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Expression of the Bacterial Hemoglobin Gene from Vitreoscilla stercoraria Increases Rifamycin B Production in Amycolatopsis mediterranei
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 106, No. 5, 493–497. 2008 DOI: 10.1263/jbb.106.493
© 2008, The Society for Biotechnology, Japan
Expression of the Bacterial Hemoglobin Gene from Vitreoscilla stercoraria Increases Rifamycin B Production in Amycolatopsis mediterranei Guerra Priscila,1 Francisco J. Fernández,1 Angel E. Absalón,1,2 Ma. del Rocío Suarez,1 Mara Sainoz,1 Javier Barrios-González,1 and Armando Mejía1* Depto. de Biotecnología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Iztapalapa, AP 55-535, México 09340, D. F. México1 and Centro de Investigación en Biotecnología Aplicada, IPN-Tlaxcala, Carretera Santa Inés-Tepetitla Km 1.5 Tepetitla, Tlaxcala, México2 Received 15 April 2008/Accepted 30 July 2008
It is well known that the culture for rifamycin B production by Amycolatopsis mediterranei requires high levels of dissolved oxygen, particularly in industrial processes. In this study, we report the construction of a vector for the expression of the bacterial hemoglobin gene (vhb) from Vitreoscilla stercoraria in a rifamycin B-overproducing strain of A. mediterranei. The effect was evaluated in the presence and absence of barbital. The vhb gene was cloned under the control of the PermE promoter, the Amycolatopsis lactamdurans plasmid pULVK2 origin of replication, the kanamycin-resistant gene (Km), the erythromycin-resistant gene (ermE) for selection, and ColE1. Industrial fermentation conditions were simulated in shake-flask cultures. Under low aeration, the transformed A. mediterranei strain with the vhb gene showed a 13.9% higher production of rifamycin B in a culture with barbital compared with the parental strain, and 29.5% higher production under the same conditions without barbital. [Key words: Actinomycetes, Amycolatopsis, barbital, bacterial hemoglobin, rifamycin, Vitreoscilla]
showed that this compound also inhibits the respiratory chain, leaving more oxygen available for biosynthesis (6). On the other hand, it is well known that Vitreoscilla stercoraria is able to grow in poorly oxygenated environments due to a kind of bacterial hemoglobin. The gene that codes for this protein, (vhb), has been expressed in other industrially important filamentous microorganisms (7, 8). Studies have shown that the vhb gene expression can significantly improve the productivity of cephalosporin C and erythromycin. The aim of this study, is to clone the vhb gene from V. stercoraria in an expression vector for A. mediterranei and evaluate its effect on the oxygen uptake and rifamycin B production.
Amycolatopsis mediterranei is a soil bacterium that produces secondary metabolites, including rifamycins that are major antibiotics in the treatment of tuberculosis, leprosy, and other mycobacterial infections. The demand for this antibiotic is increasing annually because rifamycin derivatives have also shown excellent activity against opportunistic microorganisms in the AIDS-related diseases (1). However, from an industrial point of view, there are some problems in increasing its production to meet the demand. For example, there is an extremely high oxygen requirement of this culture, as biosynthesis of the main fermentation product, rifamycin B, involves some oxidative modification steps that occur at C-34a and in the carbon chain cleavage between C-12 and C-29, or naphthoquinone formation (2). These steps utilize atmospheric oxygen, hence the need for oxygen (3, 4). When the culture reaches a stationary phase, this actinomycete, like other filamentous microorganisms, produces high viscosity, provoking low levels of dissolved oxygen. At this phase, secondary metabolism begins and the culture needs more oxygen (3). For this reason, several related and undesirable rifamycins (mainly rifamycin W) accumulate in the culture medium along with rifamycin B (5). Since 1961, to counteract this effect, 5,5-diethylbarbituric acid (barbital) has been added in industrial fermentations to direct biosynthesis preferentially towards rifamycin B. In 2003, we
MATERIALS AND METHODS Microorganism A. mediterranei Msb2 was used in this study. Msb2 is a mutant strain of M18, isolated for its higher sensitivity to barbital (9). This strain produces 3 times more rifamycin B than its parental strain (M18). However, when cultured in a fermentor or under low aeration, this strain requires barbital to produce rifamycin B exclusively (the same phenotype is displayed by modern industrial strains). V. stercoraria (ATCC 15218) was used to get the vhb gene and Saccharopolyspora erythraea (ATCC 11635) to obtain the promoter ermP. Escherichia coli DH5α strain was used as a recipient strain for plasmids. The strains of A. mediterranei were lyophilized and stored, either frozen at −20°C or maintained in Bennet agar medium. E. coli was stored at −80°C.
