Molecular breeding of a biotin-hyperproducing Serratia marcescens strain.

We previously reported that an acidomycin-resistant mutant of Serratia marcescens Sr41, SB304, and a mutant that was derived from SB304 and was resistant to a higher concentration of acidomycin, SB412, produced 5 and 20 mg of D-biotin, respectively, per liter of a medium containing sucrose and urea (N. Sakurai, Y. Imai, M. Masuda, S. Komatsubara, and T. Tosa, Appl. Environ. Microbiol. 59:2857-2863, 1993). In order to increase the productivity of D-biotin, the biotin (bio) operons were cloned from strains SB412, SB304, and 8000 (wild-type strain), and pLGM412, pLGM304, and pLGW101, respectively, were obtained through subcloning. These plasmids harbored 7.2-kb DNA fragments coding for the bioABFCD genes on a low-copy-number vector and were introduced into SB304, SB412, and 8000. Among the resulting recombinant strains, SB412(pLGM304) exhibited the highest D-biotin production (200 mg/liter) in the production medium. The plasmid was stably maintained in cells. Unexpectedly, SB412(pLGM412) grew very slowly, and the D-biotin productivity of this recombinant strain was not evaluated because pLGM412 was unstable.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Repression of Acetyl-Coenzyme A Carboxylase by Unsaturated Fatty Acids: Relationship to Coenzyme Repression

It has been reported that the level of d-biotin in the growth medium of Lactobacillus plantarum regulates the synthesis of apoacetyl-coenzyme A (CoA) carboxylase; high levels cause repression, and deficient levels effect derepression. In this study, evidence has been obtained which suggests that coenzyme repression by biotin is an indirect effect; i.e., biotin regulates the synthesis of unsaturated fatty acids which are the true repressors of the acetyl-CoA carboxylase. This was observed in an experiment in which long-chain unsaturated fatty acids were added to media containing deficient, sufficient, or excess levels of d-biotin. In every case, independently of the biotin concentration for growth, the unsaturated fatty acids caused a severe repression of the carboxylase. Saturated fatty acids were without effect. The level of oleic acid required to give maximal repression was 50 mug/ml. The free fatty acids had no adverse effect on the activity of the cell-free extracts nor on the permeation of d-biotin into the cell. Saturated and unsaturated fatty acids decreased the rate of holocarboxylase formation from d-biotin and the apoacetyl-CoA carboxylase in the extracts. It is concluded that there are at least three mechanisms that control the acetyl-CoA carboxylase in this organism: (i) indirect coenzyme repression by d-biotin, (ii) repression by unsaturated fatty acids, and (iii) regulation of the activity of the holocarboxylase synthetase by both saturated and unsaturated fatty acids.     

Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutants

Pseudomonas sp. Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate. The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture. One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells. Among the mutants, strain BA+63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h. The conversion rate of benzoate to catechol was 50% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation. The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.     

Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2

We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain. The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin. Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain. Complementation analysis with B. subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not. A bio(+) transformant, isolated from an OK2 strain, has biotin autotrophy.     

Induction of nutritional mutants ofMicrococcus glutamicus and their amino acid accumulation

Micrococcus glutamicus ATCC 13032, a glutamic acid-producing organism, was treated with 0.2M ethylmethane sulfonate, the auxotrophs isolated showing varied patterns of extracellular amino acids. Eighty auxotrophic strains were obtained, out of which 31 excreted 1.0-4.0 mg threonine per ml and all the auxotrophs required biotin for growth and production of the amino acid. Eleven auxotrophs produced 1.5 to 3.0 mg alanine per ml and these auxotrophs required amino acids for their growth. Other auxotrophs lost their excretion capacity in subsequent fermentation trials. Further mutation of the biotin-requiring auxotroph Micrococcus glutamicus EM with gamma rays resulted in the isolation of 89 auxotrophic strains, out of which 28 excreted threonine (up to 5.0 mg per ml) higher than the parent auxotroph. Exposure to X-rays yielded 97 auxotrophs, out of these 35 producing 1.0-3.0 mg methionine per ml and requiring biotin for growth and production of the amino acid. Other auxotrophs produced alanine (0.5 to 2.0 mg per ml) and threonine (2.0 to 3.3 mg per ml). Irradiation with gamma rays favoured the development of threonine producing auxotrophs while X-rays favoured methionine-producing auxotrophs.     

Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli.

