Atsuyuki Satoh

The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

SummaryWe have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.

Production of Poly(γ-glutamic acid) by Bacillus subtilis F-2-01

Production of Poly(γ-glutamic acid) by Bacillus subtilis F-2-01 Hidetoshi Kubota, Toshio Matsunobu, Kazumichi Uotani, Hidehi Takebe, Atsuyuki Satoh, Toshio Tanaka & Makoto Taniguchi a Pharmaceutical Technology Laboratories, Meiji Seika Kaisha, Ltd., 788 Kayama, Odawara-shi, Kanagawa 250, Japan b Faculty of Science, Osaka City University, 3–3–138 Sugimoto, Sumiyoshi-ku, Osaka 558, Japan. Published online: 12 Jun 2014.

Optimization and scale-up of γ-linolenic acid production by Mortierella ramanniana MM 15-1, a high γ-linolenic acid producing mutant

Abstract Optimum conditions for cultivation of Mortierella ramanniana mutant MM 15-1 were investigated with an aim of producing a lipid which contains high γ-linolenic acid (GLA). The optimum initial glucose concentration and pH control level were 300 g/ l and 4.0, respectively. Although both pellet and filamentous forms of this mutant were observed during the cultivation, the pellets accumulated lipid with higher GLA content than the filamentous forms. The effects of culture conditions on pellet formation were therefore investigated in a 30- l jar fermentor. The results showed that with an inoculum spore concentration of 5.0 × 10 3 /ml and an agitation speed of 800 rpm, 0.15–0.5 mm pellets were formed and consequently, the GLA content of the lipid increased to a very high value of 18.3%. Furthermore, when this process was scaled up to a 10-kl fermentor, using the impeller tip velocity as the scale-up parameter, both the pellet formation and the lipid production as well as the GLA content of the lipid were consistent with those obtained in the 30- l jar fermentor.

Studies on the biosynthesis of bialaphos (SF-1293) Part 3. Production of phosphinic acid derivatives, MP-103, MP-104 and MP-105, by a blocked mutant of Streptomyceshygroscopicus SF-1293 and their roles in the biosynthesis of bialaphos

During biosynthetic studies on bialaphos to reveal the formation mechanisms of carbon-phosphorous bonds in detail, three new metabolites containing a H-P-C bond structure were isolated from the fermentation broth of a mutant of Streptomyces hygroscopicus SF-1293. Based on the spectroscopic analysis, the structures of these compounds have been determined as shown in Fig. 1. Transformation experiments of these metabolites to bialaphos suggested that the reduction of the phosphorous atom in phosphate will take place at an early biosynthetic stage.

The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus

The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

γ-Linolenic acid production by a low temperature-resistant mutant of Mortierella ramanniana

As a result of breeding experiments using low temperature growth as an index, Mortierella ramanniana mutant MM 15-1, which produces lipids with a high γ-linolenic acid (GLA) content, was obtained. The results of cultivation experiments in Erlenmeyer flasks showed that although the total lipid produced by the MM15-1 mutant was about 14% lower than that of the parent strain (M. ramanniana IFO 8187), the GLA content of the lipid was 16.5%, which represents about a 2-fold increase over that of the parent strain. In particular, there was a significant increase in the GLA proportion of the phospholipid fraction in the MM15-1 mutant. Furthermore, the results of the lipid composition analysis after phospholipid fractionation by TLC showed that there was a significant increase in the GLA proportions in the phosphatidylethanolamine (PE) and phosphatidylinositol (PI) of the MM 15-1 mutant. When the MM 15-1 mutant was cultivated in a 600-l fermentor, it produced lipid with 18.3% GLA. This was a 2-fold higher than the value obtained with the parent strain under the same cultivation conditions.

Breeding of bialaphos producing strains from a biochemical engineering viewpoint

Abstract A breeding study was performed from the viewpoint of biochemical engineering to obtain strains which produce bialaphos (a herbicide) with high productivity and high yield from inexpensive substrates under low oxygen supply, with good filtration efficiency and easy downstream treatment. After induing mutagenesis using conventional chemical mutagens, first selection on agar culture was made to obtain strains which exhibited a large inhibition zone with a small colony, without being influenced by carbon catabolite regulation and with suppression of product decomposition. Subsequently, in a liquid culture system, selection was made to obtain strains which exhibited high product concentrations under low oxygen supply, with good filtration efficiency. A strain thus obtained was found to have about five hundred times higher product concentration than the wild strain. It also enabled us to use an inexpensive carbon source and to produce bialaphos under low oxygen supply, with good filtration efficiency.