* Corresponding author. e-mail: [email protected] phone: +52-55-5804-6453 fax: +52-55-5804-4712 493
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Media and culture conditions E. coli strains and plasmidcontaining transformants were grown at 37°C in Luria–Bertani medium. A. mediterranei was grown in a seed medium containing the following components, (in grams per liter): glucose, 20; soybean meal, 20; CaCO3, 2.5; MgSO4 ⋅ 7H2O, 0.4; FeSO4 ⋅ 7H2O, 0.01; ZnSO4 ⋅ 7H2O, 0.05; and CoCl2 ⋅ 6H2O, 0.003. The seed cultures were grown in a shaking incubator (150 rpm) at 28°C for 84 h. Lee production medium contained (in grams per liter): glucose, 120; Bacto-peptone, 10; yeast extract, 5; and sodium 5,5-diethylbarbiturate, 0.7. The pH was 7.2 and erythromycin (10 μg/ml) was present when transformants were used. This medium was inoculated with 20% of the seed culture and incubated at 25°C in a 250-ml Erlenmeyer flask with a 25 ml production medium (250 rpm). When low aeration conditions were required, the culture was incubated at 200 rpm in a 250-ml Erlenmeyer flask with a 70-ml production medium (3, 10). Bennet agar was composed of the following (in grams per liter): glucose, 10; meat extract, 1; NZ-amine A, 2; yeast extract, 1; and agar, 20. Analytical methods Cell growth in production medium was monitored by measuring the dry weight. Rifamycin B contents of the culture were determined using the previously described procedure (9). Total DNA isolation Small-scale preparation of plasmid DNA from E. coli was performed with a Qiagen Plasmid Kit (Qiagen, Chatsworth, CA, USA) or a standard procedure (11). Large-scale plasmid DNA from E. coli was prepared using a standard alkaline lysis protocol (12). Plasmid DNA from A. mediterranei was obtained as follows. S699 strain was cultured in 100 ml of yeast extract–malt extract (YEME) medium and incubated at 25°C, shaking for 72 h at 250 rpm. The culture was centrifuged at 3381×g for 15 min at 4°C. The pellet was washed once, suspended in 10 ml of lysozyme solution (10.3% sucrose, 25 mM Tris–HCl, pH 8.0, 25 mM EDTA, pH 8, and 10 mg/ml lysozyme) and incubated at 30°C for 2 h. Then, 3.5 ml of SDS solution (2% SDS and 0.3 M NaOH) was added and incubated at 80°C for 10 min. After this, the samples were treated according to the protocol of Moretti et al. (13). Total DNA from Vitreoscilla stercoraria was obtained according to the protocol of Holmes and Quigley (11), without the boiling step. RNA isolation Total RNA isolation was performed with RNeasy Mini Kit (Qiagen) by grinding the recommended amounts of mycelium (30 mg per minicolumn) with liquid nitrogen in a frozen mortar and pestle, taking care not to let the mycelium thaw until it was placed in the appropriate lysis buffer supplied with the kit. At this point the manufacturer’s protocol was followed. The RNA was eluted twice in a volume of 50 μl (total) of RNase-free H2O. RNA was treated for DNA contamination by extraction with acid phenol:chloroform (5 : 1 phenol : CHCl3; pH 4.7). DNA was retained in the organic phase and RNA remained in the aqueous phase. RNA was subsequently recovered by precipitation. Samples were aliquoted and stored at −80°C. Polymerase chain reaction (PCR) The vhb gene was obtained by PCR amplification from V. stercoraria genomic DNA using oligonucleotides 5′-ATGTTAGACCAGCAAACCATT-3′ as a forward primer and 5′-TAGGATCCATGCCAAGGCACACCTG AAGA-3′ as a reverse primer, which contained a BamHI site (underlined) located next to the 5′ end of the gene. The promoter PermE was obtained by PCR from the Saccharopolyspora erythraea genomic DNA using the following oligonucleotides: 5′-GCGAAGCTTATGCGAGTGTCCGTTCGAGTG-3′ as a forward primer and 5′-CGCTGGATCCTACCAACCGGC-3′ as a reverse primer. Reverse transcriptase-PCR (RT-PCR) According to the
J. BIOSCI. BIOENG.,
manufacturer’s instructions, one-step RT-PCR (Qiagen) was carried out in 50-μl reaction volumes containing 1 μl of template RNA, 0.6 μl of forward and reverse primers (16 and 17.5 μM), 10 μl of 5 ×QIagen one-step buffer (100 mM Tris–HCl, 500 mM KCl, 12.5 mM MgCl2, and pH 8.7), 2 μl of dNTPs mix (10 mM), and 2 μl of Qiagen one-step enzyme mix (reverse transcriptase and HotStart Taq DNA polymerase). Thermocycling was performed as follows: 50°C for 30 min, 95°C for 15 min, then 50 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, followed by 72°C for 10 min. Amplified products were observed after electrophoresis in ethidium bromide-stained agarose gels. β-subunit of RNA polymerase was used as the housekeeping gene. Transformation and screening A. mediterranei Msb strain was made competent, and transformed with pUAMSAG1 by biolistic process (vacuum 20 inches Hg, 6 cm target distance, 900 