US 2005O130160A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0130160 A1 Chew et al. (43) Pub. Date: Jun. 16, 2005 (54) OVER-EXPRESSION OF EXTREMOZYME (86) PCT No.: PCT/US03/04288 GENES IN PSEUDOMONADS AND CLOSELY RELATED BACTERIA (30) Foreign Application Priority Data (75) Inventors: Lawrence C. Chew, San Diego, CA Feb. 13, 2002 (US) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - USO2/04294 (US); Stacey L. Lee, San Diego, CA O O (US); Henry W. Talbot, San Diego, CA Publication Classification US (US) (51) Int. Cl." ............................ C12O 1/68; CO7H 21/04; Correspondence Address: C12N 9/22; C12N 1/21; C12N 15/74 THE DOW CHEMICAL COMPANY (52) U.S. Cl. ............................. 435/6; 435/69.1; 435/199; INTELLECTUAL PROPERTY SECTION 435/252.3; 435/320.1; 536/23.2 P. O. BOX 1967 MIDLAND, MI 48641-1967 (US) (57) ABSTRACT (73) Assignee: Dow Global Technologies Inc An extremoyZme over-expression System in which (21) Appl. No.: 10/504,505 Pseudomonads and closely related bacteria are used as host cells, and methods and kits for use thereof, extremozymes (22) PCT Filed: Feb. 13, 2003 expressed therefrom. Patent Application Publication Jun. 16, 2005 US 2005/0130160 A1 repb' RSF1 01 0-based expression vector PtaC Exogenous extremozyme CDS transcription terminator US 2005/O13016.0 A1 Jun. 16, 2005 OVER-EXPRESSION OF EXTREMOZYME GENES chiral intermediates for Synthesis of pharmaceutical N PSEUDOMONADS AND CLOSELY RELATED active ingredients, use of other proteases, lipases, and BACTERIA glycosidases having high Stability at high temperatures or in organic Solvents, BACKGROUND 0007 4. Cellulose and gum degradation and process 0001 Enzymes have long found use as biocatalysts in ing, e.g.: paper and pulp bleaching by Xylanases, cel industrial and household processes and, more recently, in lobiohydrolases, beta-glucosidases and beta-gluca medical applications. For example, enzymes are commonly nases for cellulose hydrolysis, thermostable cellulases employed in traditional industrial biotechnological pro and glucanases for degradation of biological gums used cesses Such as the catalytic liquefaction of corn Starch (e.g., in oil recovery; by amylase enzymes), in household processes Such as cata lytic Stain removal (e.g., by Subtilisins and other protease 0008 5. Food and feed processing, e.g.: pectinases, enzymes), and in medical applications Such as catalytic cellulases, and chitinases, galactosidases for lactose thrombolysis for the in vivo dissolution of clots (e.g., by hydrolysis, and phytases for dephosphorylation of urokinase enzymes). It is widely recognized that enzymes phytate in animal feed during high temperature pro having increased Stability under the conditions present in the cessing; intended use, a feature typically described in terms of the 0009. 6. Medical treatments and diagnostic devices half-life of the enzyme’s activity under Such conditions, and kits, e.g.: peroxidases, phosphatases, oxidases, have greater desirability than those with lesser stability. It is carboxylases, and dehydrogenases, also widely recognized as desirable for the enzyme to exhibit a maximal degree of catalytic activity under the 0010) 7. Detergents and household products, e.g.: ther conditions of use, a feature referred to as the enzyme’s mophilic proteases, alkalophilic proteases, and, alka “optima” (stated in the plural to reflect that the maximum line amylases, and possible level(s) of an enzyme’s catalytic activity can vary 0011 8. Other industrial applications, e.g.: biomining with different environmental parameters, e.g., temperature, and bio-leaching of minerals, bioremediation, remedia Salinity, pH, etc.). This means that it is most desirable for an tion of radioactive wastes, antioxidation Systems. enzyme to exhibit both high Stability and catalytic optima under the conditions of the intended use. 0012. The main recognized source for extremozymes is the diverse group of organisms known as extremophiles. 0002 Many intended uses for enzymes have been pro Extremophiles are organisms that have been discovered to posed wherein the environmental conditions include high or thrive under extreme environmental conditions, e.g., in or low temperature, high or low pH, high Salinity, and other near deep Sea hydrothermal vents, hot Springs, high-Salinity conditions that deviate substantially from the environmental lakes, exposed desert Surfaces, glaciers and ice packs. Mem parameterS Supporting more common living things, among bers of this group of organisms include representatives from Such “more common' biotic conditions are, e.g., tempera within each of the following categories, e.g.: prokaryotes tures of about 20-60 C., pH of about 6.0–7.5, and salinity including archaea and bacteria, and