Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli.

We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.     

Construction of a low-temperature protein expression system using a cold-adapted bacterium, Shewanella sp. strain Ac10, as the host

A recombinant protein expression system working at low temperatures is expected to be useful for the production of thermolabile proteins. We constructed a low-temperature expression system using an Antarctic cold-adapted bacterium, Shewanella sp. strain Ac10, as the host. We evaluated the promoters for proteins abundantly produced at 4 degrees C in this bacterium to express foreign proteins. We used 27 promoters and a broad-host-range vector, pJRD215, to produce beta-lactamase in Shewanella sp. strain Ac10. The maximum yield was obtained when the promoter for putative alkyl hydroperoxide reductase (AhpC) was used and the recombinant cells were grown to late stationary phase. The yield was 91 mg/liter of culture at 4 degrees C and 139 mg/liter of culture at 18 degrees C. We used this system to produce putative peptidases, PepF, LAP, and PepQ, and a putative glucosidase, BglA, from a psychrophilic bacterium, Desulfotalea psychrophila DSM12343. We obtained 48, 7.1, 28, and 5.4 mg/liter of culture of these proteins, respectively, in a soluble fraction. The amounts of PepF and PepQ produced by this system were greater than those produced by the Escherichia coli T7 promoter system.     

Expression of the nopaline synthase gene in Escherichia coli

The Agrobacterium tumor-inducing (Ti) plasmid pTiT37 encodes nopaline synthase (NOS) gene (nos) with eukaryotic promoter elements that is expressed in transformed plant cells but not in the bacterial host. We have fused the nos gene to the Escherichia coli trp promoter, and observed synthesis of NOS in E. coli. The nopaline produced by this enzyme is excreted into the culture medium. NOS is enzymatically active at 30 degrees C but not 37 degrees C, as based on nopaline production. NOS protein is produced at both temperatures, based on production in minicells.     

Non-toxic expression in Escherichia coli of a plasmid-encoded gene for phage T4 lysozyme

The phage T4 gene coding for lysozyme has been cloned into a plasmid under control of the (trp/lac) hybrid tac promoter and expressed in Escherichia coli with no significant toxic effect to actively growing cells. E. coli D1210 (lacIq) transformed with this plasmid produced active T4 lysozyme at levels up to 2% of the cellular protein after induction with isopropyl-beta-D-thiogalactoside. A strain producing active lysozyme was shown to be under a selective disadvantage when co-cultured with a similar strain producing inactive lysozyme. Purified strains, however, are reasonably stable in culture and under normal storage conditions.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.     

On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant.

Studies of two temperature-sensitive Escherichia coli topA strains AS17 and BR83, both of which were supposed to carry a topA amber mutation and a temperature-sensitive supD43,74 amber-suppressor, led to conflicting results regarding the essentiality of DNA topoisomerase I in cells grown in media of low osmolarity. We have therefore reexamined the molecular basis of the temperature sensitivity of strain AS17. We find that the supD allele in this strain had lost its temperature sensitivity. The temperature sensitivity of the strain, in media of all osmolarity, results from the synthesis of a mutant DNA topoisomerase I that is itself temperature-sensitive. Nucleotide sequencing of the AS17 topA allele and studies of its expected cellular product show that the mutant enzyme is not as active as its wild-type parent even at 30 degrees C, a permissive temperature for the strain, and its activity relative to the wild-type enzyme is further reduced at 42 degrees C, a nonpermissive temperature. Our results thus implicate an indispensable role of DNA topoisomerase I in E. coli cells grown in media of any osmolarity.     

Mass production of thermostable D-hydantoinase by batch culture of recombinant Escherichia coli with a constitutive expression system

D-Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D-amino acids. A gene encoding thermostable D-hydantoinase of Bacillus stearothermophilus SD-1 has previously been cloned and constitutively expressed by its native promoter in Escherichia coli XL1-Blue (Lee et al., 1996b). In this work, we attempted mass production of the D-hydantoinase by batch culture of the recombinant E. coli using glycerol as a carbon source. The plasmid content in cells increased in proportion to the culture temperature, which resulted in a two- or three-fold increase of the specific D-hydantoinase activity at 37 degrees C compared with that at 30 degrees C. The plasmid was stably maintained over 80 generations. When glycerol was initially added to a concentration of 100 g/L, the final biomass concentration reached about 50 g-dry cell weight/L in a 50 L-scale fermentation, resulting in the specific enzyme production of 3.8 x 10(4) unit/g-dry cell weight in a soluble form. Glycerol-using batch cultivation of recombinant E. coli was found to be a cost-effective process for the mass production of industrially useful D-hydantoinase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 449-455, 1997.     