psi, BioRad M5 tungsten particle). The treated cells were seeded on plates with Bennet agar. After incubation for 6 h at 30°C, the plates were covered with soft agar containing erythromycin (10 μg/ml). Transformants Msb-vhb were selected for their ability to grow in this medium. Measurement of specific oxygen uptake rate Oxygen uptake was measured with a Biological Oxygen Monitor, model 5300 (Yellow Springs Instruments, Yellow Springs, OH, USA), in an air-saturated 0.1 M potassium-phosphate buffer (pH 7.2) at 28°C. The electrode was calibrated with air-equilibrated water. Then the cells were harvested and washed twice by centrifugation at 3381×g for 10 min at 4°C. Of the resulting suspension 0.5 ml was added to 2.5 ml of air-saturated buffer (0.1 M glucose and 20 mM potassium–phosphate buffer, pH 7.2). Biomass concentration was calculated simultaneously by dry weight. The change in oxygen concentration, of the buffer containing the cells, was monitored for 10 min.
RESULTS AND DISCUSSION Rifamycin B biosynthesis by A. mediterranei requires high dissolved oxygen, particularly in industrial processes, where the volumes used can be higher than 120,000 l. Several attempts have been made to develop a process with better oxygen transfer, but no strategy had used the hemoglobin gene from V. stercoraria (or from a different organism) in A. mediterranei. Therefore, with this goal in mind, the vhb gene was cloned, and the vector pUAMSAG1 was constructed. Construction of hemoglobin (VHb) expression vector In order to develop a plasmid-cloning vector with an effective marker gene, the sensitivity of A. mediterranei Msb to kanamycin and erythromycin was tested in a Bennet medium. Although this strain was sensitive to kanamycin (50 μg/ml, the resistance marker (Kmr) could not be used because resistant mutants appeared spontaneously and with high frequency. Since Msb was also sensitive to erythromycin (10 μg/ml), it was chosen as the marker gene. The coding sequence of vhb gene and promoter PermE were generated by PCR amplification, using primers with BamHI and HindIII sites, respectively. The blunt ends of the vhb gene and promoter PermE were ligated and cloned in the pULVK2 vector, which contains pA-rep origin for A. mediterranei and ColE1 for E. coli. Erythromycin-resistant genes (ermE) from pIJ4026 were cloned into the unique EcoRI and XbaI sites to give the plasmid pUAMSAG1. The size of this chimeric product was determined to be about 8.162 kb, which corresponds to the expected size. The con-
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FIG. 1. Construction of pUAMSAG1. The final vector was constructed as shown, using the P ermE promoter region, the vhb gene, kanamycin (Kmr), and erythromycin (ermE) resistant genes, the origin of replication pA-rep for A. mediterranei, and ColE1 for E. coli.
struction scheme of pUAMSAG1 plasmid is shown in Fig. 1. However, A. mediterranei Msb2 strain could not be transformed by conventional procedures (14). Even electroporation of mycelium or protoplasts did not yield any transformant. This inefficient transformation could have been due to a specific characteristic of the Msb2 strain, as the transformation was possible when the S699 strain was used. Thus, it was decided to transform Msb2 strain by a biolistic process. With this procedure, Msb2 strain could successfully be transformed to pUAMSAG1. Although pUAMSAG1 is not a high copy number vector, a good stability was observed at the end of each culture in the absence of selective pressure with erythromycin. This
stability was observed during the entire batch process, including the seed culture. Effect of low aeration With the aim to compare the performance of the strain with the cloned vhb gene in A. mediterranei, rifamycin B production was evaluated. However, because the parental strain (like most industrial strains) requires barbital to produce rifamycin B exclusively, its production was evaluated with and without barbital. The transformants of A. mediterranei, Msb containing pUAMSAG1 (Msb-sag), and the strain with pUAMAE3 (lacking the Vhb expression cassette) were cultivated in shake-flasks under low aeration (as described in the Materials and Methods section). These conditions had already been tested and proved
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FIG. 2. Time course of rifamycin B production (continuous line) and growth (dotted line) by the transformed strain (Msb-sag). Flasks (250 ml) containing 70 ml of the production medium with 0.7 g/l barbital (closed triangles) and without barbital (open triangles) at 26°C, 200 rpm. A control culture of parental strain (Msb) with barbital (closed circles) and without barbital (open circles) is shown.
