Ajinomoto Co., Ltd.

Yoshimi Kikuchi

Keiichi Yokoyama

Masayo Date

Osamu Kurahashi

Yukiko Umezawa

Hiroyuki Kojima

Hiroshi Itaya

Hiroshi Matsui

Tomoko Suzuki

Kazuo Nakanishi

Tsuyoshi Nakamatsu

Yoshio Kawahara

Eiichiro Kimura

Norimasa Onishi

Yasushi Nishimura

Junichiro Kojima

Shinya Goto

Nobuharu Tujimoto

Yoshiko Kawahara

Kenzo Yokozeki

Hisashi Yasueda

Takashi Tanaka

Nami Nakamura

Yoshihiko Matsuda

Haruo Heima

Mototaka Suzuki

Ekaterina Aleksandrovna Kutukova

Noriko Tsurui

Haruki Beppu

Jurgis Antanas Vladovich Jomantas

A novel technique for improving secretory production of a heterologous protein by coryneform bacteria is described, and thereby a method for secretory production of a heterologous protein is provided. A coryneform bacterium is cultured so that it secretes a heterologous protein, the bacterium having a genetic construct which includes a promoter sequence that functions in the coryneform bacterium, a nucleic acid sequence coding for a signal peptide that functions in the coryneform bacterium, which is ligated downstream from the promoter sequence, and a nucleic acid sequence coding for a fusion protein having an amino acid sequence that includes Gln-Glu-Thr and the heterologous protein, which is ligated downstream from the nucleic acid sequence coding for the signal peptide.

Method for secretory production of protein

A method for secretory production of a heterologous protein is provided by developing a novel technique for improving ability of a coryneform bacterium to produce a heterologous protein by secretory production. By utilizing a coryneform bacterium having an ability to produce a heterologous protein by secretory production which has been modified so that the activity of a penicillin-binding protein is reduced and in which the activity of a cell surface layer protein has been reduced as an expression host, a heterologous protein is produced by secretory production.

The present invention provides a method for efficiently producing an industrially useful protein in coryneform bacteria, and more particularly, a method for efficiently producing a protein for which secretion was difficult using conventional protein secretion pathways. In particular, the present invention provides a method for efficiently producing heterologous proteins comprising culturing coryneform bacteria containing an genetic construction containing a promoter sequence which functions in coryneform bacteria, a nucleic acid sequence encoding a Tat system-dependent signal peptide region, and a nucleic acid sequence encoding a heterologous protein, in the direction from 5'-end to 3&#x2032;-end, and secretory producing the heterologous protein by coryneform bacteria.

Method of producing proteins

By culturing a methanol-assimilating bacterium which harbors a DNA construct which contains a promoter sequence that functions in the methanol-assimilating bacterium and a nucleotide sequence that encodes a polypeptide containing a signal sequence and an objective protein which is functionally connected to the promoter sequence, in a liquid medium containing methanol as a major carbon source, the bacterium is allowed to secrete the objective protein, and the secreted objective protein is recovered.

Method for producing protein

The present invention provides a neutral metalloprotease from actinomycetes which selectively cleaves a pro-structure part of a microbial protransglutaminase and a gene encoding said neutral metalloprotease. An active microbial transglutaminase having the pro-structure part cleaved can be obtained by culturing a microorganism into which a gene encoding the neutral metalloprotease from actinomycetes according to the present invention has been introduced, where by producing the neutral metalloprotease from actinomycetes, and reacting it on a microbial protransglutaminase.

Method of producing microbial transglutaminase

Provided is a method for efficiently producing a 3-amino-4-hydroxybenzoic acid-type compound by culturing a coryneform bacterium that has a gene encoding a mutated aspartokinase not subject to feedback inhibition, and that is transformed with a recombinant vector containing a DNA encoding a protein having an activity to form 3-amino-4-hydroxybenzoic acid from dihydroxyacetone phosphate and aspartate semialdehyde.

