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 at 0.4% yeast extracts, a fact that makes industrial Scale production tcp, as calculated from data presented therein); and C Pire et Significantly cost disadvantaged. In fact, for most proposed al., in FEMS Microbiol. Lett. 200(2): 221-27 (Jun. 25, 2001) uses in which extremozymes could replace existing indus (25-40 mg/L yield of a halophilic glucose dehydrogenase trial enzymes, this cost disadvantage would make it too expressed in E. coli). expensive to Supply extremozymes to the market for indus 0.015 Two exceptions to the rule of poor expression of trial use. extremozymes are reported, both for hyperthermophilic dehydrogenases expressed in E. coli, at levels of 50% tep 0020 Hence, the biotechnology industry continues to and 15% top, respectively. See, H. Connaris et al., in lack a commercially viable, industrial Scale extremozyme Biotech. Bioeng. 64(1): 38-45 (Jul. 5, 1999) (Haloferax expression System. volcanii dihydrolipoamide dehydrogenase expressed in E. coli at 50% top); and J. Diruggiero & F. T. Robb, in Appl. SUMMARY OF THE INVENTION Environ. Microbiol. 61(1): 159-164 (January 1995) (Pyro 0021. The present invention provides novel means for coccuS furiosus glutamate dehydrogenase expressed in E. overexpression of extremozymes, native to extremophilic coli at 15% tep). However, even these examples fail to organisms, on a commercial Scale. In a more Specific aspect, provide a commercially viable, industrial Scale extrem the invention teaches commercial Scale production of these Ozyme expression System for the following reasons. extremozymes by overexpression in host cell Species 0016 First, the E. coli host cells used in the expression Selected from Pseudomonads and closely related bacteria. Systems reported by Connaris and Diruggiero grow on a rich 0022. These extremozyme expression Systems according medium, which can Support a maximum cell density of to the present invention are capable of overexpressing the about 2 g/L (maximum biomass accumulation Stated in extremozymes at high levels, at greater than 5% total cell terms of dry cell weight). At Such a low cell density, even an protein, greater than 30% total cell protein, and Still higher expression level of 50% tep (total cell protein), results in a levels. These extremozyme expression Systems according to yield far too low for industrial scale production. For the present invention are capable of obtaining high cell example, with a maximum biomass of 2 g/L, the total cell densities, with a dry weight biomass of greater than 20 g/L protein content is approximately 1 g/L, thus, at a 50% top and even greater than 80 g/L, and are capable of maintaining expression level, only about 0.5 g/L of the extremozyme high levels of extremozyme expression at these high cell would be expressed. An expression System providing a total densities, thereby providing a high level of total productivity productivity of only about 0.5 g/L extremozyme is far too of extremozyme. These extremozyme expression Systems low to be considered capable of industrial Scale production. according to the present invention are also capable of This is especially highlighted when considered in light of the industrial Scale fermentation, at or above the 10-Liter Scale, bulk quantities of extremozymes required to enable market while maintaining high levels of total productivity. In addi Supply for the majority of proposed industrial processing tion, the extremozyme expression Systems according to the and household product uses (most of which are premised on present invention retain these abilities when grown on large-scale, mass production). Simple, inexpensive media, Such as carbon Source-Supple 0017 Second, the largest scale of fermentation reported mented mineral Salts media. by either of the Connaris and Diruggiero references is a 0023 The present invention also provides: one-liter (1 L) fermentation, which is far too low to be considered “industrial Scale' fermentation. Generally, the 0024. A recombinant bacterial host cell genetically engi lowest limit for any cognizable industrial Scale fermentation neered to contain an expression vector operative therein, the is about 10 L, though for most purposes this is still consid expression vector containing a nucleic acid containing an ered a Small “seed-Scale' fermentor. However, Some, Small exogenous extremozyme coding Sequence operably linked scale commercial uses can be provided by 5 L or 10 L to a control Sequence, Said host cell being capable of fermentation if the total productivity of the expression overexpressing Said coding Sequence, So as to produce Said System is Sufficiently high. Common “seed-Scale” fermen extremozyme at a total productivity of at least 1 g/L, when tors also include 20 L and 40 L fermentors, common grown on a medium under conditions permitting expression, US 2005/O13016.0 A1 Jun. 16, 2005