eukaryotes including below about 3.5% (w/v). In order to attempt to fulfill these fungi and yeasts, lichens, protists and protozoa, algae and proposed uses, “extremozymes' have been Suggested. mosses, tardigrades and fish. Because organisms of this Extremozymes are generally considered to be enzymes group naturally thrive under environmental eXtremes, they having Significant catalytic activities under extreme envi are viewed as a Source of naturally occurring extremozymes. ronmental conditions, and typically often exhibiting high Accordingly, a number of extremozymes from extremo Stability to and catalytic optima under Such extreme condi philes have been isolated and tested, and found to have the tions. desired advantageous properties of high Stability and cata 0.003 Examples of proposed uses in which extrem lytic optima under proposed, extreme conditions of use. Ozymes could offer particular advantages include, e.g., those However, while the industry has anxiously awaited the listed in Table 2 of M. W. W. Adams & R. M. Kelly, Finding expected widespread commercialization of extremozymes, and Using Hyperthermophilic Enzymes, TIBTECH 16:329 this has not been forthcoming. 332 (1998). Such proposed applications have contemplated 0013 The problem is that extremophiles have been found the use of extremozymes in: either impossible to culture, or at least too difficult to culture 0004 1. Molecular biology, e.g.: employing hyperther on a commercially significant enough Scale to permit cost mophilic DNA polymerases in the Polymerase Chain effective isolation of extremozymes in Sufficient quantity for Reaction (PCR); use of extremophilic DNA ligases in marketing purposes. As a result, genetic engineering has genetic engineering, extremophilic proteases for use in been tried wherein extremozyme genes, isolated from extre research; mophiles, have been transformed into and expressed in common expression host organisms. Chief among these 0005 2. Starch hydrolysis and processing, e.g.: using expression host organisms are E. coli and Bacillus Subtilis. alpha-amylases, beta-amylases, glucoamylases, alpha Yet, these expression hosts, which have been found So glucosidases, pullulanases, amylopululanase, cyclo reliable in producing commercial quantities of non-extrem maltodextrin glucanotransferases, glucose isomerases, Ozyme proteins, have So far been unreliable at producing, or and Xylose isomerases to produce Such products as unable to produce, commercial quantities of extremozymes. oligosaccharides, maltose, glucose Syrups, high fruc Thus, at best, in Spite of the wealth of potential applications tose Syrups, for extremozymes, their use has been limited to Specialized, 0006 3. Chemical synthesis, e.g.: ethanol production; Small-scale applications Such as thermostable DNA poly production of aspartame by thermolysin; production of merases for use in research, Significant industrial Scale use US 2005/O13016.0 A1 Jun. 16, 2005 has not yet been achieved because of the lack of a commer “pilot-scale” fermentors can range from about 50 L to 200 cially viable, industrial Scale extremozyme expression SyS L., 250 L, and even 500 L in volume. Typical industrial scale tem. productions are done in fermentors having a volume of 0.014. Many examples of such attempts at expression of 1,000 L and above; even 10,000 L and 50,000 L fermentors heterologous extremozyme genes have been reported in E. are not uncommon. coli hosts, and occasionally in Bacillus hosts, and the 0018 Thus, Scaling up a 1 L fermentation-scale expres expression levels are typically poor, i.e. less than 5% total Sion System to industrial Scale fermentation is not a trivial cell protein. Representative examples include, e.g.: G. Dong matter. Scaling it up in Such as way as to provide industrial et al., in Appl. Envir. Microbiol. 63(9): 3569-3576 (Septem Scale enzyme production is typically quite a challenge, and ber 1997) (Pyrococcus furiosus amylopullulanase expressed especially So when Starting with a low-productivity expres in E. coli at 10-28 mg/L, i.e. about 1.4% total cell protein Sion System Such as reported in the Connaris and Diruggiero (tcp)); E. Leveque et al., in FEMS Microbiol. Lett. 186(1): references. Nor do these references provide any Suggestion 67-71 (May 1, 2000) (Thermococcus hydrothermalis alpha or guidance as to how to attempt or accomplish Such a amylase expressed in E. coli at less than 5% top, as estimated Scale-up with the expression Systems they describe. from SDS-PAGE); A. Linden et al., in J. Chromatog. B 0019. Third, the use of rich media, e.g., LB medium and Biomed. Sci. Appl. 737(1-2): 253-9 (Jan. 14, 2000) (Pyro others, requires expensive additives Such as peptones and coccuS WOeSei alpha-amylase expressed in E. coli