Transgenic microbial polyhydroxyalkanoate producers

Details Transgenic microbial polyhydroxyalkanoate producers Transgenic microbial polyhydroxyalkanoate producers

1999-08-17

2003-07-15

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing oxygen-containing organic compound

C435S143000, C435S146000, C435S183000, C435S190000, C435S191000, C435S193000, C435S232000, C435S375000, C435S252300, C435S320100, C435S829000, C435S831000, C435S877000, C536S023200

Reexamination Certificate

active

06593116

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention is generally in the field of biosynthesis of poly(3-hydroxyalkanoates), and more particularly to improved microbial strains useful in commercial production of polyhydroxyalkanoates.
Poly(3-hydroxyalkanoates) (PHAS) are biological polyesters synthesized by a broad range of bacteria. These polymers are biodegradable and biocompatible thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams & Peoples,
CHEMTECH
26:38-44 (1996)). PHA biopolymers have emerged from what was originally considered to be a single homopolymer, poly-3-hydroxybutyrate (PHB) into a broad class of polyesters with different monomer compositions and a wide range of physical properties. About 100 different monomers have been incorporated into the PHA polymers (Steinbuchel & Valentin,
FEMS Microbiol. Lett.
128:219-28 (1995)).
It has been useful to divide the PHAs into two groups according to the length of their side chains and their biosynthetic pathways. Those with short side chains, such as PHB, a homopolymer of R-3-hydroxybutyric acid units, are crystalline thermoplastics, whereas PHAs with long side chains are more elastomeric. The former have been known for about seventy years (Lemoigne & Roukhelman, 1925), whereas the latter materials were discovered relatively recently (deSmet et al.,
J. Bacteriol.
154:870-78 (1983)). Before this designation, however, PHAs of microbial origin containing both (R)-3-hydroxybutyric acid units and longer side chain (R)-3-hydroxyacid units from C
5
to C
16
had been identified (Wallen & Rohweder,
Environ. Sci. Technol.
8:576-79 (1974)). A number of bacteria which produce copolymers of(R)-3-hydroxybutyric acid and one or more long side chain hydroxyacid units containing from five to sixteen carbon atoms have been identified (Steinbuchel & Wiese,
Appl. Microbiol. Biotechnol.
37:691-97 (1992); Valentin et al.,
Appl. Microbiol. Biotechnol.
3:507-14 (1992); Valentin et al.,
Appl. Microbiol. Biotechnol.
40:710-16 (1994); Abe et al.,
Int. J. Biol. Macromol.
16:115-19 (1994); Lee et al.,
Appl. Microbiol. Biotechnol.
42:901-09 (1995); Kato et al.,
Appl. Microbiol. Biotechnol.
45:363-70 (1996); Valentin et al.,
Appl. Microbiol. Biotechnol.
46:261-67 (1996); U.S. Pat. No. 4,876,331 to Doi). A combination of the two biosynthetic pathways outlined described above provide the hydroxyacid monomers. These copolymers can be referred to as PHB-co-HX (where X is a 3-hydroxyalkanoate or alkanoate or alkenoate of 6 or more carbons). A useful example of specific two-component copolymers is PHB-co-3-hydroxyhexanoate (PHB-co-3HH) (Brandl et al.,
Int. J. Biol. Macromol.
11:49-55 (1989); Amos & McInerey,
Arch. Microbiol.
155:103-06 (1991); U.S. Pat. No. 5,292,860 to Shiotani et al.).
PHA production by many of the microorganisms in these references is not commercially useful because of the complexity of the growth medium, the lengthy fermentation processes, or the difficulty of down-stream processing of the particular bacterial strain. Genetically engineered PHA production systems with fast growing organisms such as
Escherichia coli
have been developed. Genetic engineering also allows for the improvement of wild type PHA production microbes to improve the production of specific copolymers or to introduce the capability to produce different PHA polymers by adding PHA biosynthetic enzymes having different substrate-specificity or even kinetic properties to the natural system. Examples of these types of systems are described in Steinbuchel & Valentin,
FEMS Microbiol. Lett.
128:219-28 (1995). PCT WO 98/04713 describes methods for controlling the molecular weight using genetic engineering to control the level of the PHA synthase enzyme. Commercially useful strains, including
Alcaligenes eutrophus
(renamed as
Ralstonia eutropha
),
Alcaligenes latus, Azotobacter vinlandii,
