escherichia coli

A nar promoter system (a modified nar promoter in a mutant host Escherichia coli (pMW618/W3110narL(-))), which is maximally induced under microaerobic conditions, was developed and characterized through batch and fed-batch culture to see whether the modified nar promoter can be used as an oxygen-dependent inducible promoter in the absence of nitrate ion. The modified nar promoter (pMW618) derived by mutations at -10 and -35 regions of the wild-type nar promoter does not require nitrate ion for the full induction, while a mutant host E. coli, W3110narL(-), does not express nitrate-dependent regulatory protein, NARL, from the host chromosome. In this study, it was found from fed-batch culture that the specific beta-galactosidase activity expressed from the lacZ gene fused to the modified nar promoter in the absence of nitrate ion was maximal when E. coli was grown under aerobic conditions (dissolved oxygen (DO) at 80%) to absorbance at 600 nm (OD(600)) of 35, and then the modified nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. The maximal specific beta-galactosidase activity became 58,000 Miller at OD(600) of 160 with an induction ratio of 20. On the basis of these results, we conclude that the modified nar promoter system (pMW618/W3110narL(-)), requiring only reduction of DO for the full induction, provides a convenient and effective high-level expression system under conditions of fed-batch culture.     

Whole cell enzyme microencapsulation of Escherichia coli with oxygen-dependent inducible nar promoter

As a means of integrating cell growth and immobilization, Escherichia coli with the cloned nar promoter on the pBR322 plasmid, which is maximally induced under anaerobic conditions in the presence of nitrate, was immobilized in liquid-core alginate capsules and cultured to a high cell density. The total -galactosidase activity obtained by immobilized cells was about 6 fold greater than that obtained by free cells. Using the immobilized -galactosidase in the whole cells, the substrate lactose was hydrolyzed to glucose and galactose stably with a conversion efficiency of more than 80% for 15 repeated batches at 30°C for 5 days.     

Characterization of the nar promoter to use as an inducible promoter

Summary The nar promoter of Escherichia coli was characterized, which is maximally induced under anaerobic conditions in the presence of nitrate. The following results were obtained; Expression of -galactosidase was optimal at 1 % of nitrate and was not affected much by molybdate; the amount of -galactosidase per unit volume was maximal when the nar promoter was induced at OD₆₀₀ = 1.7, and when anaerobic condition was made by supplying nitrogen gas. At the optimal condition, the ratio of -galactosidase between before and after induction was approximately 250 and Miller units were approximately 7,500. The results showed that the nar promoter can be used as an inducible promoter.     

Autoregulation of the nar operon encoding nitrate reductase in Escherichia coli

Summary Nitrate reductase is demonstrated to exert an autogenous control on its own synthesis. This effect requires the participation of the molybdenum cofactor. Use of strains in which the control region of the nar operon is mutated reveals two loci in this region: one, affected in strain LCB94, is common to both autoregulation and induction by nitrate while the other, mutated in strain LCB188, is specific for the induction by nitrate. It is proposed that the autogenous control prevents the unnecessary accumulation of the nitrate reductase subunits in the cytoplasm.     

Development of anaerobically inducible nar promoter expression vectors for the expression of recombinant proteins in Escherichia coli

Dissolved oxygen (DO)-controlled nar promoter expression vectors were constructed, and their expression efficiency was compared with that of the T7 promoter pET22 expression vector by expressing human growth hormone (hGH), enhanced green fluorescence protein (EGFP), and β-tyrosinase in Escherichia coli cells. The nar promoter expression vector pRBS, which was engineered with a 5′-untranslated region and ribosomal binding site for the T7 promoter, expressed hGH at a rate of up to 32% of the total cellular proteins (TCP) in E. coli W3110narL⁻. The expression level of hGH was further enhanced, up to ∼42% of the TCP, by adding the N-terminal peptide tag of β-galactosidase to hGH, which was comparable to the expression of ∼43% of the TCP in pET-lac:hGH/BL21(DE3). A further engineered expression vector, pRBS(fnr), which coexpressed fumarate/nitrate reductase (fnr), expressed more EGFP than pET22 in BL21(DE3). In addition, recombinant β-tyrosinase was successfully expressed at a rate of up to ∼45% of the TCP in pRBS(fnr) in W3110narL⁻. From these results, the DO-controlled nar promoter system developed in this study can be considered a reliable and cost-effective expression system for protein production, especially in large-scale fermentation, as an alternative to the pET/BL(DE3) system.     