Method for producing an aminohydroxybenzoic acid-type compound

Anbacterium having dihydrodipicolinate synthase and aspartokinase, both of which are desensitized to feedback inhibition by L-lysine. The intracellular activity of dihydrodipicolinate reductase in this bacterium can also be enhanced. Furthermore, a diaminopimelate dehydrogenase gene can be introduced into this bacterium, or intracellular activities of tetrahydrodipicolinate succinylase and succinyl diaminopimelate deacylase can be enhanced. Finally, the intracellular activities of aspartate-semialdehyde dehydrogenase or phosphoenolpyruvate carboxylase can be enhanced in this bacterium. The bacterium can be cultured in a suitable medium to produce and accumulate L-lysine in culture, and the L-lysine is collected from the culture.

Method for producing L-lysine

The present invention relates to a process for secretory production of a foreign protein, in particular, transglutaminase by a coryneform bacterium. According to the present invention, a process is provided for the secretory production of a foreign protein, in particular, transglutaminase, by making a coryneform bacterium to produce an industrially useful foreign protein, in particular, transglutaminase and efficiently release the product extracellularly (i.e., secretory production). An intended foreign protein, in particular, transglutaminase, is produced by using an expression construct wherein the gene sequence of the intended foreign protein containing the pro-structure part, in particular, pro-transglutaminase gene sequence, is ligated to the downstream of a sequence encoding the signal peptide region from a coryneform bacterium, introducing this expressional genetic construct into a coryneform bacterium, culturing the thus transformed coryneform bacterium, and treating the extracellularly released protein with a protease, etc. to cleave and eliminate the pro-part.

Process for producing transglutaminase

Anbacterium (1) which harbors dihydrodipicolinate synthase of which feedback inhibition by L-lysine is desensitized and aspartokinase of which feedback inhibition by L-lysine is desensitized, (2) in which intracellular activity of dihydrodipicolinate reductase is enhanced, and (3) in which a diaminopimelate dehydrogenase gene is introduced or intracellular activities of tetrahydrodipicolinate succinylase and succinyl diaminopimelate deacylase are enhanced, wherein intracellular activity of aspartate-semialdehyde dehydrogenase or phosphoenolpyruvate carboxylase is enhanced, is cultured in a suitable medium to produce and accumulate L-lysine in culture, and the L-lysine is collected from the culture.

[object Object]

The object of the present invention is to provide a method of producing a heterologous protein by making a coryneform bacterium to produce and efficiently extracellularly secrete (secreto-production) an industrially useful heterologous protein. According to the present invention, a genetic construct is used where a gene sequence encoding an intended protein which is ligated to the downstream of a sequence encoding the signal peptide derived from a coryneform bacterium, the gene construct is introduced into a mutant coryneform bacterium which has a capacity of secreting the heterologous protein at least 2-fold higher than the wild typeATCC 13869, the mutant coryneform bacterium is cultured and the extracellularly released heterologous protein is recovered.

Methods for secretory production of proteins

A method for producing a fermentative product by utilizing a microorganism, the method comprising culturing the microorganism in a medium to produce and accumulate the fermentative product in the medium, and collecting the fermentative product, wherein the microorganism expresses a heat shock protein derived from a hyperthermophilic archaeon strain KOD-1 in a cell of the microorganism by introduction of a gene coding for the heat shock protein.

Method for producing fermentative product and stress-resistant microorganism

A microorganism is utilized to fermentatively produce useful substances such as amino acids by cultivating the microorganism in a medium to allow a fermentative product to be produced and accumulated in the medium, and collecting the fermentative product, wherein the microorganism to be used is modified by introduction of at least one of a gene coding for a heat shock protein and a gene coding for a &sgr; factor which specifically functions for the heat shock protein gene to enhance expression amount of the heat shock protein in cells, whereby the microorganism is allowed to have added resistance to stress which would otherwise restrain growth of the microorganism and/or production of the fermentative product.

Growing community of inventors

Kawasaki, Japan

Yoshimi Kikuchi