Masato Terasawa

Depression of by-product formation during l-isoleucine production by a living-cell reaction process

SummaryTwo unnatural and unwanted amino acids, norvaline (Nva) and O-ethylhomoserine (O-EH) are formed as by-products in l-isoleucine production by Brevibacterium flavum AB-07 using a new process named the living cell reaction process. Nva formation was depressed by using a leucine auxotrophic mutant (AB-07-Leu-2) derived from strain AB-07. It was found that Nva formation was closely related to leucine biosynthesis. O-EH formation was repressed by addition of l-methionine to the reaction mixture. However, the homoserine-O-acetyltransferase of AB-07-Leu-2 was not subject to either inhibition or repression by addition of l-methionine. Furthermore, the O-EH-forming enzyme, which converts O-acetylhomoserine to O-EH, was speculated to be repressed by l-methionine.

Living cell reaction process forl-isoleucine andl-valine production

SummaryA new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and α-ketobutyric acid (α-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l−1 · day−1 (molecular yield to α-KB: 95%) and 300 mmol · l−1 · day−1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.

Cloning and nucleotide sequencing of the tyrosine phenol lyase gene fromEscherichia intermedia

SummaryThe tyrosine phenol lyase (TPL) gene was cloned from the genomic DNA of aEscherichia intermedia strain and the nucleotide sequence of the TPL structural gene was determined. The 1801 bpHincll-Nrul DNA fragment containing the TPL gene had an open reading frame of 1368 bp and the deduced amino acid sequence was 456 residues long with a molecular weight of 51,441 daltons.

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

SummaryIn anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Apr). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.

A novel industrial process for l-aspartic acid production using an ultrafiltration-membrane

Abstract A novel industrial process for the production of l -aspartic acid was developed. This process is characterized by the use of intact cells of a coryneform bacterium, Brevibacterium flavum MJ-233, which does not undergo autolysis, and by the use of an ultrafiltration-membrane for the separation and recycling of the cells. Unwanted formation of by-product, l -malic acid, was selectively eliminated by heat treating cells prior to reaction. Improvement of the process is in progress based on genetic engineering techniques.

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

SummaryThe application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; Ptna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of Ptna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-PtnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h.

Biochemical Production by Living Cell Reaction Processes

We have proposed a new bio-process named living cell reaction(LCR) process[1,2]. LCR process is a kind of enzymatic process using viable whole cells. In this process, intracellular multi-step enzyme reactions function while cell division and growth are repressed. In this view point, LCR process differs from the conventional fermentation process. Its advantages are, increase of product yield due to decrease of energy loss for cell growth and decrease of by-products formation generally coincided with cell growth. To design LCR process, we used coryneform bacterium Brevibacterium flavum MJ233. This strain strictly requires biotin, an essntial factor related to cell wall biosynthesis, for growth. Consequently, cell division and growth are repressed simply by using minimum medium without biotin as the reaction solution. This strain does not show cell-lysis even under non-growing condition. We report here, as a confirmation of advantages of LCR process, the production of L-isoleucine(L-Ile) and the solution of problems derived along the study.