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.     

Increase in Isoleucine Accumulation by α-Aminobutyric Acid-Resistant Mutants of Serratia marcescens

Several alpha-aminobutyric acid-resistant (Abu-r) mutants of Serratia marcescens were found to be superior to the parent strain in converting d-threonine to l-isoleucine. One of them accumulated 1.5 times more l-isoleucine that the parent strain. The level of acetohydroxy acid (AHA) synthetase in this mutant increased twofold above that of the parent strain. In the parent strain, AHA synthetase was repressed and l-isoleucine accumulation was decreased by either l-valine or l-leucine, whereas in the mutant the AHA synthetase level and l-isoleucine accumulation were not affected by these amino acids. AHA synthetase of the Abu-r mutant was feedback-inhibited by l-valine to the same extent as that of the parent strain. The level of d-threonine dehydratase in both strains was only slightly affected by several amino acids tested. l-Threonine dehydratase of the parent strain and of the mutant was almost completely inhibited by l-isoleucine. These results indicate that the increase in l-isoleucine accumulation by Abu-r mutants is due to the genetic derepression of AHA synthetase.     

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived from Brevibacterium lactofermentum 2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256.

In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered.

The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.     

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.     

Role of Histidine Transfer Ribonucleic Acid in Regulation of Synthesis of Histidyl-Transfer Ribonucleic Acid Synthetase of Salmonella typhimurium

The role of histidine transfer ribonucleic acid (tRNA) in repression of synthesis of histidyl-tRNA synthetase was examined in two strains of Salmonella typhimurium, one of which was a histidine tRNA (hisR) mutant possessing 52% of the wild-type (hisR(+)) histidine tRNA and a derepressed level of the histidine biosynthetic enzymes during histidine-unrestricted growth. Histidine-restricted growth caused a derepression of the rate of formation of histidyl-tRNA synthetase in both strains. In the case of the wild-type strain, addition of histidine to the derepressed culture caused a repression of synthesis of histidyl-tRNA synthetase for at least one generation of growth. In contrast, when histidine was restored to the derepressed hisR mutant culture, synthesis of histidyl-tRNA synthetase was continued at the initial derepressed rate. These results suggest that histidine must be attached to histidine tRNA for repression of synthesis of histidyl-tRNA synthetase.     

Valine accumulation by alpha-aminobutyric acid-resistant mutants of Serratia marcescens

alpha-Aminobutyric acid, norvaline, and norleucine, which are analogues of branched-chain amino acids, inhibited the growth of Serratia marcescens. The inhibitory effect of these three analogues was counteracted by branched-chain amino acids. A number of mutants resistant to these analogues were isolated. alpha-Aminobutyric acid-resistant (abu-r) mutants markedly accumulated l-valine in the culture medium, but the other analogue-resistant mutants did not. Acetohydroxy acid synthetase, which seems to be rate-limiting for the biosynthesis of l-valine, was derepressed in abu-r mutants. One of the abu-r mutants, no. 140, which accumulated over 8 mg of l-valine per ml, had about a 20-fold increase in the enzyme level. Most of the abu-r mutants had acetohydroxy acid synthetase activity which was sensitive to feedback inhibition by l-valine to the same extent as in the parent strain. However, the enzyme of two of abu-r mutants was less sensitive to l-valine, and one of the two was the best valine accumulator.     

Mutants of Salmonella typhimurium with an Altered Leucyl-Transfer Ribonucleic Acid Synthetase

Two trifluoroleucine-resistant mutants of Salmonella typhimurium, strains CV69 and CV117, had an altered leucyl-transfer ribonucleic acid (tRNA) synthetase. The mutant enzymes had higher apparent K(m) values for leucine (ca. 10-fold) and lower specific activities (ca. twofold) than the parent enzyme when tested in crude extracts. Preparations of synthetase purified ca. 60-fold from the parent and strain CV117 differed sixfold in their leucine K(m) values. In addition, the mutant enzyme was inactivated faster than the parent enzyme at 50 C. The growth rates of strains CV69 and CV117 at 37 C were not significantly different from that of the parent, whereas at 42 C strain CV69 grew more slowly than the parent. Leucine-, valine-, and isoleucine-forming enzymes were partially derepressed when the mutants were grown in minimal medium; the addition of leucine repressed these enzymes to wild-type levels. During growth in minimal medium, the proportion of leucine tRNA that was charged in the mutants was about 75% of that in the parent. The properties of strain CV117 were shown to result from a single mutation located near gal at minute 18 on the genetic map. These studies suggest that leucyl-tRNA synthetase is involved in repression of the enzymes required for the synthesis of branched-chain amino acids.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Enhancement of bacitracin biosynthesis by branched-chain amino acids in a regulatory mutant ofBacillus licheniformis