characterized in that the bacterial host cell is Selected from a quantity of a bacterial host cell Selected from the the Pseudomonads and closely related bacteria. Pseudomonads and closely related bacteria; a quantity of an 0.025. An extremozyme overexpression system compris expression vector operative in Said bacterial host cell and ing a recombinant bacterial host cell, an expression vector containing a control Sequence and an exogenous extrem operative in Said host cell, the expression vector containing Ozyme coding Sequence operably linked thereto; instructions a nucleic acid containing an exogenous extremozyme coding for transforming Said expression vector into Said bacterial Sequence operably linked to a control Sequence, Said expres host cell to form a recombinant bacterial host cell; and Sion System being capable of overexpressing Said coding instructions for growing Said recombinant bacterial host cell Sequence So as to produce Said extremozyme at a total on a medium under conditions permitting expression; and productivity of at least 1 g/L when grown on a medium optionally, a quantity of Said medium; and optionally, a under conditions permitting expression, characterized in that quantity of an inducer for a regulated promoter where Said the bacterial host cell is selected from the Pseudomonads control Sequence utilizes Said regulated promoter. and closely related bacteria. 0031) Any of the above wherein the extremozyme is a 0026. A process for overexpressing an extremozyme at a hydrolase. Any of the above wherein the extremozyme is a total productivity of at least 1 g/L, comprising the Steps of cellulase or amylase; or a peptidase. Any of the above providing (a) a bacterial host cell Selected from the wherein the extremozyme is an amylase, or a Serine Pseudomonads and closely related bacteria, (b) an expres endopeptidase or aspartic endopeptidase. Any of the above Sion vector operative in Said host cell and containing a wherein the extremozyme is an alpha-amylase, or a pyrol nucleic acid containing an exogenous extremozyme coding ySin or thermopsin. The extremozyme expressed according Sequence operably linked to a control sequence, and (c) a to any of the above. Use of an extremozyme expressed medium; transforming Said expression vector into Said bac according to any of the above in a biocatalytic process. terial host cell to form a recombinant bacterial host cell; and 0032) Any of the above wherein the host cell is a growing Said recombinant bacterial host cell on the medium Pseudomonas species. Any of the above wherein the host under conditions permitting expression; and optionally lyS cell is a fluorescent Pseudomonas Species. Any of the above ing the host cell and Separating, isolating, or purifying the wherein the host cell is Pseudomonas fluorescens. extremozyme therefrom. 0033) Any of the above wherein the expression vector is 0027. A method for overexpressing an extremozyme, at a RSF1010 or a derivative thereof. Any of the above wherein total productivity of at least 1 g/L, comprising: (1) trans the heterologous extremozyme promoter is P forming an expression vector, containing a nucleic acid tact containing an exogenous extremozyme coding Sequence 0034) Any of the above wherein the extremozyme is operably linked to a control Sequence, into a bacterial host expressed in an inclusion body within the host cell and the cell selected from the Pseudomonads and closely related inclusion body is solubilized. Any of the above wherein the bacteria to produce a recombinant bacterial host cell; and (2) extremozyme is refolded using a refolding Step. growing Said recombinant bacterial host cell on a medium under conditions permitting expression; and optionally lyS BRIEF DESCRIPTION OF DRAWINGS ing the host cell and Separating, isolating, or purifying the 0035 FIG. 1 presents a plasmid map of an RSF1010 extremozyme therefrom. based expression vector useful in expressing extremozyme 0028. Use, in a method for overexpressing an extrem genes according to the present invention. Ozyme at a total productivity of at least 1 g/L from a recombinant bacterial host cell grown on a medium under DETAILED DESCRIPTION OF PREFERRED conditions permitting expression, of a recombinant bacterial EMBODIMENTS host cell selected from the Pseudomonads and closely 0036) The present invention provides a