263
Biotin
S-8-6
Production DNA
O .IFUKU,
Shiseido
Research
Center,
S.HAZE,
by
Using
Recombinant
Technology J. KISHIMOTO,
1050, Nippa-cho,
and
Kohoku-ku,
M.YANAGI
Yokohama
223 (Japan)
I.
INTRODUCTION Biotin, or vitamin H is one of B-group vitamins and an essential component for animals, plants and many microorganisms. Until now biotin has been commercially produced only by chemical synthetic methods. However, these procedures are so complicated that biochemical methods have been sought for a long time , since biotin is one of the most expensive vitamins. We have tried to establish the bio-production of biotin by using genetic engineering. We chose an Escherichia coli as a host bacterial strain because of its extremely well known genetic information. In the genetic map of E.coli it is known that the biotin operon at min 17 .5, which consists of a cluster of 5 genes (bioA, B,F ,C,D), and bioH gene at min 75 are concerned in the biotin biosynthesis. B1rA gene at min 85 is also known as a regulator gene for the biotin biosynthesis. Within the biotin biosynthetic pathway in E.coli, three enzymes, 7-Keto-8-aminopelargonic acid synthetase , 7,8-Diaminopelargonic acid aminotransferase and Dethiobiotin(DTB) synthetase are coded by bioF, bioA and bioD respectively, have already been clarified . It is suggested that two genes bioC and bioH, whose function still remains unclear , serve the reaction f or the formation of Pimeloyl-CoA. Also an enzyme coded by bioB is believed to catalyze the conversion of DTB to biotin, the last step of biotin biosynthesis . It is well known that biotin itself acts to tightly regulate biotin biosynthesis by a mechanism of feedback repression. The protein coded by birA , which directly acts as the biotin repressor in this mechanism, has mainly two different functions . That is, the repressor protein has both the repressor activity and the holoenzyme synthetase activity. Because of the later function, complete defect of birA gene is lethal for microorganisms.
II.
PRINCIPLE OF BREEDING OF BIOTIN-HYPERPRODUCING STRAINS We succeeded in obtaining biotin-hyperproducing strains by two different techniques, a chemical mutagenesis and a recombinant DNA technique . As I mentioned above, it is necessary for the biotin bio-production to remove the strong feedback repression by biotin. That is, first of all we obtained derepression mutants as a host strain by mutagenesis . We also obtained the biotin operon from chromosomal DNA in E .coli, then it was inserted into a extrachromosomal DNA, called a plasmid. The resultant recombinant plasmid is generally multiplied up to several dozens of copies when it is introduced into a E.coli host cell (derepression mutant). This multiplication , called "gene dosage effect" results in over expression of biotin operon and is expected to cause biotin overproduction. To shorten a complicated procedure , the breeding of a bi otin-hyperproducing strain was accomplished by the combination of these two techniques.
III.
SCREENING OF DEREPRESSION MUTANTS Pai's procedure 1 1 was applied for screening derepression mutants . Strain W3110, a wild strain of E.coli K-12, was mutated by treatment with N-methyl-N'-
Symposium
264
(8) Biotechnology
nitro-N-nitrosoguanidine(NTG) and then plated on selection agar plates . These plates consist of two biotin-free agar medium layers, the bottom layer containing strain JSO4, a biotin-requiring mutant lacking bioB, therefore only if the mutated colony produces a larger amount of biotin by releasing the repression , can the JSO4 strain just under this colony grow and form a growing spot . In this way we screened and obtained derepression mutants . DRK332 strain, one of these mutants produced more than 1000 times as much biotin as the wild strain, however, the level of its accumulation was still quite low, just about several scores Atgil . The biotin concentration was quantitatively measured by microbiological assay with Lactobacillus plantarum. We assume the mutated chromosomal region of the DRK332 strain is within the operator-binding domain of the birA protein which has mainly two different functions reported by Eisenberg [2]. We also confirmed this mutation is not referred to the operator region itself by sequencing the regulatory region of the biotin operon.
N.