Scale-up of bialaphos production

Abstract Scale-up of bialaphos production from a 3 l jar fermentor to a 300 kl fermentor was investigated. When the scale-up was based on power consumption (for agitation) per unit volume of the reactor, bialaphos production in the 300 kl fermentor was remarkably lower than that obtained with the 3 l jar fermentor. The reason for the unsuccessful scale-up was investigated by considering the major differences between the 3 l jar and the 300 kl fermentor. It was found that variation in the shear stress had no significant effect on bialaphos production. A special pressure-proof fermentor with pressure control system was used to study the effect of liquid pressure gradient in the 300 kl fermentor. When the cells in the special fermentor were subjected to cyclic variation in pressure (between 1,013 and 1,994 HPa) as though they are circulated between the top and the bottom of a 10 m liquid level in the 300 kl fermentor, bialaphos production was as low as that obtained in the 300 kl fermentor. The optimum DO concentration for bialaphos production in the 3 l fermentor was found to be 0.5 ppm and above this concentration, an inverse relationship between the DO concentration and bialaphos production was observed. The inhibition of bialaphos production at high pressure was therefore attributed to the increase in the DO concentration at high liquid pressures. When the scale-up was based on the DO concentration and the DO at the middle of the 300 kl fermentor was controlled at 0.5 ppm, bialaphos production increased to 85 % of that obtained with the 3 l jar fermentor. By taking the DO concentration gradient in the 300 kl fermentor into consideration, the DO at the bottom of the fermentor was maintained at 0.5 ppm. This resulted in a successful scale-up wherein bialaphos production increased to 96% of the maximum production in the 3 l jar fermentor.

Effect of oxygen partial pressure on bialaphos synthesis, sugar metabolism and the activity of tricarboxylic acid cycle enzymes in submerged culture of Streptomyces hygroscopicus☆

Abstract Bialaphos [a herbicide, 2-amino-4-(hydroxy)(methyl)-phospinoylbutyryl-alanyl-alanine] produced by Streptomyces hygroscopicus increased significantly as the oxygen supply decreased during both growth and production phases of batch culture. Under low oxygen partial pressure, there were decreases in sugar consumption by the cells, cell concentration and the activity of tricarboxylic acid cycle enzymes in the cells. On the other hand, the activity of glyoxylic acid cycle enzymes in the cells increased. These phenomena were more prominent in high producing than in low producing strains. From these results, it is suggested that in high bialaphos producing strains, low oxygen partial pressure suppresses the activity of tricarboxylic acid enzymes so that both acetyl-CoA and pyruvate (the substrates for bialaphos synthesis) are used mainly for secondary metabolism. This results in a high production of bialaphos.

Relationship between sugar consumption and tricarboxylic acid cycle enzyme activity in a high bialaphos-producing strain☆

Abstract The biosynthesis of bialaphos [a herbicide, 2-amino-4(hydroxy)(methyl)-phosphinoylbutyryl-alanylalanine] produced by Streptomyces hygroscopicus , proceeds through the degradation of glucose to phosphoenol pyruvate, the formation of a CP bond, acetic addition by acetyl-CoA and alanine addition. Therefore, bialaphos formation is considered to be closely related to glucose metabolism. Based on this hypothesis, sugar consumption and the activities of the tricarboxylic acid cycle enzymes were examined using a highly productive strain and a strain of the lower productivity. It was clear that the highly productive strain has a lower sugar consumption rate and lower yield of cells, compared with the lower productivity strain. The activities of the tricarboxylic acid cycle enzymes of the highly productive strain were lower than those of the lower productivity strain, while the activities of the glyoxylic acid cycle enzymes of the highly productive strain were higher. From these findings, it is suggested that the highly productive strain suppresses the flow from acetyl-CoA and pyruvate (as substrates of bialaphos) to the tricarboxylic acid cycle, and efficiently directs these substrates to the secondary metabolism by activation of the glyoxylic acid cycle, resulting in a high rate of bialaphos production in this strain.