commercial scale related bacteria. production System for extremozymes in which 0029. A commercial kit for overexpressing an extrem Pseudomonads and closely related bacteria are used as host Ozyme at a total productivity of at least 1 g/L, comprising: cells to over-express the extremozymes. Pseudomonas spp. a quantity of a bacterial host cell Selected from the have previously been use as expression Systems. See, e.g., Pseudomonads and closely related bacteria; a quantity of an U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat. No. 5,128, expression vector operative in Said bacterial host cell and 130 to Gilroy et al.; U.S. Pat. No. 5,281,532 to Rammler et containing a control Sequence; instructions for inserting into al; U.S. Pat. Nos. 5,527,883 and 5,840,554 to Thompson et Said expression vector a nucleic acid containing an exog al.; U.S. Pat. Nos. 4,695,455 and 4,861,595 to Barnes et al.; enous extremozyme coding Sequence, So as to operably link U.S. Pat. No. 4,755,465 to Gray et al.; and U.S. Pat. No. the coding Sequence to the control Sequence, thereby pre 5,169,760 to Wilcox. However, in none of these references paring the expression vector; instructions for Subsequently has it been Suggested that Pseudomonads and closely related transforming Said expression vector into Said bacterial host bacteria would be particularly advantageous at over-express cell to form a recombinant bacterial host cell; and instruc ing extremozymes in commercial quantities, as defined by tions for growing Said recombinant bacterial host cell on a the present invention. medium under conditions permitting expression; and option ally, a quantity of Said medium; and optionally, a quantity of 0037 Glossary an inducer for a regulated promoter where Said control 0038 A and An Sequence utilizes said regulated promoter. 0039. As used herein and in the appended claims, the 0.030. A commercial kit for overexpressing an extrem singular forms “a”, “an', and “the” include both singular and Ozyme at a total productivity of at least 1 g/L, comprising: plural referents unless the context clearly dictates otherwise. US 2005/O13016.0 A1 Jun. 16, 2005

Thus, for example, reference to “a host cell” literally defines tocols in Molecular Biology (1995) (John Wiley & Sons); both those embodiments employing only a Single host cell Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning and those employing a plurality of Such host cells. (1989) (Cold Spring Harbor Laboratory Press, NY); Berger & Kimmel, Methods in Enzymology 152: Guide to Molecu 0040. In and On lar Cloning Techniques (1987) (Academic Press); and 0041 AS used herein in regard to growing organisms by Bukhari et al. (eds.), DNA Insertion Elements, Plasmids and use of a growth medium, the organisms may be said to be Episomes (1977) (Cold Spring Harbor Laboratory Press, grown “in” or “on” the medium. In the expression systems NY). of the present invention, the medium is a liquid medium. 0052 X-gal means 5-bromo-4-chloro-3-indolyl-beta Thus, in this context, the terms “in” and “on” are used D-galactoside Synonymously with one another to indicate growth of the host cells in contact with the medium and generally within 0053 IPTG means Isopropylthio-beta-D-galactoside the bulk of the medium, though Some incidental cell growth 0054 ORF means Open reading frame. at, in, or upon the Surface of the medium is also contem plated. 0.055 tep and % tep 0.042 Comprising 0056. As used herein, the term “tcp” means “total cell protein’ and is a measure of the approximate mass of 0.043 AS used herein, the term “comprising” means that expressed cellular protein per liter of culture. AS used herein, the Subject contains the elements enumerated following the the term “% tep” means “percent total cell protein’ and is a term “comprising as well as any other elements not So measure of the fraction of total cell protein that represents enumerated. In this, the term “comprising is to be construed the relative amount of a given protein expressed by the cell. as a broad and open-ended term; thus, a claim to a Subject “comprising enumerated elements is to be construed inclu 0057 Exogenous and Heterologous Sively, i.e. construed as not limited to the enumerated 0058. The term “exogenous” means “from a source exter elements. Therefore, the term “comprising can be consid nal to a given cell or molecule. The term "heterologous' ered Synonymous with terms Such as, e.g., “having,”“con means “from a source different from a given cell or taining,” or “including.” molecule. In the present application, as is common use in the 0044) The invention, as described herein, is spoken of