CLONING OF BIOTIN OPERON We directly cloned the biotin operon from the chromosomal DNA of the derepressed mutant DRK332 by using biotin auxotroph JSO4 as a host strain . In this process we realized that a plasmid containing the biotin operon was highly unstable in terms of gene deletion. We finally developed a stable plasmid by eliminating the entire uvrB promotor region including a 9 by inverted repeat sequence, which accidentally located downstream of the biotin operon in E . coli. van den Berg et al. [3] suggest its repeat sequence has extensive homology to recognition sites for the dnaA protein which regulates initiation of DNA replication, and the presence of the putative DnaA box in the regulatory region of uvrB might be related to the instability of the uvrB gene on a high-copy plasmid . Therefore, we think our result must be the same phenomenon as van den Berg mentioned. We constructed a tetracycline resistant plasmid pXBA312 containing the complete biotin operon and sequenced the complete biotin operon region with the plasmid by DNA chain termination techniques. Since the sequence of the biotin regulatory region was identical to Otsuka's report [4] (corrected part of it by Barker [5]), we assume our isolated derepression mutant DRK332 is not an operator mutant but a repressor one. We also constructed several plasmids in which we deleted each bio gene within the biotin operon and transformed the derepression mutant(DRK332) with each plasmid. It is clear that strains holding the bioC- or bioB plasmid have poor biotin productivity, and this indicates that it is the early and last steps of the biotin biosynthetic pathway that mainly contribute to the biotin biosynthesis . V.
CLONING OF BIOH GENE We also cloned and sequenced the bioH gene which is mapped for the biotin biosynthesis besides the biotin operon in E.coli. We found no homology of the bioH promotor region with the regulatory region of the biotin operon. This suggests the expression of the bioH gene is not controlled by the same regulation as the biotin operon, in other words, is not regulated by feedback repression with biotin. We introduced this bioH fragment to several plasmids containing the biotin operon and also obtained recombinant DRK332 strains transformed with these plasmids. Contrary to our expectation, the biotin productivity of all these recombinant strains in which the bioH gene had been introduced were reduced . This result may suggest something about the pimeloyl-CoA biosynthesis , however, we concluded that over expression of the bioH gene results in reduction of the biotin precursor(s) in spite of its obligation for the biotin biosynthesis . Therefore we decided to use only the biotin operon for the biotin bio-production .
O. IFUKU
et
al.
265
VI.
DEVELOPMENT OF HIGH EXPRESSION SYSTEMS FOR THE BIOTIN OPERON In order to develop the high expression systems for the biotin operon, we first screened biotin analog-resistant mutants from one of derepression mutants by treatment with NTG. We chose two biotin analogs, Actithiazic acid(ACM) and 5-(2-Thienyl)-Valeric acid(TVA). Biotin analogs compete with biotin in the microorganism, therefore the parent strain usually can not grow on the agar medium containing these analogs. On the other hand, resistant strains are expected to produce higher amounts of biotin, enough to overcome these analogs. We obtained 8 analog-resistant mutants which clearly demonstrated the increase of biotin productivity compared with the parent strain. To specify the mutated region we cloned the biotin operons from the chromosomal DNA of all these mutants and analyzed by sequence. All mutation points were found in or near the regulatory region of the biotin operon and were classified into three groups(Fig 1). Only one strain showed no difference from the wild type sequence. We suppose that it has a different mutation excluding the biotin operon region . The presumptive operator region of the biotin operon can form the extremely large imperfect palindrome structure around a base of position 1, and bi-directional transcription is repressed sterically by binding of the repressor to this operator region. Type 9 mutation makes its secondary structure unstable and leads to activation of transcription in terms of releasing the repression . Moreover, this mutation point is within the pribnow box, which is a functional sequence to be bound by RNA polymerase for the rightward transcription , and elevates the rightward promoter activity. Therefore , we expect the type 9 mutation to have double effects to activate transcription. We assumed the region including the mutation points of type 7 and type 6 forms very stable secondary structure by computer analysis. These mutations could break down its structure and open the putative ribosome binding site of the bioB gene, hence these types of mutation might contribute to activate translation rather than transcription . Although the type 6 mutation is located within the bioB structural gene, we do not suppose the. ino acid transition from alanine to threonine by this mutation influences the protein stability and the enzyme activity of the bioB product . We have just started to consider for application of these mutated DNAs for biotin production. Up to now the utilization of these DNAs on high-copy plasmid vector has been unsuccessful because we have a problem of transformant instability. We assume this may result from over expression of the biotin operon . We have now tried to obtain a stable transformant by means of low copy vector plasmid.