to provide low aeration (3, 10). The results were compared with the parental Msb strain (Fig. 2). Results showed that under low aeration conditions (70 ml production medium and 200 rpm), rifamycin B production decreased by about 50% in relation to the culture under high aeration conditions (25 ml of production medium, 250 rpm) when the strain was cultivated without barbital. However, when barbital was added to the culture under low aeration conditions, the production of rifamycin B was almost as high as in the culture under high aeration conditions (Fig. 2). Rifamycin B production of the transformant strain, Msb-sag, with barbital was practically the same compared with the parental strain. However, production of Msb-sag did not decrease without barbital. These results suggest that this strain could take up oxygen more efficiently under low aeration conditions, helping to confirm the theory that barbital increases O2 availability for rifamycin B biosynthesis by decreasing oxygen requirements in the respiratory chain (6). It is important to note, that no significant differences were found in the growth; therefore, the difference between specific productions was almost the same. To confirm that higher production of rifamycin B by the transformed strain was due to its ability to consume more oxygen, we simultaneously compared the rates of oxygen uptake between strains. Cultures in the production medium were harvested at 96 h (in idiophase) and oxygen uptake was measured from the washed cells of each strain. Results indicated that the rate of oxygen uptake of the transformed strain (Msb-sag) was about 1.5 times faster than that of the parental strain (Fig. 3). Detection of mRNA by reverse transcriptase (RT-PCR) To confirm that the higher oxygen uptake by the Msb-sag strain was due to the expression of the cloned vhb gene, RTPCR was used. RNA was extracted from idiophase cells of the parental and transformed strains, and RT-PCR experiments were carried out using a primer for a 503 bp fragment
FIG. 3. Measurement of the rate of oxygen uptake. Cell preparations were obtained from parental (Msb) and transformed strain (Msbsag) carrying the cloned vhb gene. Experimental details are given in Materials and Methods. Continuous lines indicate averages of three individual observations.
FIG. 4. RT-PCR analysis. Amplification of a 503-kb fragment from vhb RNA during the idiophase of Msb-sag strain (lane 2) and of Msb strain (lane 1). Nucleotide sequence of the primers is the following: RTvhbF, 5′-GCCACTGTTCCTGTATTGAAGGAGC-3′; RTvhbR, 5′TTTTGGCCAACAGCCAAACTGCTGC-3′.
of the vhb gene. No transcripts in the parental strain (Msb) were detectable (Fig. 4, lane 1), while the transformed strain (Msb-sag) clearly produced the expected fragment, which corresponded to vhb RNA (Fig. 4, lane 2). These results show that VHb expression improved the oxygen uptake and had a beneficial effect on the production of rifamycin B under low aeration conditions, or from another point of view, they indicate that the transformant with the vhb gene does not need barbital to continue production under low aeration conditions. ACKNOWLEDGMENTS The authors wish to thank the Consejo Nacional de Ciencia y Tecnología (CONACyT, México) for financial support (grant SEP2003-C02-44911).