art, these two terms are used interchangeably, as Synonyms. using the terms “comprising” and “characterized in that.' Both of these terms are used herein to indicate that a given However, words and phrases having narrower meanings object is foreign to the cell or molecule, i.e. not found in than these are also useful as Substitutes for these open-ended nature in the cell or not found in nature with or connected to terms in describing, defining, or claiming the invention more the molecule. narrowly. For example, as used herein, the phrase “consist 0059) Extremophilic ing of means that the Subject contains the enumerated elements and no other elements. In this, the phrase “con 0060) Extremophilic is defined as any condition falling Sisting of is to be construed as a narrow and closed-ended within the parameters listed in Table 1. term. Therefore, the term “consisting of can be considered Synonymous with, e.g.: “containing only or “having TABLE 1. solely”. Parameters Defining “Extremophilic' 0045 Depositories Extremophilic Condition Approximate Definition 0.046 ACAM-Australian Collection of Antarctic Hyperthermophilic 70-130+ C. Microorganisms, Cooperative Research Centre for Antarctic Psychrophilic -2-20 C. And Southern Ocean Environment, University of Tasmania, Halophilic 2-5M salt GPO Box 252C, Hobart, Tasmania 7001, Australia. Acidophilic pH s (4.5 + 0.5) Alkalophilic pH 2 (9 + 0.5) 0047 ATCC-American Type Culture Collection, 10801 Piezophilic 10-80- MPa. University Boulevard, Manassas, Va. 20110-2209, U.S.A. Xerophilic aw < 0.85 *Xerophilic is defined by a dimensionless quantity known as “water 0048 NCIMB-National Collection of Industrial and potential: aw = (Vapor Pressure of Water in Liquid Solution)/(Vapor Marine Bacteria, National Collections of Industrial, Food Pressure of Pure Water), wherein “Liquid Solution' indicates any aqueous and Marine Bacteria, 23 Machar Drive, Aberdeen, AB24 medium or aqueous environment, whether intracellular or extracellular. 3RY, Scotland. 0061 Extremophiles are thus defined as those organisms 0049 UQM-Culture Collection, Department of Micro that readily Survive or thrive under extracellular environ biology, University of Queensland, St. Lucia, Queensland mental conditions falling within these listed parameters. 4067, Australia. Extremophilic enzymes, or extremozymes, are likewise 0050 General Materials & Methods defined with reference to the conditions defined in Table 1, 0051. Unless otherwise noted, standard techniques, vec and these may be either intracellular or extracellular condi tors, control Sequence elements, and other expression System tions. elements known in the field of molecular biology are used 0062). In Some cases, chemophilic (e.g., metalophilic) and for nucleic acid manipulation, transformation, and expres radiophilic conditions are also recognized in the art as Sion. Such Standard techniques, Vectors, and elements can be classes of extremophilic conditions, although these depend found, for example, in: Ausubel et al. (eds.), Current Pro on the type of chemical (e.g., a specific metal or a organic US 2005/O13016.0 A1 Jun. 16, 2005

compound) and the type of radiation, and thus no uniform niques described in: U.S. Pat. Nos. 5,958,672, 6,057,103, definition is included in the present definition of “extremo and 6,280,926 to Short; U.S. Pat. No. 6,261,842 to and WO philic.” 01/81567 of Handelsman et al.; U.S. Pat. No. 6,090,593 to Fleming & Sayler; or in L. Diels et al., Use of DNA probes 0063) Enzymes and plasmid capture in a Search for new interesting envi 0064. As used herein, the term “enzymes' includes: ronmental genes, Sci. Of the Total Environ. 139-140: 471-8 (Nov. 1, 1993). In addition, the techniques described in the 0065 1. Oxidoreductases (IUBMB EC 1: including, following references may also be used: S. Jorgensen et al., e.g., monooxygenases, cytochromes, dioxygenases, in J. Biol. Chem. 272(26): 16335-42 (Jun. 27, 1997); EP dehydrogenases, metalloreductases, ferredoxins, Patent No. 577257 B1 to Laderman & Anfinsen; EP Patent thioredoxins); No. 579360B1 to Asada et al.; EP 648843A1 of Taguchi et 0066 2. Transferases (IUBMB EC 2: including, e.g., al.; WO 98/45417 of Zeikus et al.; U.S. Pat. No. 6,100,073 glycosyltransferases, alkyltransferases, acyltrans to Deweer & Amory; and G. Dong et al., in Appl. Environ. ferases, carboxyltransferases, fatty acyl Synthases, Microbiol. 