Fig. 1. Mutations in the regulatory region of biotin operon Lines above and below the DNA sequence delineate the imperfect palindrome . Sequences that form a secondary structure of RNA are underlined by dotted and the first AUG sequences in each RNA are enclosed in boxes.
lines
Symposium
266
(8)Biotechnology
VII. BIO-PRODUCTION OF BIOTIN A recombinant strain DRK332/pXBA312 was cultivated in batch culture in a 51 mini-jar fermentor. The medium used consisted of phosphate, ammonium sulfate, yeast extract, peptone, alanine and so on [6] . The result indicated that biotin production depends on the cell growth, that is, biotin is no longer produced after the logarithmic growth phase. Hence we adopted fed-batch culture in order to keep the growth activity higher until high-cell density. We fed glucose with DO-stat as a carbon source and ammonia with pH-stat as an inorganic nitrogen source. We kept dissolved oxygen above 3ppm during fermentation by supplying pure oxygen gas or oxygen gas mixed with air. Concentration of glucose in the fermentation medium was kept below lgn during the feeding period following the initial consumption of glucose (approximately 4 hours after cultivation started). After 24 hours of cultivation, the dry cell weight was 27g/l and accumulated biotin reached 42m/l. However, despite the fact that nutrient and the concentration of dissolved oxygen were sufficient for cell growth, the cell growth rate began to decrease after 12 hours of cultivation. After investigation we found a large amount of acetate(approximately 18g//) in the culture broth as by products, it was a sufficient level to completely inhibit the growth of E. coli. To avoid this problem, we tried to block the pathway from Acetyl-CoA to Acetate through Acetyl-phosphate, which is a major biosynthetic pathway of acetate. As its reaction is reversible we screened resistant mutants against fluoro acetate, which is a halogen analog of acetate, and obtained a low-acetate producing mutant strain DRK3323. The acetate productivity of this mutant was greatly diminished, less than one tenth the productivity of the parent strain(DRK332). Recombinant strain DRK3323/pXBA312 showed apparent improvement on both cell growth activity and biotin productivity in the fed-batch culture with the same fermentation system. Accumulated acetate and dry cell weight were 6g/l and 55g/l respectively, and accumulated biotin reached to 105mg// after 24 hours of cultivation.
VIII.
FINALLY
We summarized the traditional mutagenesis Until commercial
now
we have production
good place conventional lead
to
industrial
bio-production and recent not succeeded of biotin or
to start, techniques success
and
we and
of genetic
biotin by engineering
in applying this useful other vitamins. However,
hope the co-operation new technology, such
of biotin
recombinant techniques.
or
other
vitamin
as
E.coli
using
new technology our attempt
and integration genetic engineering,
production
in the
near
both
for the may be a between can future.
REFERENCES [1] Pai, C. H. (1972): J. Bacteriol., 112, 1280-1287. [2] Eisenberg, M. A., Prakash, O. and Hsiung, S-C. (1982): J. Biol. Chem., 257, 15167-15173. [3] van den Berg, E. A., Geerse, R. H., Memelink, J., Bovenberg, R. A. L., Magnee, F. A. and van de Putte, O. (1985): Nucleic Acid Res., 13, 1829-1840. [4] Otsuka, A. and Abelson, J. (1978): Nature, 276, 689-694. [5] Barker, D. F., Kuhn, J., and Campbell, A. M. (1981): Gene, 13, 89-102. [6] European Patent Application Publn, No.0316229
Biotin
S-8-6
Production DNA
O .IFUKU,
Shiseido
Research
Center,
S.HAZE,
by
Using
Recombinant
Technology J. KISHIMOTO,
1050, Nippa-cho,
and
Kohoku-ku,
M.YANAGI
Yokohama
223 (Japan)
I.