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REFERENCES 1. Dickinson, J. and Mitchison, D.: In vitro activity of new rifamycins against rifampicin-resistant M. tuberculosis, MAIS complex mycobacteria. Tubercle, 68, 177–182 (1987). 2. August, P., Tang, L., Ion, Y., Ning, S., Muller, R., Yu, T., Taylor, M., Hoffmann, D., Kim, C., Zhang, X., Hutchinson, R., and Floss, H.: Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699. Chem. Biol., 5, 69–79 (1998). 3. Virgilio, A., Marcelli, E., and Agrimino, A.: Aerationagitation studies on the rifamycin fermentation. Biotechnol. Bioeng., 6, 271–283 (1964). 4. Jin, Z., Lin, J., and Cen, P.: Scale-up of rifamycin B fermentation with Amycolatopsis mediterranei. J. Zhejiang Univ. Sci., 5, 1590–1596 (2004). 5. Mejía, A., Gelista, A., Resendiz, B., and Rodriguez, F.: A strain of Nocardia mediterranei that produces a mixture of rifamycin B and W. Biotechnol. Lett., 12, 283–288 (1990). 6. Mejía, A., Viniegra-González, G., and Barrios-González, J.: Biochemical mechanism of the effect of barbital on rifamycin B biosynthesis by Amycolatopsis mediterranei (M18 strain). J. Biosci. Bioeng., 95, 288–292 (2003). 7. DeModena, J., Gutiérrez, S., Velasco, J., Fernández, F., Fachini, R., Galazzo, J., Hughes, D., and Martín, J. F.: The production of cephalosporin C by Acremonium chrysogenum
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is improved by the intracellular expression of a bacterial hemoglobin. Bio/Technology, 11, 926–929 (1993). Brünker, P., Minas, W., Kalio, P., and Bailey, J.: Genetic engineering of an industrial strain of Saccharopolyspora erythraea for stable expression of the Vitreoscilla hemoglobin gene (vhb). Microbiology, 144, 2441–2448 (1998). Mejía, A., Barrios-González, J., and Viniegra-González, G.: Overproduction of rifamycin B by Amycolatopsis mediterranei and its relationship with the toxic effect of barbital on growth. J. Antibiot., 51, 58–63 (1998). Krishna, P., Venkateswarlu, G., and Rao, L.: Studies on fermentative production of rifamycin using Amycolatopsis mediterranei. World J. Microbiol. Biotechnol., 14, 689–691 (1998). Holmes, D. and Quigley, M.: A rapid boiling method for the preparation of bacteria plasmids. Anal. Biochem., 114, 193– 197 (1981). Sambroock, J. and Russell, D.: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001). Moretti, P., Hintermann, G., and Hütter, R.: Isolation and characterization of an extrachromosomal element from Nocardia mediterranei. Plasmid, 14, 126–133 (1985). Lal, R., Khanna, R., Dhingra, N., Khanna, M., and Lal, S.: Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei). J. Antibiot., 51, 161–169 (1997).
© 2008, The Society for Biotechnology, Japan
Expression of the Bacterial Hemoglobin Gene from Vitreoscilla stercoraria Increases Rifamycin B Production in Amycolatopsis mediterranei Guerra Priscila,1 Francisco J. Fernández,1 Angel E. Absalón,1,2 Ma. del Rocío Suarez,1 Mara Sainoz,1 Javier Barrios-González,1 and Armando Mejía1* Depto. de Biotecnología, División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Iztapalapa, AP 55-535, México 09340, D. F. México1 and Centro de Investigación en Biotecnología Aplicada, IPN-Tlaxcala, Carretera Santa Inés-Tepetitla Km 1.5 Tepetitla, Tlaxcala, México2 Received 15 April 2008/Accepted 30 July 2008
It is well known that the culture for rifamycin B production by Amycolatopsis mediterranei requires high levels of dissolved oxygen, particularly in industrial processes. In this study, we report the construction of a vector for the expression of the bacterial hemoglobin gene (vhb) from Vitreoscilla stercoraria in a rifamycin B-overproducing strain of A. mediterranei. The effect was evaluated in the presence and absence of barbital. The vhb gene was cloned under the control of the PermE promoter, the Amycolatopsis lactamdurans plasmid pULVK2 origin of replication, the kanamycin-resistant gene (Km), the erythromycin-resistant gene (ermE) for selection, and ColE1. Industrial fermentation conditions were simulated in shake-flask cultures. Under low aeration, the transformed A. mediterranei strain with the vhb gene showed a 13.9% higher production of rifamycin B in a culture with barbital compared with the parental strain, and 29.5% higher production under the same conditions without barbital. [Key words: Actinomycetes, Amycolatopsis, barbital, bacterial hemoglobin, rifamycin, Vitreoscilla]
showed that this compound also inhibits the respiratory chain, leaving more oxygen available for biosynthesis (6). On the other hand, it is well known that Vitreoscilla stercoraria is able to grow in poorly oxygenated environments due to a kind of bacterial hemoglobin. The gene that codes for this protein, (vhb), has been expressed in other industrially important filamentous microorganisms (7, 8). Studies have shown that the vhb gene expression can significantly improve the productivity of cephalosporin C and erythromycin. The aim of this study, is to clone the vhb gene from V. stercoraria in an expression vector for A. mediterranei and evaluate its effect on the oxygen uptake and rifamycin B production.