63(9): 3577-84 (September 1997). kinases, RNA and DNA polymerases, reverse tran 0075 Once obtained, the extremozyme-encoding nucleic Scriptases, nucleic acid integrases); acids may be altered and expressed to obtain an extrem 0067 3. Hydrolases (IUBMB EC 3: including, e.g., Ozyme exhibiting improvement in or toward a desired cata glycosylases, glycosidases, glucohydrolases, gluca lytic property. Such alteration may be accomplished by use nases, amylases, cellulases, peptidases and proteases, of one or more rounds of nucleic acid mutagenesis and/or nucleases, phosphatases, lipases, nucleic acid recom recombination, resulting in formation of a library compris ing altered nucleic acids, followed by or, if desired when binases); using multiple rounds, regularly or intermittently alternating 0068 4. Lyases (IUBMB EC 4: including, e.g., decar with) expression of the library and Screening of the resulting boxylases, RUBISCOS, adenylate cyclases); enzymes. The nucleic acid mutagenesis and recombination technique(s) Selected may be in vitro techniques or in vivo 0069) 5. Isomerases (IUBMB EC 5: including, e.g., or in cyto techniques, and may be random techniques racemases, epimerases, mutases, topo-isomerases, (random mutagenesis, random recombination) or directed gyrases, foldases); and techniques (e.g., oligonucleotide-directed mutagenesis, site 0070) 6. Ligases (IUBMB EC 6: including, e.g., car directed recombination). Many Such mutagenesis and boxylases, acyl Synthetases, peptide Synthetases, recombination techniques are commonly known in the art. nucleic acid ligases). For example, any of the techniques described in U.S. Pat. No. 5,830,696, 5,965,408, or 6,171,820 to Short; in U.S. Pat. 0.071) Extremozymes Nos. 5,605,793 and 5,811,238 to Stemmer et al.; and WO 0.072 A wide range of extremozymes are known in the art 98/42832 to Arnold et al. may be used; in addition, mutagen See, e.g., references 11-20. AS used herein, the term esis may be performed by use of the technique, Error-Prone “extremozyme” means an enzyme exhibiting an optimum of PCR (also referred to as Low-Fidelity PCR). at least one catalytic property under at least one extremo 0076. In a preferred embodiment, the extremozyme will philic condition as defined in Table 1, and encoded by either: be selected from among any of the classes, IUBMB EC 1-6. 1) nucleic acid obtained from an extremophilic organism; or In a preferred embodiment, the extremozyme will be 2) nucleic acid obtained from an extremophilic organism selected from among any of the classes, IUBMB EC 2-6. In and further altered by mutagenesis and/or recombination as a preferred embodiment, the extremozyme will be Selected described below. In a preferred embodiment, the extremo from among any of the classes, IUBMB EC 2-5. In a philic organism will be an extremophilic Archaeon, extre preferred embodiment, the extremozyme will be selected mophilic bacterium, or extremophilic eukaryote. Particu from among either of the classes, IUBMB EC2-3. In a larly preferred extremophilic eukaryotes include preferred embodiment, the extremozyme will be selected extremophilic fungi and extremophilic yeasts. In a particu from among any of the enzymes within IUBMB EC 3, i.e. larly preferred embodiment, the organism will be an extre extremophilic hydrolases. In a preferred embodiment, the mophilic Archaeon or an extremophilic bacteria. extremozyme will be Selected from among any of the enzymes within IUBMB EC 3.1-3.8. In a preferred embodi 0.073 Whether the extremozyme-encoding nucleic acid is ment, the extremozyme will be selected from among any of native or altered, the codons of the coding sequence(s) of the the enzymes within IUBMB EC 3.1-3.2. In a preferred nucleic acid may be optimized according to the codon usage embodiment, the extremozyme will be Selected from among frequency of a host cell in which it is to be expressed. The any of the enzymes within IUBMB EC 3.2, i.e. extremo catalytic property in which the optimum is exhibited may be, philic glycosylases. In a preferred embodiment, the extrem e.g.: catalytic activity per Se or enzymatic throughput, a Ozyme will be Selected from among any of the enzymes metric Such as K., k, k, kii, or V, or stability (catalytic within IUBMB EC 3.2.1, i.e. extremophilic glycosidases. In half-life) under conditions of use or proposed use. In addi a preferred embodiment, the extremozyme will be Selected tion, the term "extremozyme,” as used herein in reference to from among any of the following enzymes within IUBMB extremozyme expression Systems of the present invention, is EC 3.2.1: amylases, amyloglucosidases, and glucoamylases, restricted to those extremozymes that are heterologous to a cellulases, cellobiohydrolases, endoglucanases, and hemi Selected host cell chosen for expression thereof. cellulases, and beta-glucosidases. In a preferred embodi 0.074) Nucleic