INTRODUCTION Biotin, or vitamin H is one of B-group vitamins and an essential component for animals, plants and many microorganisms. Until now biotin has been commercially produced only by chemical synthetic methods. However, these procedures are so complicated that biochemical methods have been sought for a long time , since biotin is one of the most expensive vitamins. We have tried to establish the bio-production of biotin by using genetic engineering. We chose an Escherichia coli as a host bacterial strain because of its extremely well known genetic information. In the genetic map of E.coli it is known that the biotin operon at min 17 .5, which consists of a cluster of 5 genes (bioA, B,F ,C,D), and bioH gene at min 75 are concerned in the biotin biosynthesis. B1rA gene at min 85 is also known as a regulator gene for the biotin biosynthesis. Within the biotin biosynthetic pathway in E.coli, three enzymes, 7-Keto-8-aminopelargonic acid synthetase , 7,8-Diaminopelargonic acid aminotransferase and Dethiobiotin(DTB) synthetase are coded by bioF, bioA and bioD respectively, have already been clarified . It is suggested that two genes bioC and bioH, whose function still remains unclear , serve the reaction f or the formation of Pimeloyl-CoA. Also an enzyme coded by bioB is believed to catalyze the conversion of DTB to biotin, the last step of biotin biosynthesis . It is well known that biotin itself acts to tightly regulate biotin biosynthesis by a mechanism of feedback repression. The protein coded by birA , which directly acts as the biotin repressor in this mechanism, has mainly two different functions . That is, the repressor protein has both the repressor activity and the holoenzyme synthetase activity. Because of the later function, complete defect of birA gene is lethal for microorganisms.
II.
PRINCIPLE OF BREEDING OF BIOTIN-HYPERPRODUCING STRAINS We succeeded in obtaining biotin-hyperproducing strains by two different techniques, a chemical mutagenesis and a recombinant DNA technique . As I mentioned above, it is necessary for the biotin bio-production to remove the strong feedback repression by biotin. That is, first of all we obtained derepression mutants as a host strain by mutagenesis . We also obtained the biotin operon from chromosomal DNA in E .coli, then it was inserted into a extrachromosomal DNA, called a plasmid. The resultant recombinant plasmid is generally multiplied up to several dozens of copies when it is introduced into a E.coli host cell (derepression mutant). This multiplication , called "gene dosage effect" results in over expression of biotin operon and is expected to cause biotin overproduction. To shorten a complicated procedure , the breeding of a bi otin-hyperproducing strain was accomplished by the combination of these two techniques.
III.
SCREENING OF DEREPRESSION MUTANTS Pai's procedure 1 1 was applied for screening derepression mutants . Strain W3110, a wild strain of E.coli K-12, was mutated by treatment with N-methyl-N'-
Symposium
264
(8) Biotechnology
nitro-N-nitrosoguanidine(NTG) and then plated on selection agar plates . These plates consist of two biotin-free agar medium layers, the bottom layer containing strain JSO4, a biotin-requiring mutant lacking bioB, therefore only if the mutated colony produces a larger amount of biotin by releasing the repression , can the JSO4 strain just under this colony grow and form a growing spot . In this way we screened and obtained derepression mutants . DRK332 strain, one of these mutants produced more than 1000 times as much biotin as the wild strain, however, the level of its accumulation was still quite low, just about several scores Atgil . The biotin concentration was quantitatively measured by microbiological assay with Lactobacillus plantarum. We assume the mutated chromosomal region of the DRK332 strain is within the operator-binding domain of the birA protein which has mainly two different functions reported by Eisenberg [2]. We also confirmed this mutation is not referred to the operator region itself by sequencing the regulatory region of the biotin operon.
N.