Amycolatopsis mediterranei is a soil bacterium that produces secondary metabolites, including rifamycins that are major antibiotics in the treatment of tuberculosis, leprosy, and other mycobacterial infections. The demand for this antibiotic is increasing annually because rifamycin derivatives have also shown excellent activity against opportunistic microorganisms in the AIDS-related diseases (1). However, from an industrial point of view, there are some problems in increasing its production to meet the demand. For example, there is an extremely high oxygen requirement of this culture, as biosynthesis of the main fermentation product, rifamycin B, involves some oxidative modification steps that occur at C-34a and in the carbon chain cleavage between C-12 and C-29, or naphthoquinone formation (2). These steps utilize atmospheric oxygen, hence the need for oxygen (3, 4). When the culture reaches a stationary phase, this actinomycete, like other filamentous microorganisms, produces high viscosity, provoking low levels of dissolved oxygen. At this phase, secondary metabolism begins and the culture needs more oxygen (3). For this reason, several related and undesirable rifamycins (mainly rifamycin W) accumulate in the culture medium along with rifamycin B (5). Since 1961, to counteract this effect, 5,5-diethylbarbituric acid (barbital) has been added in industrial fermentations to direct biosynthesis preferentially towards rifamycin B. In 2003, we
MATERIALS AND METHODS Microorganism A. mediterranei Msb2 was used in this study. Msb2 is a mutant strain of M18, isolated for its higher sensitivity to barbital (9). This strain produces 3 times more rifamycin B than its parental strain (M18). However, when cultured in a fermentor or under low aeration, this strain requires barbital to produce rifamycin B exclusively (the same phenotype is displayed by modern industrial strains). V. stercoraria (ATCC 15218) was used to get the vhb gene and Saccharopolyspora erythraea (ATCC 11635) to obtain the promoter ermP. Escherichia coli DH5α strain was used as a recipient strain for plasmids. The strains of A. mediterranei were lyophilized and stored, either frozen at −20°C or maintained in Bennet agar medium. E. coli was stored at −80°C.
* Corresponding author. e-mail: [email protected] phone: +52-55-5804-6453 fax: +52-55-5804-4712 493
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Media and culture conditions E. coli strains and plasmidcontaining transformants were grown at 37°C in Luria–Bertani medium. A. mediterranei was grown in a seed medium containing the following components, (in grams per liter): glucose, 20; soybean meal, 20; CaCO3, 2.5; MgSO4 ⋅ 7H2O, 0.4; FeSO4 ⋅ 7H2O, 0.01; ZnSO4 ⋅ 7H2O, 0.05; and CoCl2 ⋅ 6H2O, 0.003. The seed cultures were grown in a shaking incubator (150 rpm) at 28°C for 84 h. Lee production medium contained (in grams per liter): glucose, 120; Bacto-peptone, 10; yeast extract, 5; and sodium 5,5-diethylbarbiturate, 0.7. The pH was 7.2 and erythromycin (10 μg/ml) was present when transformants were used. This medium was inoculated with 20% of the seed culture and incubated at 25°C in a 250-ml Erlenmeyer flask with a 25 ml production medium (250 rpm). When low aeration conditions were required, the culture was incubated at 200 rpm in a 250-ml Erlenmeyer flask with a 70-ml production medium (3, 10). Bennet agar was composed of the following (in grams per liter): glucose, 10; meat extract, 1; NZ-amine A, 2; yeast extract, 1; and agar, 20. Analytical methods Cell growth in production medium was monitored by measuring the dry weight. Rifamycin B contents of the culture were determined using the previously described procedure (9). Total DNA isolation Small-scale preparation of plasmid DNA from E. coli was performed with a Qiagen Plasmid Kit (Qiagen, Chatsworth, CA, USA) or a standard procedure (11). Large-scale plasmid DNA from E. coli was prepared using a standard alkaline lysis protocol (12). Plasmid DNA from A. mediterranei was obtained as follows. S699 strain was cultured in 100 ml of yeast extract–malt extract (YEME) medium and incubated at 25°C, shaking for 72 h at 250 rpm. The culture was centrifuged at 3381×g for 15 min at 4°C. The pellet was washed once, suspended in 10 ml of lysozyme solution (10.3% sucrose, 25 mM Tris–HCl, pH 8.0, 25 mM EDTA, pH 8, and 10 mg/ml lysozyme) and incubated at 30°C for 2 h. Then, 3.5 ml of SDS solution (2% SDS and 0.3 M NaOH) was added and incubated at 80°C for 10 min. After this, the samples were treated according to the protocol of Moretti et al. (13). Total DNA from Vitreoscilla stercoraria was obtained according to the protocol of Holmes and Quigley (11), without the boiling step. RNA isolation Total RNA isolation was performed with RNeasy Mini Kit (Qiagen) by grinding the recommended amounts of mycelium (30 mg per minicolumn) with liquid nitrogen in a frozen mortar and pestle, taking