The technique developed by Casadaban (M. J. Casadaban, J. Mol. Biol. 104: 541-555, 1976) has been employed to construct Escherichia coli K-12 derivatives in which the genes determining lactose utilization are fused to the regulatory region of the biotin operon. Fusions of the lac genes to either arm of this divergently transcribed operon have been isolated. When the operon is derepressed, expression of the lac genes is sufficient to permit growth on lactose minimal medium. Repressing conditions prevent growth on lactose. This property of bio-lac fusion strains, as well as the ease of determining the level of operon expression by assaying beta-galactosidase, was used for the isolation and characterization of mutants defective in repression. Preliminary analyses of several newly isolated regulatory mutants are presented. For the several birA mutants examined, there appeared to be no direct correlation between effects on minimum biotin requirement and alterations in repressibility, suggesting a possible dual function for the gene. Parallel attempts to obtain fusions of lac to bioH were unsuccessful, indicating lack of direct biotin control at the bioH locus.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.     

Increase in arginine and citrulline production by 6-azauracil-resistant mutants of Bacillus subtilis.

In the arginine producer AHr-5, an L-arginine hydroxamate-resistant mutant of Bacillus subtilis, accumulation of N8-acetyl-L-ornithine increased as the level of L-arginine accumulation increased in the medium containing L-glutamic acid. Ornithine carbamoyltransferase of this strain was genetically derepressed. These results suggested that carbamoylphosphate might be deficient in vivo. With the intention to increase endogenous carbamoylphosphate, pyrimidine analogs inhibiting growth were selected and the mutants resistant to these compounds were derived from the AHr-5 mutant. Of the resistant mutants derived, the 6-azauracil-resistant mutant AAr-9 produced 28 mg of L-arginine per ml, which corresponded to more than twofold the amount produced by the parent strain. Derivation of an arginine-requiring mutant from the double-resistant mutant AAr-9 provides a new advantageous method for the production of L-citrulline. The increase in arginine and citrulline production is discussed.     

Improvement of an l-Leucine-Producing Mutant of Brevibacterium lactofermentum 2256 by Genetically Desensitizing It to alpha-Acetohydroxy Acid Synthetase

Genetic improvement of l-leucine productivity in strain 218, an ile 2-thiazolealanine-resistant mutant of Brevibacterium lactofermentum 2256, was attempted. In strain 218, which produced 28 mg of l-leucine per ml from 13% glucose, alpha-isopropylmalate synthetase was genetically desensitized and derepressed to the effect of l-leucine, whereas alpha-acetohydroxy acid synthetase remained unaltered, although it could be derepressed phenotypically by limiting the isoleucine concentration in the culture. From strain 218 we isolated 103 mutants resistant to beta-hydroxyleucine (4 mg/ml). Among these, three were found to produce mere l-leucine than the parent. The alpha-acetohydroxy acid synthetase of all three mutant strains was found to be genetically desensitized to all of the branched-chain amino acids l-isoleucine, l-valine, and l-leucine. The repression mechanism in alpha-acetohydroxy acid synthetase formation was the same as in the parent strain. The improved strains typically produced 34 mg of l-leucine per ml, the highest productivity ever reported.     

Analysis of Ribosomes from Viomycin-Sensitive and -Resistant Strains of Mycobacterium smegmatis

Viomycin-resistant strains were isolated from Mycobacterium smegmatis. Ribosomes were isolated and tested for drug resistance in subcellular systems containing poly(U) as messenger ribonucleic acid. Resistance to viomycin in these strains was due to altered ribosomes. Further analysis showed that viomycin resistance of two mutants with low level resistance (20 mug/ml) was due to altered 30S ribosomal subunits. Another mutant that was highly resistant to viomycin (1 mg/ml), however, had altered 50S ribosomal subunits.     

The Controlling Action of Actithiazic Acid on the Biosynthesis of Biotin-vitamers by Various Microorganisms

By the addition of actithiazic acid, or acidomycin (ACM), to culture media, the accumulation of desthiobiotin by various microorganisms was enhanced from two-fold to about seventyfold, while that of biotin was markedly reduced. Especially, Bacillus sphaericus accumulated 350 μg per ml of biotin-vitamers assayed with Saccharomyces cerevisiae. ACM was not incorporated into the desthiobiotin molecule by resting cells of B. sphaericus. The amount of biotin-vitamers assayed with S. cerevisiae which was synthesized from pimelic acid by the resting cells grown with ACM was twice as great as that synthesized by the cells grown without ACM. From these results, the mechanism of the controlling action of ACM on biotin biosynthesis was discussed.     

Threonine production by ethionine-resistant mutants of Serratia marcescens.

