High growth methanotropic bacterial strain

Details High growth methanotropic bacterial strain High growth methanotropic bacterial strain

2001-08-22

2004-02-10

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Utilizing media containing lower alkanol

C435S252100, C435S248000, C435S232000, C435S071100, C435S250000, C536S024100

Reexamination Certificate

active

06689601

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the field of microbiology. More specifically, the invention relates to the use of a novel methanotrophic bacterial strain capable of utilizing a central carbon pathway for more efficient production of commercially useful products.
BACKGROUND OF THE INVENTION
Methanotrophic bacteria are defined by their ability to use methane as their sole source of carbon and energy. Although methanol is an obligate intermediate in the oxidation of methane, the ability to grow on methanol alone is highly variable among the obligate methanotrophs due to its toxicity (Green, Peter. Taxonomy of Methylotrophic Bacteria. In: Methane and Methanol Utilizers (Biotechnology Handbooks 5) J. Colin Murrell and Howard Dalton eds. 1992 Pleanum Press NY, pp. 23-84). Methane monooxygenase is the enzyme required for the primary step in methane activation and the product of this reaction is methanol (Murrell et al.,
Arch. Microbiol.
(2000), 173(5-6), 325-332). This reaction occurs at ambient temperatures and pressures, whereas chemical transformation of methane to methanol requires temperatures of hundreds of degrees and high pressure (Grigoryan, E. A.,
Kinet. Catal.
(1999), 40(3), 350-363; WO 2000007718; U.S. Pat. No. 5,750,821). It is this ability to transform methane under ambient conditions along with the abundance of methane that makes the biotransformation of methane a potentially unique and valuable process.
The commercial applications of biotransformation of methane have historically fallen broadly into three categories: 1) Production of single cell protein, (Sharpe D. H. BioProtein Manufacture (1989). Ellis Horwood series in applied science and industrial technology. New York: Halstead Press) (Villadsen, John,
Recent Trends Chem. React. Eng
., [Proc. Int. Chem. React. Eng. Conf.], 2nd (1987), Volume 2, 320-33. Editor(s): Kulkarni, B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher: Wiley East., New Delhi, India; Naguib, M., Proc. OAPEC Symp. Petroprotein, [Pap.] (1980), Meeting Date 1979, 253-77 Publisher: Organ. Arab Pet. Exporting Countries, Kuwait, Kuwait); 2) epoxidation of alkenes for production of chemicals (U.S. Pat. No. 4,348,476); and 3) biodegradation of chlorinated pollutants (Tsien et al.,
Gas, Oil, Coal, Environ. Biotechnol.
2, [Pap. Int. IGT Symp.
Gas, Oil, Coal, Environ. Biotechnol.],
2nd (1990), 83-104. Editor(s): Akin, Cavit; Smith, Jared. Publisher: Inst. Gas Technol., Chicago, Ill.; WO 9,633,821; Merkley et al.,
Biorem. Recalcitrant Org
., [Pap. Int. In Situ On-Site Bioreclam. Symp.], 3rd (1995), 165-74. Editor(s): Hinchee, Robert E; Anderson, Daniel B.; Hoeppel, Ronald E. Publisher: Battelle Press, Columbus, Ohio: Meyer et al.,
Microb. Releases
(1993), 2(1), 11-22). Only epoxidation of alkenes has experienced little commercial success due to low product yields, toxicity of products and the large amount of cell mass required to generate products.
Large-scale protein production from methane, termed single cell protein or SCP has been technically feasible and commercialized at large scale (Villadsen supra). However, SCP has been less than economically successful due to the relatively high cost to produce microbial protein compared to agriculturally derived protein (i.e. soy protein). Single cell protein is a relatively low value product and therefore economic production cannot tolerate heavy bioprocessing costs. For this reason the yield of the methanotrophic strain may be critical to the overall economic viability of the process. Microbial biomass produced by methanotrophic bacteria is typically very high in protein content (~70-80% by weight), which can restrict the direct use of this protein to certain types of animal feed.