care not to let the mycelium thaw until it was placed in the appropriate lysis buffer supplied with the kit. At this point the manufacturer’s protocol was followed. The RNA was eluted twice in a volume of 50 μl (total) of RNase-free H2O. RNA was treated for DNA contamination by extraction with acid phenol:chloroform (5 : 1 phenol : CHCl3; pH 4.7). DNA was retained in the organic phase and RNA remained in the aqueous phase. RNA was subsequently recovered by precipitation. Samples were aliquoted and stored at −80°C. Polymerase chain reaction (PCR) The vhb gene was obtained by PCR amplification from V. stercoraria genomic DNA using oligonucleotides 5′-ATGTTAGACCAGCAAACCATT-3′ as a forward primer and 5′-TAGGATCCATGCCAAGGCACACCTG AAGA-3′ as a reverse primer, which contained a BamHI site (underlined) located next to the 5′ end of the gene. The promoter PermE was obtained by PCR from the Saccharopolyspora erythraea genomic DNA using the following oligonucleotides: 5′-GCGAAGCTTATGCGAGTGTCCGTTCGAGTG-3′ as a forward primer and 5′-CGCTGGATCCTACCAACCGGC-3′ as a reverse primer. Reverse transcriptase-PCR (RT-PCR) According to the
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manufacturer’s instructions, one-step RT-PCR (Qiagen) was carried out in 50-μl reaction volumes containing 1 μl of template RNA, 0.6 μl of forward and reverse primers (16 and 17.5 μM), 10 μl of 5 ×QIagen one-step buffer (100 mM Tris–HCl, 500 mM KCl, 12.5 mM MgCl2, and pH 8.7), 2 μl of dNTPs mix (10 mM), and 2 μl of Qiagen one-step enzyme mix (reverse transcriptase and HotStart Taq DNA polymerase). Thermocycling was performed as follows: 50°C for 30 min, 95°C for 15 min, then 50 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, followed by 72°C for 10 min. Amplified products were observed after electrophoresis in ethidium bromide-stained agarose gels. β-subunit of RNA polymerase was used as the housekeeping gene. Transformation and screening A. mediterranei Msb strain was made competent, and transformed with pUAMSAG1 by biolistic process (vacuum 20 inches Hg, 6 cm target distance, 900 psi, BioRad M5 tungsten particle). The treated cells were seeded on plates with Bennet agar. After incubation for 6 h at 30°C, the plates were covered with soft agar containing erythromycin (10 μg/ml). Transformants Msb-vhb were selected for their ability to grow in this medium. Measurement of specific oxygen uptake rate Oxygen uptake was measured with a Biological Oxygen Monitor, model 5300 (Yellow Springs Instruments, Yellow Springs, OH, USA), in an air-saturated 0.1 M potassium-phosphate buffer (pH 7.2) at 28°C. The electrode was calibrated with air-equilibrated water. Then the cells were harvested and washed twice by centrifugation at 3381×g for 10 min at 4°C. Of the resulting suspension 0.5 ml was added to 2.5 ml of air-saturated buffer (0.1 M glucose and 20 mM potassium–phosphate buffer, pH 7.2). Biomass concentration was calculated simultaneously by dry weight. The change in oxygen concentration, of the buffer containing the cells, was monitored for 10 min.
RESULTS AND DISCUSSION Rifamycin B biosynthesis by A. mediterranei requires high dissolved oxygen, particularly in industrial processes, where the volumes used can be higher than 120,000 l. Several attempts have been made to develop a process with better oxygen transfer, but no strategy had used the hemoglobin gene from V. stercoraria (or from a different organism) in A. mediterranei. Therefore, with this goal in mind, the vhb gene was cloned, and the vector pUAMSAG1 was constructed. Construction of hemoglobin (VHb) expression vector In order to develop a plasmid-cloning vector with an effective marker gene, the sensitivity of A. mediterranei Msb to kanamycin and erythromycin was tested in a Bennet medium. Although this strain was sensitive to kanamycin (50 μg/ml, the resistance marker (Kmr) could not be used because resistant mutants appeared spontaneously and with high frequency. Since Msb was also sensitive to erythromycin (10 μg/ml), it was chosen as the marker gene. The coding sequence of vhb gene and promoter PermE were generated by PCR amplification, using primers with BamHI and HindIII sites, respectively. The blunt ends of the vhb gene and promoter PermE were ligated and cloned in the pULVK2 vector, which contains pA-rep origin for A. mediterranei and ColE1 for E. coli. Erythromycin-resistant genes (ermE) from pIJ4026 were cloned into the unique EcoRI and XbaI sites to give the plasmid pUAMSAG1. The size of this chimeric product was determined to be about 8.162 kb, which corresponds to the expected size. The con-
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FIG. 1. Construction of pUAMSAG1. The final vector was constructed as shown, using the P ermE promoter region, the vhb gene, kanamycin (Kmr), and erythromycin (ermE) resistant genes, the origin of replication pA-rep for A. mediterranei, and ColE1 for E. coli.