DL-4-Azaleucine-resistant mutant of Bacillus licheniformis azlr-1 isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, was a better bacitracin producer than the parent strain. In the minimal medium, the antibiotic biosynthesis was 4 times higher in the mutant than in the parent strain and less dependent on L-leucine addition. In the complex fermentation medium, the yield was 18-20% higher in the mutant strain. Transaminase B activity measured in the crude extract revealed that the branched-chain amino acid biosynthetic enzymes were 5-10 times derepressed supplying bacitracin synthetase with enhanced quantity of isoleucine and leucine, the building units of bacitracin molecule.     

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.     

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.     

Regulation of histidyl-transfer ribonucleic acid synthetase formation in a histidyl-transfer ribonucleic acid synthetase mutant of Salmonella typhimurium

Control of formation of the histidyl-transfer ribonucleic acid (tRNA) synthetase with an increased K(m) for histidine was studied in a hisS mutant of Salmonella typhimurium. Histidine restriction of both the hisS and hisS(+) strains resulted in a derepression of synthesis of histidyl-tRNA synthetase. When grown in a concentration less than the K(m) (100 mug/ml) of l-histidine, the hisS mutant maintained a higher level of histidyl-tRNA synthetase than the hisS(+) strain. Addition of excess amounts of l-histidine to the growth medium of the hisS mutant culture grown with 100 mug of l-histidine per ml resulted in a repression of histidyl-tRNA synthetase formation to equal that of the hisS(+) strain grown in 100 mug of l-histidine per ml. These data confirm previous findings that histidine tRNA is involved in the repression of synthesis of histidyl-tRNA synthetase.     

Suppressors of a genetic regulatory mutation affecting isoleucine-valine biosynthesis in Escherichia coli K-12.

Escherichia coli K-12 mutant PS187 carries a mutation, ilvA538, in the structural gene for the biosynthetic L-threonine deaminase that leads to a leucine-sensitive growth phenotype, an isoleucine- and leucine-hypersensitive L-threonine deaminase, and pleiotropic effects resulting in abnormally low and invariant expression of some of the isoleucine-valine biosynthetic enzymes. Fifty-eight derivatives of strain PS187 were isolated as resistant to growth inhibition by leucine, by valine, or by valine plus glycly-valine and were biochemically, genetically, and physiologically characterized. All of these derivatives produced the feedback-hypersensitive L-threonine deaminase, and thus presumably possess the ilvA538 allele of the parent strain. Elevated synthesis of L-threonine deaminase was observed in 41 of the 58 isolates. Among 18 strains analyzed genetically, only those with mutations linked to the ilv gene clusters at 83 min produced elevated levels of L-threonine deaminase. One of the strains, MSR91, isolated as resistant to valine plus glycyl-valine, was chosen for more detailed study. The locus in strain MSR91 conferring resistance was located in four factor crosses between ilvE and rbs, and is in or near the ilvO gene postulated to be a site controlling the expression of the ilvEDA genes. Synthesis of the ilvEDA gene products in strain MSR91 is constitutive and derepressed approximately 200-fold relative to the parent strain, indicating that the genetic regulatory effects of the ilvA538 allele have been suppressed. Strain MSR91 should be suitable for use in purification of the ilvA538 gene product, since enzyme synthesis is fully derepressed and the suppressor mutation is clearly not located within the ilvA gene.     

一种基于途径分析提高丙酮酸产量的育种策略

为进一步提高光滑球拟酵母发酵生产丙酮酸的水平 ,在途径分析的基础上提出了一种组成型降低丙酮酸脱酸酶、但增强乙酰辅酶A合成酶活性的育种策略。通过亚硝基胍诱变 ,获得 1株乙酸需求型突变株CCTCCM2 0 2 0 19,在外加乙酸的培养基中表现出高于出发株 2 1%的丙酮酸生产能力和良好的遗传稳定性。检测突变株CCTCCM2 0 2 0 19中丙酮酸代谢相关酶的活性发现 :(1)丙酮酸脱羧酶活性降低了 4 0 % ;(2 )外加乙酸与否的条件下 ,乙酰辅酶A合成酶的活性分别提高了 10 3 5 %和 5 7 4 % ;(3)添加乙酸和突变对丙酮酸羧化酶、丙酮酸脱氢酶系、乙醇脱氢酶和乙醛脱氢酶的活性没有显著影响。在含有乙酸的培养基中突变株细胞干重比出发株高 2 1 7% ,可能是因为乙酰辅酶A合成酶活性的提高 ,补充了因丙酮酸脱羧酶活性降低而引起的胞质乙酰辅酶A短缺。在 7L罐中含有 6g L乙酸钠的培养基中发酵 6 2h ,丙酮酸产量达到 6 8 7g L ,对葡萄糖的产率为 0 6 5 1g g。     

Studies on gramicidin S synthetase. Purification of the heavy enzyme obtained from some mutants of Bacillus brevis.