and Pseudomonads, for producing PHAs are disclosed in Lee,
Biotechnology
&
Bioengineering
49:1-14 (1996) and Braunegg et al., (1998), J. Biotechnology 65: 127-161.
The development of recombinant PHA production strains has followed two parallel paths. In one case, the strains have been developed to produce copolymers, a number of which have been produced in recombinant
E. coli.
These copolymers include poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB-co-4HB), poly(4-hydroxybutyrate) (P4HB) and long side chain PHAs comprising 3-hydroxyoctanoate units (Madison and Huisman, 1999. Strains of
E. coli
containing the phb genes on a plasmid have been developed to produce P(3HB-3HV) (Slater, et al.,
Appl. Environ. Microbiol.
58:1089-94 (1992); Fidler & Dennis,
FEMS Microbiol. Rev.
103:231-36 (1992); Rhie & Dennis,
Appl. Environ. Micobiol.
61:2487-92 (1995); Zhang, H. et al.,
Appl. Environ. Microbiol
60:1198-205 (1994)). The production of P(4HB) and P(3HB-4HB) in
E. coli
is achieved by introducing genes from a metabolically unrelated pathway into a P(3HB) producer (Hein, et al.,
FEMS Microbiol. Lett.
153:411-18 (1997); Valentin & Dennis,
J. Biotechnol.
58:33-38 (1997)).
E. coli
also has been engineered to produce medium short chain polyhydroxyalkanoates (msc-PHAs) by introducing the phaC1 and phaC2 gene of
P. aeruginosa
in a fadB::kan mutant (Langenbach, et al.,
FEMS Microbiol. Lett.
150:303-09 (1997); Qi, et al.,
FEMS Microbiol. Lett.
157:155-62 (1997)).
Although studies demonstrated that expression of the
A. eutrophus
PHB biosynthetic genes encoding PHB polymerase, -ketothiolase, and acetoacetyl-CoA reductase in
E. coli
resulted in the production of PHB (Slater, et al.,
J. Bacteriol.
170:4431-36 (1988); Peoples & Sinskey,
J. Biol. Chem.
264:15298-303 (1989); Schubert, et al.,
J. Bacteriol.
170:5837-47 (1988)), these results were obtained using basic cloning plasmid vectors and the systems are unsuitable for commercial production since these strains lacked the ability to accumulate levels equivalent to the natural producers in industrial media.
For commercial production, these strains have to be made suitable for large scale fermentation in low cost industrial medium. The first report of recombinant P(3HB) production experiments in fed-batch cultures used an expensive complex medium, producing P(3HB) to 90 g/L in 42 hours using a pH-stat controlled system (Kim, et al,
Biotechnol. Lett.
14:811-16 (1992)). Using stabilized plasmids derived from either medium- or high-copy-number plasmids, it was shown that
E. coli
XL1-Blue with the latter type plasmid is required for substantial P(3HB) accumulation (Lee, et al.,
Ann. N.Y. Acad. Sci.
721:43-53 (1994)). In a fed-batch fermentation on 2% glucose/LB medium, this strain produced 81% P(3HB) at a productivity of 2.1 g/L-hr (Lee, et al.,
J. Biotechnol.
32:203-11 (1994)). The P(3HB) productivity was reduced to 0.46 g/L-hr in minimal medium, but could be recovered by the addition of complex nitrogen sources such as yeast extract, tryptone, casamino acids, and collagen hydrolysate (Lee & Chang,
Adv. Biochem. Eng. Biotechnol.
52:27-58 (1995); Lee, et al.,
J. Ferment. Bioeng.
79:177-80 (1995)).
Although recombinant
E. coli
XL 1-blue is able to synthesize substantial levels of P(3HB), growth is impaired by dramatic filamentation of the cells, especially in defined medium (Lee, et al.,
Biotechnol. Bioeng.
44:1337-47 (1994); Lee,
Biotechnol. Lett.
16:1247-52 (1994); Wang & Lee,
Appl. Environ. Microbiol.
63:4765-69 (1997)). By overexpression of FtsZ in this strain, biomass production was improved by 20% and P(3HB) levels were doubled (Lee & Lee,
J. Environ. Polymer Degrad.
4:131-34 (1996)). This recombinant strain produced 104 g/L P(3HB) in defined medium corresponding to 70% of the cell dry weight. The volumetric productivity of 2 g/L-hr, however, is lower than achievable with
R. eutropha.
Furthermore, about 15% of the cells lost their ability to produce PHB by the end of the fermentation (Wang & Lee,
Biotechnol. Bioeng.
58:325-28 (1998)).
Re

Affiliated with

Also associated with

Rating

Transgenic microbial polyhydroxyalkanoate producers does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Transgenic microbial polyhydroxyalkanoate producers, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Transgenic microbial polyhydroxyalkanoate producers will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3018549