CLONING OF BIOTIN OPERON We directly cloned the biotin operon from the chromosomal DNA of the derepressed mutant DRK332 by using biotin auxotroph JSO4 as a host strain . In this process we realized that a plasmid containing the biotin operon was highly unstable in terms of gene deletion. We finally developed a stable plasmid by eliminating the entire uvrB promotor region including a 9 by inverted repeat sequence, which accidentally located downstream of the biotin operon in E . coli. van den Berg et al. [3] suggest its repeat sequence has extensive homology to recognition sites for the dnaA protein which regulates initiation of DNA replication, and the presence of the putative DnaA box in the regulatory region of uvrB might be related to the instability of the uvrB gene on a high-copy plasmid . Therefore, we think our result must be the same phenomenon as van den Berg mentioned. We constructed a tetracycline resistant plasmid pXBA312 containing the complete biotin operon and sequenced the complete biotin operon region with the plasmid by DNA chain termination techniques. Since the sequence of the biotin regulatory region was identical to Otsuka's report [4] (corrected part of it by Barker [5]), we assume our isolated derepression mutant DRK332 is not an operator mutant but a repressor one. We also constructed several plasmids in which we deleted each bio gene within the biotin operon and transformed the derepression mutant(DRK332) with each plasmid. It is clear that strains holding the bioC- or bioB plasmid have poor biotin productivity, and this indicates that it is the early and last steps of the biotin biosynthetic pathway that mainly contribute to the biotin biosynthesis . V.
CLONING OF BIOH GENE We also cloned and sequenced the bioH gene which is mapped for the biotin biosynthesis besides the biotin operon in E.coli. We found no homology of the bioH promotor region with the regulatory region of the biotin operon. This suggests the expression of the bioH gene is not controlled by the same regulation as the biotin operon, in other words, is not regulated by feedback repression with biotin. We introduced this bioH fragment to several plasmids containing the biotin operon and also obtained recombinant DRK332 strains transformed with these plasmids. Contrary to our expectation, the biotin productivity of all these recombinant strains in which the bioH gene had been introduced were reduced . This result may suggest something about the pimeloyl-CoA biosynthesis , however, we concluded that over expression of the bioH gene results in reduction of the biotin precursor(s) in spite of its obligation for the biotin biosynthesis . Therefore we decided to use only the biotin operon for the biotin bio-production .
O. IFUKU
et
al.
265
VI.
DEVELOPMENT OF HIGH EXPRESSION SYSTEMS FOR THE BIOTIN OPERON In order to develop the high expression systems for the biotin operon, we first screened biotin analog-resistant mutants from one of derepression mutants by treatment with NTG. We chose two biotin analogs, Actithiazic acid(ACM) and 5-(2-Thienyl)-Valeric acid(TVA). Biotin analogs compete with biotin in the microorganism, therefore the parent strain usually can not grow on the agar medium containing these analogs. On the other hand, resistant strains are expected to produce higher amounts of biotin, enough to overcome these analogs. We obtained 8 analog-resistant mutants which clearly demonstrated the increase of biotin productivity compared with the parent strain. To specify the mutated region we cloned the biotin operons from the chromosomal DNA of all these mutants and analyzed by sequence. All mutation points were found in or near the regulatory region of the biotin operon and were classified into three groups(Fig 1). Only one strain showed no difference from the wild type sequence. We suppose that it has a different mutation excluding the biotin operon region . The presumptive operator region of the biotin operon can form the extremely large imperfect palindrome structure around a base of position 1, and bi-directional transcription is repressed sterically by binding of the repressor to this operator region. Type 9 mutation makes its secondary structure unstable and leads to activation of transcription in terms of releasing the repression . Moreover, this mutation point is within the pribnow box, which is a functional sequence to be bound by RNA polymerase for the rightward transcription , and elevates the rightward promoter activity. Therefore , we expect the type 9 mutation to have double effects to activate transcription. We assumed the region including the mutation points of type 7 and type 6 forms very stable secondary structure by computer analysis. These mutations could break down its structure and open the putative ribosome binding site of the bioB gene, hence these types of mutation might contribute to activate translation rather than transcription . Although the type 6 mutation is located within the bioB structural gene, we do not suppose the. ino acid transition from alanine to threonine by this mutation influences the protein stability and the enzyme activity of the bioB product . We have just started to consider for application of these mutated DNAs for biotin production. Up to now the utilization of these DNAs on high-copy plasmid vector has been unsuccessful because we have a problem of transformant instability. We assume this may result from over expression of the biotin operon . We have now tried to obtain a stable transformant by means of low copy vector plasmid.