The heavy enzyme of gramicidin S synthetase was purified to an almost homogeneous state by a combination of ammonium sulfate fractionation, ornithine-Sepharose 4B chromatography, DEAE-cellulose chromatography, and Ultrogel AcA 22 chromatography. The enzyme was proved to be essentially homogeneous by ultracentrifugation and polyacrylamide disc gel electrophoresis. The heavy enzymes of gramicidin S synthetase from various groups of mutant strains lacking the ability to form gramicidin S were also purified to a similar extent. The sedimentation rates of the purified enzymes from a wild strain and the mutant strains (BI-3, BII-3, BI-9) were studied by analytical centrifugation and sucrose density gradient centrifugation. The enzymes from the wild strain and these mutant strains were all found to have an S20,W value of 12.2 at a protein concentration of 2.5 mg per ml. These results strongly suggest that the failure of specific amino acid activation in the heavy enzyme of these gramicidin-lacking mutants might be due to some modification at the active center of the corresponding amino acid-activating enzyme rather than to a complete absence of the amino acid-activating enzyme protein in the heavy enzyme.     

flrB, a Regulatory Locus Controlling Branched-Chain Amino Acid Biosynthesis in Salmonella typhimurium

Salmonella typhimurium strain CV123 (ara-9 gal-205 flrB1), isolated as a mutant resistant to trifluoroleucine, has derepressed and constitutive levels of enzymes forming branched-chain amino acids. This strain grows more slowly than the parent at several temperatures, both in minimal medium and nutrient broth. It overproduces and excretes sizeable amounts of leucine, valine, and isoleucine in comparison with the parental strain. Both leuS (coding for leucyl-transfer ribonucleic acid [tRNA]synthetase) and flrB are linked to lip (min 20 to 25) by P1 transduction, whereas only leuS is linked to lip by P22 transduction. Strain CV123 containing an F' lip(+) episome from Escherichia coli has repressed levels of leucine-forming enzymes, indicating that flrB(+) is dominant to flrB. Leucyl-tRNA synthetase from strain CV123 appears to be identical to the leucyl-tRNA synthetase in the parent. No differences were detected between strain CV123 and the parent with respect to tRNA acceptor activity for a number of amino acids. Furthermore, there was no large difference between the two strains in the patterns of leucine tRNA isoaccepting species after fractionation on several different columns. Several other flrB strains exhibited temperature-sensitive excretion of leucine, i.e., they excreted leucine at 37 C but not 25 C. In one such strain, excretion at 37 C was correlated with derepression of some enzymes specified by ilv and leu. These latter results suggest that flrB codes for a protein.     

Repression of 3-deoxy-D-arabinoheptulosonic acid-7-phosphate synthetase (trp) and enzymes of the tryptophan pathway in Escherichia coli K-12

Mutant strains of Escherichia coli K-12 have been isolated in which the synthesis of 3-deoxy-d-arabinoheptulosonic acid-7-phosphate (DAHP) synthetase (trp) is partially constitutive. The mutation causing derepression is closely linked to aroH [the structural gene for DAHP synthetase (trp)] and occurs in a locus designated aroJ. The aroJ mutation is not recessive in an aroJ(+)/aroJ(-) diploid strain, as the synthesis of DAHP synthetase (trp) is still derepressed in this strain. On the basis of its close linkage to aroH and its continued expression in an aroJ(+)/aroJ(-) diploid, it is postulated that aroJ is an operator locus controlling the expression of the structural gene aroH. In support of this conclusion, the synthesis of anthranilate synthetase is still normally repressible in aroJ(-) strains, whereas, in trpR(-) strains, both DAHP synthetase (trp) and anthranilate synthetase are synthesized constitutively. The synthesis of DAHP synthetase (trp) remains repressible in an operator-constitutive mutant of the tryptophan operon. In two trpS mutants which possess defective tryptophanyl transfer ribonucleic acid synthetase enzymes, neither DAHP synthetase (trp) nor anthranilate synthetase derepress under conditions in which the defective synthetase causes a decrease in growth rate. On the other hand, an effect of the trpS mutant alleles on the level of anthranilate synthetase has been observed in strains which are derepressed for the synthesis of this enzyme, because of a mutation in the gene trpR. Possible explanations for this effect are presented.