Development of HIV-1 protease expression methods using the T7 phage promoter system

New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21(DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E.␣coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli. Received: 5 September 1996 / Received last revision: 1 November 1996 / Accepted: 15 November 1996     

Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol

An electrochemical reactor employing activated carbon fibers (ACF) was constructed for the disinfection of bacteria in drinking water. The application of an alternating potential of 1.0 V and −0.8 V versus a saturated calomel electrode, for disinfecting and desorbing bacteria, enabled reactor operation for 840 h. Drinking water was passed through the reactor in stop/flow mode: 300 ml/min flow for 12 h and no flow for 12 h, alternately. The bacterial cell density in treated water was always been less than 20 cells/ml. It was also found that the formation of biofilm on the ACF reactor caused an increase in current, enabling the self-detection of microbial fouling. Received: 19 February 1996 / Received last revision: 23 July 1996 / Accepted: 2 September 1996     

Catabolite-repressor-like protein regulates the expression of a gene under the control of the Escherichia coli lac promoter in the plant pathogen Xanthomonas campestris pv. campestris

  Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression in the clp -deficient mutant. Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI ^q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide). Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996     

Isolation and characterization of Rhodococcus rhodochrous for the degradation of the wastewater component 2-hydroxybenzothiazole

2-Hydroxybenzothiazole (OBT) is present in wastewaters from the industrial production of the rubber vulcanization accelerator 2-mercaptobenzothiazole (MBT). We have achieved the first isolation of axenic bacterial cultures capable of the degradation of OBT and growth on this substrate as the sole source of carbon, nitrogen and energy. All isolates had similar characteristics corresponding to one particular isolate, which was studied in more detail and identified as Rhodococcus rhodochrous. The strains were also capable of degrading benzothiazole (BT) but not MBT or benzothiazole-2-sulphonate (BTSO₃). OBT was degraded at a concentration of up to 600 mg · l⁻¹. BT was toxic above 300 mg · l⁻¹. MBT inhibited OBT degradation. Growth on OBT was not significantly different at pH values of between 6.3 and 7.9 or salt concentrations between 1 % and 3 %. In shake flasks the cells clumped together, which resulted in a lower rate of oxygen transfer and slower degradation as compared to cells grown on OBT in a stirred reactor. Received: 22 August 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Improved efficiency and stability of multiple cloned gene insertions at the δ sequences of Saccharomyces cerevisiae

Two δ-integration vectors were evaluated for the insertion of an inducible expression cassette (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene, CUP1p-lacZ) and a bacterial neomycin-resistance gene (neo) into the genome of Saccharomyces cerevisiae via homologous recombination. Cells containing integrations were selected by resistance to the aminoglycoside G418. The first vector was a traditional construct containing only one δ sequence; with this vector, the transformation efficiency and the number of integrations per cell were quite low. The second carried two δ sequences flanking the desired insert, and the unneeded bacterial sequences were removed by restriction-enzyme digestion immediately before transformation. When this double δ vector was employed, the integrated copy number was more than doubled relative to the single δ system and final β-galactosidase levels exceeded those obtained with the 2μ-based plasmid. Furthermore, the integrations appeared more stable in long-term sequential culture (both with and without induction of the lacZ gene) than those obtained via the single δ vector. Received: 2 December 1996 / Received revision: 21 March 1997 / Accepted: 13 April 1997     

Extracellular pH affects regulation of the pcbAB gene in Penicillium chrysogenum

The effect of extracellular pH and dissolved oxygen on regulation of the pcbAB gene in P.␣chrysogenum was examined, using Northern analysis and a reporter gene fusion. It was found that ambient pH markedly affected levels of pcbAB mRNA whereas maintenance of dissolved oxygen concentration above 10 % had no detectable effect. The presence of a DNA-binding protein, which binds upstream of the pcbAB translational start codon, was also related to ambient pH. In all fermentations, pcbAB mRNA was most abundant at around the late exponential/early stationary phase of a culture. Received: 10 May 1996 / Received revision: 14 October 1996 / Accepted: 25 October 1996     

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B.␣lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine. Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996     

Plasmids for efficient single-copy gene cloning into gdh2 and trpC of Bacillus megaterium DSM319 and QM B1551

We constructed integrative plasmids to place xylA-lacZ indicator gene fusions into two different loci of the Bacillus megaterium chromosome, gdh2 and trpC, in lac mutants of strains DSM 319 and QM B1551, which differ markedly. Single-crossover integration was achieved in all cases while double crossovers occurred only in gdh2 of DSM 319 and QM B1551 and in trpC of QM B1551. Neither of the loci affected regulation of the xylA-lacZ fusions. These results confirm the suitability of the two gene loci for single-copy cloning. Received: 28 October 1996 / Received revision: 29 December 1996 / Accepted: 4 January 1997     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Phycocyanin,a new elicitor for capsaicin and anthocyanin accumulation in plant cell cultures

