Hisashi Yamagata

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.

Characterization of alcohol-assimilating photosynthetic purple non-sulfur bacteria and cloning of molecular chaperones from a purple non-sulfur bacterium

Abstract We screened several strains which exhibited significant photoorganotrophic growth on a chemically defined synthetic medium containing alcohols. Samples from sewage, rice fields, or other places were inoculated under anaerobic-light conditions at 30°C. After multiple enrichments, the growth characteristics of three fast growing isolates were investigated. We describe also the cloning of molecular chaperones from a purple non-sulfur bacterium.

Cloning of dnaK and dnaJ homologous genes from a purple non-sulfur bacterium Rhodopseudomonas species.

The dnaK and dnaJ genes were isolated as a cluster from a purple non-sulfur phototrophic bacterium, Rhodopseudomonas species No. 7 by a polymerase chain reaction (PCR) based method. The deduced products of dnaK (631 amino acids) and dnaJ (379 amino acids) were 67% and 56% identical to the respective Escherichia coli gene products. The functions of DnaK and DnaJ could be confirmed by complementation of the respective E. coli mutants.

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.