Acetic acid bacterium, plasmid derived from said bacterium and s

Details Acetic acid bacterium, plasmid derived from said bacterium and s Acetic acid bacterium, plasmid derived from said bacterium and s

1994-12-23

1996-12-03

Vogel, Nancy T.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

4353201, 4351723, C12N 120, C12N 1574, C12N 1500

Patent

active

055807828

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to Acetobacter sp. strain BPR 2001, a plasmid named pAH4 carried by said strain and shuttle vectors constructed from said plasmid and an E. coli-derived plasmid.


DISCUSSION OF THE BACKGROUND

Microorganisms belonging to Acetobacter xylinum have been long known as acetic acid bacteria that produce bacterial cellulose. Recently, bacterial cellulose produced by acetic acid bacteria including Acetobacter xylinum has received attention as material for, for example, medical pads or loudspeaker diaphragms because its cellulose microfibrils are more uniformly oriented as compared with plant-derived cellulose so that it is homogeneous and mechanically strong.
However, the cellulose productivity of acetic acid bacteria is low. Therefore, some attempts were made to improve the productivity by varying incubation conditions or introducing mutation. In addition to these conventional means, the modern recombinant gene technology is also being applied in the studies to improve the productivity. In order to bring about gene recombination in cellulose-producing acetic acid bacteria, it is necessary to develop, first of all, a host-vector system for the object bacterium.
It has been known that such microorganisms produce not only cellulose but also acetan which is a water-soluble polysaccharide. In view of the utility of acetan as starting material for food-based thickeners and various chemical products, it is also useful to develop such a host-vector system. Seven vector plasmids carried by Acetobacter aceti subsp. xylinum IFO 3288 were already reported (JPA1-199580).
Plasmids carried by acetic acid bacteria were also reported in JPW4-503456 and Proc. Natl. Acad. Sci. USA, Vol. 87, pp. 8130-8134 (1990).
On the other hand, it would be very convenient for gene recombination of acetic acid bacterial plasmids, if plasmid vectors which can replicate not only in acetic acid bacteria but also other host cells such as E. coli (hereinafter referred to as "shuttle vectors") are available.
Such shuttle vectors were previously reported in, for example, JPA1-199580 cited above and BIOTECHNOLOGY LETTERS, Vol. 14, No. 7 (July, 1922), pp. 539-542.


SUMMARY OF THE INVENTION

In this state of arts, the present inventors have studied to provide a plasmid derived from a novel cellulose-producing acetic acid bacterium and novel shuttle vectors constructed from said plasmid and an E. coli-derived plasmid, and succeeded in newly isolating a cellulose-producing bacterium, which was classified as an Acetobacter according to the method described in Bergey's Manual of Systematic Bacteriology and designated as Acetobacter sp. strain BPR 2001. The Acetobacter sp. strain BPR 2001 is taxonomically characterized as a rod-shaped, gram-negative, non-spore-forming and aerobic bacterium, and proves to be positive for catalase, negative for oxidase, positive for acetic acid production from ethanol, positive for oxidation of acetate and positive for oxidation of lactate. The inventors have further found that this strain is novel and carries a novel plasmid, and thus completed this invention.
Accordingly, this invention provides Acetobacter sp. strain BPR 2001 and endogenous plasmids derived from said strain, especially pAH4.


BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction of pSA19 and pSA7.
FIG. 2 illustrates the construction of pK5.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This plasmid pAH4 has a relatively small size of about 4.0 kb, and restriction endonuclease sites which make it suitable for use as vector, as shown in Example 1.
Further, the plasmid pAH4 of this invention has the base sequence of about 4002 bp shown as SEQ ID NO: 1 in the SEQUENCE LISTING annexed.
This invention also provides shuttle vectors constructed from said plasmid and an E. coli-derived plasmid such as pUC18.
Typical examples of such shuttle vectors include pSA19, pSA7 and pK5, which are all able to replicate either in the host cells of cellulose-producing acetic acid bacteria or those of E. coli,

REFERENCES:
Tonouchi et al., Biosci. Biotech. Biochem. 58(10):1899-1901 (1994).
Fukaya et al., Agric. Biol. Chem. 49(5):1349-1355 (1985).
Agric. Biol. Chem. 49(7), pp. 2083-2090, 1985, Masahiro Fukaya, et al., "Construction of New Shuttle Vectors for Acetobacter".
Agric. Biol. Chem. 49 (4), pp. 1011-1017, 1985, Hajime Okumura, et al., "Construction of Plasmid Vectors and a Genetic Transformation System for Acetobacter Aceti".

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