Ethionine reduced both the growth rate and the final growth level of Serratia marcescens Sr41. Growth inhibition was completely reversed by methionine. Strain D-315, defective in homoserine dehydrogenase I, was more sensitive to ethionine-mediated growth inhibition than was the wild-type strain. Ethionine-resistant mutants were isolated from cultures of strain D-316, which was derived from strain D-315 as a threonine deaminase-deficient mutant. Of 60 resistant colonies, 7 excreted threonine on minimal agar plates. One threonine-excreting strain, ETr17, was highly resistant to ethionine and, moreover, insensitive to methionine-mediated growth inhibition, whereas the parent strain was sensitive. When cultured in minimal medium with or without excess methionine, strain ETr17 had a higher homoserine dehydrogenase level than did strain D-316. The homoserine dehydrogenase activity was not inhibited by threonine or methionine. Transductional analysis revealed that the ethionine-resistant (etr-1) mutation carried by strain ETr17 was located in the metBM-argE region and caused the derepressed synthesis of homoserine dehydrogenase II. Strain ETr17 had a higher aspartokinase level than did the parent strain. By transductional cross with the argE+ marker, the etr-1 mutation was transferred into strain D-562 which was derived from D-505, a strain defective in aspartokinases I and III. The constructed strain had a higher aspartokinase level than did strain D-505 in medium with or without excess methionine, indicating that the etr-1 mutation led to the derepressed synthesis of aspartokinase II. Strain ETr17 produced about 8 mg of threonine per ml of medium containing sucrose and urea.     

Synthesis of Desthiobiotin from 7,8-Diaminopel-argonic Acid in Biotin Auxotrophs of Escherichia coli K-12

The synthesis of desthiobiotin from 7,8-diaminopelargonic acid (DAP) was demonstrated in resting cell suspensions of Escherichia coli K-12 bioA mutants under conditions in which the biotin locus was derepressed. The biosynthetically formed desthiobiotin was identified by chromatography, electrophoresis, and by its ability to support the growth of yeast and those E. coli biotin auxotrophs that are blocked earlier in the biotin pathway. Optimal conditions for desthiobiotin synthesis were determined. Desthiobiotin synthetase activity was repressed 67% when partially derepressed resting cells were incubated in the presence of 3 ng of biotin per ml. Serine, bicarbonate, and glucose stimulated desthiobiotin synthesis apparently by acting as sources of CO(2). The results of this study are consistent with an earlier postulated pathway for biotin biosynthesis in E. coli: pimelic acid --> 7-oxo-8-aminopelargonic acid --> DAP --> desthiobiotin --> biotin.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

L-Histidine production by histidase-less regulatory mutants of Serratia marcescens constructed by transduction.

2-Methylhistidine (2MH) and 1,2,4-triazole-3-alanine (TRA) inhibited the growth of Serratia marcescens. These inhibitory effects were counteracted by L-histidine. Enzymatic studies showed that 2MH acts as a false feedback inhibitor and TRA acts as both a false feedback inhibitor and a repressor. Mutants resistant to each analog were isolated from a histidase-less mutant, because the wild-type strain possesses a potent histidase activity. 2MH-resistant mutants had a feedback-insensitive phosphoribosyltransferase, but they produced only small amounts of L-histidine. TRA-resistant mutants were divided into two types according to their histidine productivity. A mutant of one type produced about 8 mg of L-histidine per ml and had about a 10-fold increase in the enzyme levels of histidine biosynthesis. Moreover, this mutant had a partially feedback-insensitive phosphoribosyltransferase. A mutant of the second type produced only a small amount of L-histidine and had only derepressed enzyme levels. Accordingly, strains possessing the genetic alterations in both 2MH- and TRA-resistant mutants were constructed by PS20-mediated transduction. They had both feedback-insensitive phosphoribosyltransferase and derepressed enzyme levels. The representative strain HT-2604 produced about 17 mg of L-histidine per ml.     

Cloning the gyrA gene of Bacillus subtilis.

We have isolated an eight kilobase fragment of Bacillus subtilis DNA by specific integration and excision of a plasmid containing a sequence adjacent to ribosomal operon rrn O. The genetic locus of the cloned fragment was verified by linkage of the integrated vector to nearby genetic markers using both transduction and transformation. Functional gyrA activity encoded by this fragment complements E. coli gyrA mutants. Recombination between the Bacillus sequences and the E. coli chromosome did not occur. The Bacillus wild type gyrA gene, which confers sensitivity to nalidixic acid, is dominant in E. coli as is the E. coli gene. The cloned DNA precisely defines the physical location of the gyrA mutation on the B. subtilis chromosome. Since an analogous fragment from a nalidixic acid resistant strain has also been isolated, and shown to transform B. subtilis to nalidixic acid resistance, both alleles have been cloned.