  Elicitors of both fungal and bacterial origin that is, polysaccharides, proteins and fatty acids, are widely used for enhancement of secondary metabolites in plant cell cultures. In the present study, phycocyanin – a natural blue pigment that is the major light-harvesting biliprotein in the blue-green alga Spirulina platensis– was used as an elicitor to enhance the accumulation of capsaicin and anthocyanin in Capsicum frutescens and Daucus carota cell cultures respectively. Phycocyanin at 0.3, 0.6 and 1.2 mg% in capsicum cell cultures elicited a more than two-fold increase in capsaicin content with maximum productivity of 192 μg/g fresh weight. Similarly in Daucus carota cell cultures a two-fold increase in anthocyanin content was obtained at 0.3 mg% with a maximum productivity of 24.8 mg% on a dry-weight basis. In both the systems, phycocyanin showed an early elicitation of secondary metabolites. Received: 15 December 1995 / Received last revision: 15 July 1996 / Accepted: 18 July 1996     

Improvement of culture conditions to overproduce β-galactosidase from Escherichia coli in Bacillus subtilis

The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated, as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration. Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996     

The effect of oxidative stress on the production of the recombinant protein, interferon γ, produced by Chinese hamster ovary cells in stirred-batch culture

The CHO320 cell line, engineered to produce human interferon γ was investigated with regard to its susceptibility to oxidative stress. Batch cultures of the cells grown in a bench-top bioreactor exhibited no marked response to changes in oxygen concentration between 6% and 14% whereas cell growth and recombinant protein production were inhibited by increasing the oxygen to 20%. High concentrations of hydrogen peroxide (in excess of 200 μM) were required to inhibit growth of the CHO320 cells whereas concentrations of 50 μm and 100 μM had no effect on recombinant protein production. Buthionine sulphoximine (50 μM and 100 μM) completely depleted the cells of glutathione within 24 h; however, no quantitative effect on recombinant protein production was seen. It is concluded that the CHO320 cells are, possibly as a consequence of the long selection process they have undergone, very resistant to oxidative stress. Received: 14 November 1996 / Received last revision: 14 April 1997 / Accepted: 19 April 1997     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

High volumetric yields of functional dimeric miniantibodies in Escherichia coli,using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl⁻¹ in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-termi-nal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hok/sok suicide system, improving plasmid maintenance, and the inducible lac p/o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l⁻¹ after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fed-batch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 g l⁻¹, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions. Received: 26 February 1996 / Received revision: 1 August 1996 / Accepted: 12 August 1996     

Use of immobilized bacteria to treat industrial wastewater containing a chlorinated pyridinol

 Pseudomonas sp. strain M285 immobilized on diatomaceous earth beads was used to remove 3,5,6-trichloro-2-pyridinol (TCP) from industrial wastewater. Batch studies showed that immobilized Pseudomonas sp. strain M285 mineralized [2,6-¹⁴C]TCP rapidly; about 75% of the initial radioactivity was recovered as ¹⁴CO₂. Transformation of TCP was inhibited by high concentrations of salt, and addition of osmoprotectants (proline and betaine at 1 mM) did not reduce the adverse effect of salt. TCP-containing wastewater (60–140 mg/l) was passed through columns containing immobilized Pseudomonas sp. strain M285 at increasing flow rates and increasing TCP concentrations; TCP removal of 80%–100% was achieved. Addition of nutrients, such as glucose and yeast extract, retarded TCP degradation. Growing cell cultures were found to be better inocula for immobilization than resting cells. Received: 5 February 1996 / Received last revision: 12 August 1996 / Accepted: 24 August 1996     

Cell-dislodging methods under serum-free conditions

In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml⁻¹), equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity. Received: 23 September 1996 / Received revision: 27 December 1996 / Accepted: 30 December 1996     

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10⁻⁵–10⁻⁷ exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50 % of larvae (LC₅₀) being 2.1 × 10⁵ and 1.3 × 10⁵ cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth. Received: 23 July 1996 / Received revision: 11 November 1996 / Accepted: 15 November 1996     

Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans

A gene for a synthetic protein-based polymer, G-(VPGVG)₁₁₉-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study represents the first attempt to express a synthetic gene (with no natural analog) in a fungus. Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria

The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated. Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles. Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein⁻¹) and also had the highest affinity for 1,3-DCP (K _m, 0.1 mM). Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (k ₁) of 1.13 h⁻¹ . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations. However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl⁻¹. Received: 30 April 1996 / Received revision: 30 July 1996 / Accepted: 5 August 1996