Cloning, sequencing, and expression of the N-acyl-D-mannosamine dehydrogenase gene from Flavobacterium sp. strain 141-8 in Escherichia coli.

The gene coding for N-acyl-D-mannosamine dehydrogenase (NAM-DH) from Flavobacterium sp. strain 141-8 was cloned and expressed under the control of a lac promoter in Escherichia coli JM109. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide composed of 272 amino acid residues (Mr, 27,473) was identified. The E. coli transformants which showed over 200-fold higher NAM-DH activity than did the Flavobacterium strain produced the enzyme as a protein fused with beta-galactosidase. Despite being a fusion, NAM-DH produced by E. coli transformants appeared unchanged in pH optimum, Km, and substrate specificity from Flavobacterium sp. strain 141-8. This newly recombinant enzyme may be applicable to the quantitative determination of sialic acid in serum.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-beta-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more beta-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in beta-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Initiation of chromosome replication in Escherichia coli after induction of dnaA gene expression from a lac promoter.

Escherichia coli HB282 carries a dnaA46(Ts) allele on the chromosome, a wild-type dnaA allele under the control of the lacUV5 promoter on the multicopy plasmid pBC32, and an overproducing lac repressor allele on an F' factor. When the plasmid dnaA gene is repressed, the strain is thermosensitive. After a temporary deficiency in active dnaA protein at nonpermissive temperature, the addition of isopropyl-beta-D-thiogalactopyranoside to the culture was found to produce a burst of initiations within 5 to 10 min at 30% of the origins in 90% of the cells. Initiations then continued at a rate slightly faster than the mass-doubling time such that after 2 h the origin-to-mass ratio of the control culture was restored.     

Cloning and DNA sequencing of xyaA, a gene encoding an endo-beta-1,4-xylanase from an alkalophilic Bacillus strain (N137).

The gene xyaA encoding an alkaline endo-beta 1,4-xylanase from an alkalophilic Bacillus sp. strain (N137) isolated in our laboratory was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1,656-bp DNA fragment containing xyaA was determined, revealing one open reading frame of 993 bp that encodes a xylanase (XyaA) of 39 kDa. This xylanase lacks a typical putative signal peptide, yet the protein is found in the Bacillus culture supernatant. In Escherichia coli, the active protein is located mainly in the periplasmic space. The xylanase activity of the cloned XyaA is an endo-acting enzyme that shows optimal activity at pH 8 and 40 degrees C. This activity is stable at a pH between 6 and 11. Incubations of XyaA at 40 degrees C for 1 h destroyed 45% of the activity.     

Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli

A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transcriptional analysis of the nitrile-degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli– T7 expression system

Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli-T7 expression system by lowering the growth temperature to 29 degrees C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1.

In common with other plant symbionts, Frankia spp., the actinomycete N2-fixing symbionts of certain nonleguminous woody plants, synthesize two glutamine synthetases, GSI and GSII. DNA encoding the Bradyrhizobium japonicum gene for GSII (glnII) hybridized to DNA from three Frankia strains. B. japonicum glnII was used as a probe to clone the glnII gene from a size-selected KpnI library of Frankia strain CpI1 DNA. The region corresponding to the Frankia sp. strain CpI1 glnII gene was sequenced, and the amino acid sequence was compared with that of the GS gene from the pea and glnII from B. japonicum. The Frankia glnII gene product has a high degree of similarity with both GSII from B. japonicum and GS from pea, although the sequence was about equally similar to both the bacterial and eucaryotic proteins. The Frankia glnII gene was also capable of complementing an Escherichia coli delta glnA mutant when transcribed from the vector lac promoter, but not when transcribed from the Frankia promoter. GSII produced in E. coli was heat labile, like the enzyme produced in Frankia sp. strain CpI1 but unlike the wild-type E. coli enzyme.     

Low temperature cultivation of Escherichia coli carrying a rice lipoxygenase L-2 cDNA produces a soluble and active enzyme at a high level

Using a T7 RNA polymerase promoter system, rice lipoxygenase L-2 cDNA was expressed in E. coli as a fusion protein with 18 amino acid residues at the amino terminal end of the original enzyme. Incubation at 37 degrees C for 3 h in the presence of the inducer resulted in the production of inactive lipoxygenase. However, when induction was carried out at 15 degrees C for 16 h, active lipoxygenase, amounting to 3% of the total soluble protein, was produced. The enzyme was purified by ammonium sulfate precipitation and Mono-Q column chromatography to homogeneity at a yield of 80%. Expression of this protein should permit future site-directed mutagenesis of the gene and crystallization of the enzyme.