The conversion of C1 compounds to complex molecules with C—C bonds is a difficult and capital intensive process by traditional chemical synthetic routes. Traditionally, methane is first converted to synthesis gas (mixtures of hydrogen, carbon monoxide and carbon dioxide), which is then used to produce other small molecular weight industrial precursors. Typically these are “commodity” type chemicals such as acetate, formaldehyde, or methanol. The basic problem is activation of the methane molecule which is thermodynamically very difficult to achieve by chemical means. “Activation” refers to the process of making the chemically unreactive methane molecule more reactive.
Methanotrophic bacteria contain enzymes (methane monooxygenases) which are capable of methane activation at ambient temperatures and pressures. Methane activation consists of oxygen insertion into methane to form methanol which is much more readily metabolized to more complex molecules within the cell. Two types of methane monooxygenase are found in methanotrophic bacteria. A particulate methane monooxygenase (pMMO) has a narrow substrate specificity and is incapable of oxygen insertion into more complex molecules. Some, but not all methanotrophs may also contain a soluble methane monooxygenase (sMMO). This enzyme has been the subject of much investigation and proprietary claims due to its ability to oxygenate, or functionalize, a wide variety of aliphatic and aromatic molecules. This characteristic has been utilized for co-metabolic production processes where methanotrophs are fed both methane and a more complex molecule to be transformed by the sMMO. Numerous examples are reported of processes requiring both methane and, typically, a petroleum-derived feedstock such as toluene, naphthalene, or decane, where sMMO plays a role. However, the art is silent with respect to using methanotrophs for net synthesis of chemicals from methane as opposed to these co-metabolic transformations. For net synthesis, only inexpensive methane is required along with the ability to genetically engineer the strain to produce the desired chemical.
Methanotrophic cells can further build the oxidation products of methane (i.e. methanol and formaldehyde) into more complex molecules such as protein, carbohydrate and lipids. For example, under certain conditions methanotrophs are known to produce exopolysaccharides (Ivanova et al.,
Mikrobiologiya
(1988), 57(4), 600-5; Kilbane, John J., II
Gas, Oil, Coal, Environ. Biotechnol.
3, [Pap. IGT's Int. Symp.], 3rd (1991), Meeting Date 1990, 207-26. Editor(s): Akin, Cavit; Smith, Jared. Publisher: IGT, Chicago, Ill.). Similarly, methanotrophs are known to accumulate both isoprenoid compounds and carotenoid pigments of various carbon lengths (Urakami et al.,
J. Gen. Appl. Microbiol.
(1986), 32(4), 317-41). Although these compounds have been identified in methanotrophs, they have not been microbial platforms of choice for production as these organisms have very poorly developed genetic systems, thereby limiting metabolic engineering for chemicals.
A necessary prerequisite to metabolic engineering of methanotrophs is a full understanding, and optimization, of the carbon metabolism for maximum growth and/or product yield. Obligate methanotrophs are typically thought to channel carbon from methane to useful products and energy via the Entner-Douderoff Pathway which utilizes the keto-deoxy phosphogluconate aldolase enzyme (Dijkhuizen, L., P. R. Levering, G. E. DeVries 1992. In: Methane and Methanol Utilizers (Biotechnology Handbooks 5) J. Colin Murrell and Howard Dalton eds. 1992 Pleanum Press NY pp 149-181). This pathway is not energy-yielding as is the case for the Embden-Meyerhof pathway. Thus, utilization of the Entner-Douderoff pathway results in lower cellular production yields and a greater proportion of the carbon produced as carbon dioxide compared to organisms that use the Embden-Meyerhof pathway. Therefore, a more energy efficient carbon processing pathway would greatly enhance the commercial viability of a methanotrophic platform for the generation of materials.
As noted above, methanotrophic bacteria possess the potential to be commercially effective production platforms for materials such as single cell protein, exopolysaccharides, and long chain carbon molec

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