acids encoding extremozymes may be ment, the extremozyme will be selected from among any of obtained, e.g., directly from environmental Samples using the following enzymes within IUBMB EC 3.2.1: amylases techniques commonly available in the art, e.g., the tech and cellulases. In a preferred embodiment, the extremozyme US 2005/O13016.0 A1 Jun. 16, 2005 will be Selected from among any of the amylases within 0082 Further examples of useful Pseudomonas expres IUBMB EC 3.2.1, i.e. extremophilic amylases. In a pre Sion vectors include those listed in Table 2. ferred embodiment, the extremozyme wilt be selected from among any of the alpha-amylases within IUBMB EC 3.2.1 TABLE 2 (ie., the enzymes of IUBMB EC 3.2.1.1), thus, the extre mophilic alpha-amylases. Some Examples of Useful Expression Vectors. Promoters, and Inducers 0077. In a preferred embodiment, the extremozyme will Replicon Vector(s) Promoter Inducer Reference be selected from among any of the enzymes within IUBMB pPS10 PCN39, pCN51 None 1. EC 3.4. In a preferred embodiment, the extremozyme will be RSF1010 PKT261-3 2 selected from among any of the enzymes within IUBMB EC PMMB66EH P. IPTG 3 3.4.21 or 3.4.23, i.e. extremophilic Serine peptidases and PEB8 PT7 IPTG 4 extremophilic aspartic endopeptidases. In a preferred PPLGN1 pR Temperature 5 PERD2O/21 Pin Benzoate 6 embodiment, the extremozyme will be selected from among RK2/RP1 PRK415 7 any of the following enzymes within IUBMB EC 3.4.21 and PJB653 8 3.4.23: pyrolysins and thermopsins. pRO1600 PUCP 9 PBSP 1O 0078. In a preferred embodiment, the extremozyme is at least one of hyperthermophilic, psychrophilic, acidophilic, alkalophilic, and halophilic. In a preferred embodiment, the 0083) The expression plasmid, RSF1010, is described, extremozyme is at least one of hyperthermophilic, psychro e.g., by F Heffron et al., in Proc. Nat'l Acad. Sci. USA 7209): philic, acidophilic, and alkalophilic. In a preferred embodi 3623-27 (September 1975), and by K Nagahari & K Sak ment, the extremozyme is at least one of hyperthermophilic, aguchi, in J. Bact. 133(3): 1527-29 (March 1978). Plasmid acidophilic, and alkalophilic. In a preferred embodiment, the RSF1010 and derivatives thereof are particularly useful extremozyme is at least hyperthermophilic. Particularly pre vectors in the present invention. Exemplary, useful deriva ferred are at least hyperthermophilic extremozymes. tives of RSF1010, which are known in the art, include, e.g., 0079. In the extremozyme expression systems of the pKT212, pKT214, pKT231 and related plasmids, and present invention, the extremozyme-encoding nucleic acid pMYC1050 and related plasmids (see, e.g., U.S. Pat. Nos. will be operably linked to a control Sequence, and optionally 5,527,883 and 5,840,554 to Thompson et al.), such a, e.g., other element(s), to form an expression construct (also pMYC1803. Other particularly useful vectors include those called an “expression cassette'), and the resulting expres described in U.S. Pat. No. 4,680,264 to Puhler et al. Sion construct will be inserted into an expression vector; alternatively, the expression cassette can be constructed 0084. In a preferred embodiment, an expression plasmid within the vector by inserting the elements of the expression is used as the expression vector. In a preferred embodiment, cassette into the vector in any other Series of Steps. The RSF1010 or a derivative thereof is used as the expression expression vector will be then be transformed into a bacte vector. In a preferred embodiment, pMYC1050 or a deriva rial host cell according to the present invention, followed by tive thereof, or pMYC1803 or a derivative thereof, is used expression of the extremozyme. as the expression vector. 0080 Vectors 0085 Control Sequences 0.081 Agreat many bacterial vectors are known in the art 0086 The term “control sequence” is defined herein as as useful for expressing proteins in the Gram(-) Proteobac the Set of all elements which are necessary, and optionally teria, and these may be used for expressing the extrem other elements that are advantageous, for the expression of Ozymes according to the present invention. Such vectors an extremozyme in the host cells according to the present include, e.g., plasmids, cosmids, and phage expression Vec invention. Each control Sequence element may be native or tors. Examples of useful plasmid vectors include the expres foreign to the nucleic acid encoding the extremozyme and sion plasmids pMB9, pBR312, pBR322, pML122, RK2, may be native or foreign to the host cell. Such control RK6, and RSF1010. Other examples of such useful vectors Sequence elements include, but are not limited to: promot include those described by, e.g.: N Hayase, in Appl. Envir: ers; transcriptional enhancers, ribosome binding sites (also Microbiol. 