Fig. 1. Mutations in the regulatory region of biotin operon Lines above and below the DNA sequence delineate the imperfect palindrome . Sequences that form a secondary structure of RNA are underlined by dotted and the first AUG sequences in each RNA are enclosed in boxes.
lines
Symposium
266
(8)Biotechnology
VII. BIO-PRODUCTION OF BIOTIN A recombinant strain DRK332/pXBA312 was cultivated in batch culture in a 51 mini-jar fermentor. The medium used consisted of phosphate, ammonium sulfate, yeast extract, peptone, alanine and so on [6] . The result indicated that biotin production depends on the cell growth, that is, biotin is no longer produced after the logarithmic growth phase. Hence we adopted fed-batch culture in order to keep the growth activity higher until high-cell density. We fed glucose with DO-stat as a carbon source and ammonia with pH-stat as an inorganic nitrogen source. We kept dissolved oxygen above 3ppm during fermentation by supplying pure oxygen gas or oxygen gas mixed with air. Concentration of glucose in the fermentation medium was kept below lgn during the feeding period following the initial consumption of glucose (approximately 4 hours after cultivation started). After 24 hours of cultivation, the dry cell weight was 27g/l and accumulated biotin reached 42m/l. However, despite the fact that nutrient and the concentration of dissolved oxygen were sufficient for cell growth, the cell growth rate began to decrease after 12 hours of cultivation. After investigation we found a large amount of acetate(approximately 18g//) in the culture broth as by products, it was a sufficient level to completely inhibit the growth of E. coli. To avoid this problem, we tried to block the pathway from Acetyl-CoA to Acetate through Acetyl-phosphate, which is a major biosynthetic pathway of acetate. As its reaction is reversible we screened resistant mutants against fluoro acetate, which is a halogen analog of acetate, and obtained a low-acetate producing mutant strain DRK3323. The acetate productivity of this mutant was greatly diminished, less than one tenth the productivity of the parent strain(DRK332). Recombinant strain DRK3323/pXBA312 showed apparent improvement on both cell growth activity and biotin productivity in the fed-batch culture with the same fermentation system. Accumulated acetate and dry cell weight were 6g/l and 55g/l respectively, and accumulated biotin reached to 105mg// after 24 hours of cultivation.
VIII.
FINALLY
We summarized the traditional mutagenesis Until commercial
now
we have production
good place conventional lead
to
industrial
bio-production and recent not succeeded of biotin or
to start, techniques success
and
we and
of genetic
biotin by engineering
in applying this useful other vitamins. However,
hope the co-operation new technology, such
of biotin
recombinant techniques.
or
other
vitamin
as
E.coli
using
new technology our attempt
and integration genetic engineering,
production
in the
near
both
for the may be a between can future.
REFERENCES [1] Pai, C. H. (1972): J. Bacteriol., 112, 1280-1287. [2] Eisenberg, M. A., Prakash, O. and Hsiung, S-C. (1982): J. Biol. Chem., 257, 15167-15173. [3] van den Berg, E. A., Geerse, R. H., Memelink, J., Bovenberg, R. A. L., Magnee, F. A. and van de Putte, O. (1985): Nucleic Acid Res., 13, 1829-1840. [4] Otsuka, A. and Abelson, J. (1978): Nature, 276, 689-694. [5] Barker, D. F., Kuhn, J., and Campbell, A. M. (1981): Gene, 13, 89-102. [6] European Patent Application Publn, No.0316229
