Masaki Sugiura

Properties of Isoleucine Hydroxamate-resistant Mutants of Serratia marcescens

SUMMARY: Isoleucine hydroxamate-resistant mutants of Serratia marcescens have been isolated. Biochemical examination of the isoleucine-valine biosynthetic enzymes allowed the mutants to be grouped into three classes: I, mutants with increased enzyme levels; II, mutants with feedback inhibition-insensitive L-threonine dehydratase; III, mutants without any alteration in either feedback inhibition or enzyme levels. Of these mutants, class II accumulated isoleucine outside the cells in medium containing L-threonine. Strain IHr313, whose L-threonine dehydratase was about 500-fold less sensitive to feedback inhibition when compared with wild-type enzyme, accumulated up to 7 mg. of isoleucine per ml. of medium.

Improvement of a proline-producing strain of Serratia marcescens by subcloning of a mutant allele of the proline gene

Abstract A hybrid plasmid pY1333 bearing a mutant allele, dpr-1 , which is responsible for proline overproduction, was not stable in a proline-hyperproducing strain SP187 during subcultures. A 7 kb fragment covering over a part of dpr-1 allele DNA and a segment for spectinomycin resistance of mini-F plasmid vector pKP1154 tended to be deleted in the hybrid plasmid pY1333. We subcloned the dpr-1 allele into an improved plasmid vector pKPT1124 in which the segment for spectinomycin resistance of plasmid vector pKP1154 was substituted for a segment for kanamycin resistance of a broad-host-range vector pKT240, resulting in an improved hybrid plasmid pY1350. Strain SP187 carrying the hybrid plasmid pY1350 was quite stable during cultivation and produced 75 mg of L-proline per ml. This amount was 1.5 times larger than that of plasmid-free host strain.

Osmoregulation in a Proline-producing Strain of Serratia marcescens

Summary: A proline-producing strain of Serratia marcescens grew more rapidly than the wild-type strain in a medium of high osmolarity due to high concentrations of NaCl, KCl, Na2SO4, (NH4)2 HPO4, sodium glutamate, glucose or sucrose. Growth inhibition by NaCl was partially reversed by proline in the wild-type strain, and by glutamate and proline in the proline-producing strain. Intracellular proline and glutamate concentrations under conditions of high osmolarity were studied. The wild-type strain accumulated endogenously synthesized glutamate, and concentrated proline taken up from the external medium. In contrast, the proline-producing strain accumulated a large amount of endogenously synthesized proline. This increased proline content contributes to the osmotolerance of the proline-producing strain. The growth inhibition by NaCl was also reversed by glycinebetaine in S. marcescens wild-type and proline-producing strains.

Stability and instability of a cloned mutant allele of the proline gene in Serratia marcescens. Use of a mini-F plasmid in the proline-overproducing mutant

Abstract For the purpose of improving proline production by a Serratia marcescens mutant, a mutant allele of the proline gene, dpr-1, that is responsible for proline overproduction, was cloned from the mutant genomic DNA into a mini-F plasmid. Furthermore, the dpr-1 allele was subcloned into plasmids pLG339 and pBR322 to investigate the effect of copy number of the hybrid plasmids on proline productivity and the plasmid stability. Strains carrying the dpr-1 hybrid plasmids grew more slowly than the control strain, indicating that the hybrid plasmids were deleterious to host cells. The dpr-1 alleles cloned on pBR322 and pLG339 were genetically unstable in the wild-type and the proline-overproducing strain, respectively. On the other hand, the allele cloned on the mini-F plasmid with the lowest copy number was considerably stable even in the proline-overproducing mutant. The mutant carrying the cloned dpr-1 on the mini-F plasmid produced 62 mg ml−1 of proline, which was approximately 10% more than the yield of the control strain.

Transductional Construction of Amino Acid-hyperproducing Strains of Serratia Marcescens

Transformation, conjugation, and transduction were found for bacteria several decades ago. In these phenomena, a chromosomal fragment of donor cells is transferred into recipient cells, subsequently, it is hybridized to the recipient chromosome at the homologous DNA region, and finally it is integrated into the recipient chromosome by crossing-over, resulting in genetic recombination, In transduction, bacteriophage is an important tool and transfers a chromosomal fragment of donor into recipient. Application of these phenomena as gene manipulation techniques enables us to combine a certain property carried by a donor strain and another specific property carried by a recipient strain. Therefore, these techniques have been used for genetic analysis of several microorganisms and contributed to establishment of molecular genetics. Here we call these techniques intracellular genetic recombination techniques. At present, studies of strain construction play an important role in industrial microbiology. The reason is that production costs depend mainly on characteristics of strains used for microbial process. Accordingly, industrial microbiologists have endeavored to obtain more advantageous strains through genetic methods. The intracellular genetic recombination techniques had been considered to provide more rational approaches for strain construction, but had not been applied to construction of industrially available strains until recent years. We have been developing various amino acid-hyperproducing strains of Serratia rnarcescens by using transduction, one of the intracellular genetic recombination techniques’ and, for the first time anywhere, succeeded in industrialization of transductionally constructed strains. Here we describe our approaches for strain construction and profiles of our strains.

Proline production via the arginine biosynthetic pathway: transfer of regulatory mutations of arginine biosynthesis into a proline-producing strain of Serratia marcescens

SummaryProline production via a part of the arginine biosynthetic pathway was examined. About 20 mg/ml ofl-proline was produced by using arginine biosynthetic enzymes. Accordingly, three mutations of arginine biosynthesis, namely, derepression of arginine biosynthetic enzymes (assigned byargR2), feedback inhibition-resistant N-acetylglutamate synthase (assigned byargA2) and defectiveness in N-acetylornithine aminotransferase (assigned byargD−) were introduced by three transductional crosses into a proline-producing strain which produced about 55 mg/ml ofl-proline. The constructed strain produced 62 mg/ml ofl-proline, although about 10 mg/ml ofl-arginine and 1 mg/ml of N-acetylglutamate-γ-semialdehyde were produced as by-products.