Construction in vitro of a cloned nar operon from Escherichia coli.

To clone the nar operon of Escherichia coli without an effective selection procedure for the nar+ phenotype, a strategy utilizing nar::Tn5 mutants was employed. Partial segments of the nar operon containing Tn5 insertions were cloned into plasmid pBR322 by using the transposon resistance character for selection. A hybrid plasmid was constructed in vitro from two of these plasmids and isolated by a procedure that involved screening a population of transformed nar(Ts) mutant TS9A for expression of thermal stable nitrate reductase activity. A detailed restriction site map of the resulting plasmid, pSR95, corresponded closely to the composite restriction endonuclease map deduced for the nar region from maps of the cloned nar::Tn5 fragments. When transformed with pSR95, wild-type strain PK27 overproduced the alpha, beta, and gamma subunits of nitrate reductase, although nitrate reductase activity was only slightly increased. The alpha and beta subunits were overproduced about 5- to 10-fold and accumulated mostly as an inactive aggregate in the cytoplasm; the gamma subunit overproduction was detected as a threefold increase in the specific content of cytochrome b555 in the membrane fraction. Functional nitrate reductase and the cytochrome spectrum associated with functional nitrate reductase were restored in the nar::Tn5 mutant EE1 after transformation with pSR95. Although the specific activity of nitrate reductase in this case was less than that of the wild type, both the alpha and beta subunits appeared to be overproduced in an inactive form. In both strains PK27(pSR95) and EE1(pSR95), the formation of nitrate reductase activity and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the accumulation of inactive subunits were repressed during aerobic growth. From these observations and the demonstration that pSR95 contains a functional nor operon that encodes the alpha, beta, gamma subunits of nitrate reductase.     

Bacterial catalysis of nitrosation: involvement of the nar operon of Escherichia coli.

We have developed a rapid and sensitive fluorimetric method, based on the formation of a fluorescent product from nitrosation of 2,3-diaminonaphthalene, for measuring the ability of bacteria to catalyze nitrosation of amines. We have shown in Escherichia coli that nitrosation can be induced under anaerobic conditions by nitrite and nitrate, that formate is the most efficient electron donor for this reaction, and that nitrosation may be catalyzed by nitrate reductase (EC 1.7.99.4). The narG mutants defective in nitrate reductase do not catalyze nitrosation, and the fnr gene is essential for nitrosation. Induction by nitrite or nitrate of nitrosation, N2O production, and nitrate reductase activity all require the narL gene.     

Alteration by mutation of the control by oxygen of the nar operon in Escherichia coli

Summary A nar-lac operon fusion was used to isolate a mutant in which the expression of the nar operon was no longer repressed by oxygen. The nar ^d mutation, located upstream of the nar structural genes, was found to be cis dominant; it led to independence from the Fnr protein which, in the wild-type strain, exerts a strict positive control on the nar operon. Both other known controls, nitrate induction and autoregulation, were unaffected. It is proposed that molecular oxygen controls the expression of nar via Fnr and that the nar ^d mutation affects the Fnr binding site of the narGHI control region.     

Evidence for an internal promoter in the Escherichia coli threonine operon.

Auxotrophic mutants of Halobacterium volcanii generated by chemical mutagenesis were used to demonstrate a native genetic transfer system in this extremely halophilic member of the class Archaeobacteria.     

Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter

From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of 'cassettes' carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence.     

narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes.

In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon. From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively. Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated. The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined. The nar operon therefore contains four genes designated and ordered as narGHJI.