60(9): 3336-42 (September 1994); A A Lushni called "Shine Delgarno Sequences”); translational enhancers kov et al., in Basic Life Sci. 30: 657-62 (1985); S Graupner (see, e.g., U.S. Pat. No. 5,232,840 to Olins); leader peptide & WWackernagel, in Biomolec. Eng. 17(1): 11-16. (October encoding Sequences, e.g., for targeting peptides or Secretion 2000); H P Schweizer, in Curr. Opin. Biotech. 12(5): 439-45 Signal peptides, pro-peptide-coding Sequences, transcrip (October 2001); M Bagdasarian & K N Timmis, in Curr. tional and translational Start and Stop signals, polyadenyla Topics Microbiol. Immunol. 96:47-67 (1982); T Ishii et al., tion Signals, and transcription terminators. in FEMS Microbiol. Lett. 116(3): 307-13 (Mar. 1, 1994); IN Olekhnovich & Y KFomichev, in Gene 140(1): 63-65 (Mar. 0087. At a minimum, the control sequence(s) will include 11, 1994); M Tsuda & T Nakazawa, in Gene 136(1-2): a promoter, a ribosome binding Site, and transcriptional and 257-62 (Dec. 22, 1993); C Nieto et al., in Gene 87(1): translational Start and Stop Signals and a transcription ter 145-49 (Mar. 1, 1990); J D Jones & N Gutterson, in Gene minator. The control Sequence elements, vector, and extrem 61(3): 299-306 (1987); M Bagdasarian et al., in Gene Ozyme coding Sequence may be attached to, or extended to 16(1-3): 237-47 (December 1981); H P Schweizer et al., in add, linkers or tails for the purpose of introducing specific Genet. Eng. (NY) 23:69-81 (2001); P Mukhopadhyay et al., Sequences (e.g., restriction sites) facilitating assembly (e.g., in J. Bact. 172(1): 477-80 (January 1990); DO Wood et al., via ligation, recombination, or PCR overlap extension) of in J. Bact. 145(3): 1448-51 (March 1981); and R Holtwick the control sequence elements with the coding sequence(s) et al., in Microbiology 147(Pt 2): 337-44 (February 2001). of the nucleic acid encoding an extremozyme, and with the US 2005/O13016.0 A1 Jun. 16, 2005

vector. The term “operably linked,” as used herein, refers to 0094. Where a negatively regulated promoter is used, the any configuration in which the elements of the control expression System will also contain, or will be genetically Sequence are covalently attached to the coding Sequence in engineered to contain, a gene encoding a repressor protein Such disposition(s), relative to the coding sequence, that in therefor, which gene is expressed, preferably constitutively and by action of the host cell, the control Sequence can direct expressed, in the host cell. The repressor-protein-encoding the expression of the coding Sequence. gene may be contained on the same vector as, or a different vector from, the vector containing the extremozyme-encod 0088 Promoters ing nucleic acid (or it may be contained within the host cell 0089. The promoter may be any nucleic acid sequence chromosome). Examples of useful repressors, and genes that exhibits transcriptional activity in the host cell of encoding them, include those described in U.S. Pat. Nos. choice, and may be a native, mutant, truncated, or hybrid 5,210,025 and 5,356,796 to Keller. promoter, native promoters may be obtained from polypep tide-encoding genes that are either native or heterologous to 0095 Many negatively regulated promoters and nega the host cell. If desired, the nucleic acid containing the tively regulated promoter-repressor combinations are well promoter may remain linked to a ribosome binding site known in the art. Examples of preferred negatively regulated found attached thereto, and optionally to at least part of the promoters include the E. coli tryptophan promoter (P), the coding Sequence controlled thereby, as found in its native E. coli lactose promoter (P) and derivatives thereof (e.g., configuration. (This native coding sequence or portion the tac, tacII, and trc promoters, Pa., Pae, and Ps, thereof, if retained, will be attached to the extremozyme described in U.S. Pat. No. 4,551,433 to DeBoer), the phage coding Sequence, ultimately resulting in expression of an T7 promoter (PT), lambda phage promoters (e.g., WPL, wer), extremozyme-fusion protein.) and the recA promoter from Rhodobacter capsulates. All of the Pa, Pa., Pae, P, and P7 promoters are repressed by 0090 Any of the many promoters known in the art as the lac repressor (lacI). capable of directing transcription in the host cells of the present invention may be selected for use therein. See, e.g., 0096. Where a regulated promoter is used, at an appro Sambrook et