struction scheme of pUAMSAG1 plasmid is shown in Fig. 1. However, A. mediterranei Msb2 strain could not be transformed by conventional procedures (14). Even electroporation of mycelium or protoplasts did not yield any transformant. This inefficient transformation could have been due to a specific characteristic of the Msb2 strain, as the transformation was possible when the S699 strain was used. Thus, it was decided to transform Msb2 strain by a biolistic process. With this procedure, Msb2 strain could successfully be transformed to pUAMSAG1. Although pUAMSAG1 is not a high copy number vector, a good stability was observed at the end of each culture in the absence of selective pressure with erythromycin. This
stability was observed during the entire batch process, including the seed culture. Effect of low aeration With the aim to compare the performance of the strain with the cloned vhb gene in A. mediterranei, rifamycin B production was evaluated. However, because the parental strain (like most industrial strains) requires barbital to produce rifamycin B exclusively, its production was evaluated with and without barbital. The transformants of A. mediterranei, Msb containing pUAMSAG1 (Msb-sag), and the strain with pUAMAE3 (lacking the Vhb expression cassette) were cultivated in shake-flasks under low aeration (as described in the Materials and Methods section). These conditions had already been tested and proved
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FIG. 2. Time course of rifamycin B production (continuous line) and growth (dotted line) by the transformed strain (Msb-sag). Flasks (250 ml) containing 70 ml of the production medium with 0.7 g/l barbital (closed triangles) and without barbital (open triangles) at 26°C, 200 rpm. A control culture of parental strain (Msb) with barbital (closed circles) and without barbital (open circles) is shown.
to provide low aeration (3, 10). The results were compared with the parental Msb strain (Fig. 2). Results showed that under low aeration conditions (70 ml production medium and 200 rpm), rifamycin B production decreased by about 50% in relation to the culture under high aeration conditions (25 ml of production medium, 250 rpm) when the strain was cultivated without barbital. However, when barbital was added to the culture under low aeration conditions, the production of rifamycin B was almost as high as in the culture under high aeration conditions (Fig. 2). Rifamycin B production of the transformant strain, Msb-sag, with barbital was practically the same compared with the parental strain. However, production of Msb-sag did not decrease without barbital. These results suggest that this strain could take up oxygen more efficiently under low aeration conditions, helping to confirm the theory that barbital increases O2 availability for rifamycin B biosynthesis by decreasing oxygen requirements in the respiratory chain (6). It is important to note, that no significant differences were found in the growth; therefore, the difference between specific productions was almost the same. To confirm that higher production of rifamycin B by the transformed strain was due to its ability to consume more oxygen, we simultaneously compared the rates of oxygen uptake between strains. Cultures in the production medium were harvested at 96 h (in idiophase) and oxygen uptake was measured from the washed cells of each strain. Results indicated that the rate of oxygen uptake of the transformed strain (Msb-sag) was about 1.5 times faster than that of the parental strain (Fig. 3). Detection of mRNA by reverse transcriptase (RT-PCR) To confirm that the higher oxygen uptake by the Msb-sag strain was due to the expression of the cloned vhb gene, RTPCR was used. RNA was extracted from idiophase cells of the parental and transformed strains, and RT-PCR experiments were carried out using a primer for a 503 bp fragment
FIG. 3. Measurement of the rate of oxygen uptake. Cell preparations were obtained from parental (Msb) and transformed strain (Msbsag) carrying the cloned vhb gene. Experimental details are given in Materials and Methods. Continuous lines indicate averages of three individual observations.
FIG. 4. RT-PCR analysis. Amplification of a 503-kb fragment from vhb RNA during the idiophase of Msb-sag strain (lane 2) and of Msb strain (lane 1). Nucleotide sequence of the primers is the following: RTvhbF, 5′-GCCACTGTTCCTGTATTGAAGGAGC-3′; RTvhbR, 5′TTTTGGCCAACAGCCAAACTGCTGC-3′.
of the vhb gene. No transcripts in the parental strain (Msb) were detectable (Fig. 4, lane 1), while the transformed strain (Msb-sag) clearly produced the expected fragment, which corresponded to vhb RNA (Fig. 4, lane 2). These results show that VHb expression improved the oxygen uptake and had a beneficial effect on the production of rifamycin B under low aeration conditions, or from another point of view, they indicate that the transformant with the vhb gene does not need barbital to continue production under low aeration conditions. ACKNOWLEDGMENTS The authors wish to thank the Consejo Nacional de Ciencia y Tecnología (CONACyT, México) for financial support (grant SEP2003-C02-44911).
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