al. (1989), Supra. The promoter selected may priate time during the host cell growth cycle, an inducer will be either a constitutive promoter or a regulated promoter, be added to activate or de-repress the regulated promoter. provided that where the extremozyme is expressed intrac Many positively regulated promoter-activator protein-in ellularly (ie., where it is not secreted or otherwise delivered ducer combinations and many negatively regulated pro to a point beyond the host cell's cytoplasm) a constitutive moter-repressor protein-inducer combinations, effective in promoter is preferably not used. the host cells of the present invention are well known in the 0.091 Where a regulated promoter is selected, it may be art. For example, in the case of P, benzoate will serve as either a positively or negatively regulated promoter. A an inducer, and in the case of Pa, Pa., Pae, P, and P7, positively regulated promoter is one that is regulated, via one preferred inducer is IPTG. Also see Table 2. Where an transcriptional activation by an activator protein, to begin extremozyme is expressed intracellularly within the host transcribing mRNA upon induction. A negatively regulated cell, preferably the inducer for the regulated promoter will promoter is one that is repressed by a repressor protein and be added upon, or shortly prior to, achievement of maximum which permits transcription of mRNA only upon de-repres host cell proliferation, i.e. maximum “cell density.” Espe Sion upon induction. Either a reversibly-inducible or irre cially preferred is to add the inducer at about the mid-log versibly-inducible regulated promoter may be selected. phase of cell proliferation. 0092. Where a positively regulated promoter is used, the 0097. In a preferred embodiment of the present invention, expression System will also contain, or will be genetically a regulated promoter is Selected. In a preferred embodiment, engineered to contain, a gene encoding an activator protein a positively regulated promoter is Selected, preferably P. In therefor, which gene is expressed, preferably constitutively a preferred embodiment, a negatively regulated promoter is expressed, in the host cell. The activator protein-encoding Selected, preferably P. In a preferred embodiment, a gene is preferably contained within the host cell chromo negatively regulated promoter is Selected for use in an Some, or it may be contained on the same vector as, or a intracellular extremozyme expression System according to different vector from, the vector containing the extrem the present invention. In a preferred embodiment, the nega Ozyme-encoding nucleic acid). Many Such positively regu tively regulated promoter is P. and the promoter-repressor lated promoters and positively regulated promoter-activator inducer combination in which the regulated promoter is utilized will be P, -lacI-IPTG. protein combinations are know in the art. For example, See: tac U.S. Pat. Nos. 5,670,350, 5,686,283, and 5,710,031 to 0098. A secreted protein expression system can use either Gaffney et al.; U.S. Pat. No. 5,686,282 to Lam et al.; constitutive or regulated promoters. In a Secreted protein Albright et al., in Annual Rev. Genet. 23:311-336 (1989); expression System, either an extremozyme or an extrem Bourret et al., in Annual Rev. Biochem. 60:401-441 (1991); Ozyme-fusion protein is Secreted from the host cell. A and Mekalanos, J. Bact. 174: 1-7 (1992). regulated promoter for a Secreted protein expression System 0.093 Examples of positively regulated promoters can be Selected from, e.g., any of those regulated promoters include, e.g.: the “meta promoter” (P) from the meta described above. A constitutive promoter for a Secreted operon of the toluene-catabolic-pathway-encoding plasmid protein expression System can be Selected from among any pWW0 of Pseudomonas putida (see N Hugouvieux-Cotte of the large number of constitutive promoters known in the Pattat et al., in J. Bact. 172(12): 6651-60 (December 1990)); art as effective for protein expression in the host cells of the and the araB promoter, which is inducible by addition of present invention. A particularly useful constitutive pro L-arabinose which interacts with the activator (the product moter is the neomycin phosphotransferase II promoter (P- of the arac gene), as described in U.S. Pat. No. 5,028,530. tI) obtained from transposon Tn5. See, e.g., DW Bauer & US 2005/O13016.0 A1 Jun. 16, 2005

A Collmer, Mol. Plant Microbe Interact. 10(3): 369-79 “Gram-Negative Aerobic Rods and Cocci” by R. E. Bucha (April 1997); and C Casavant et al., A novel genetic system nan and N. E. Gibbons (eds.), Bergey's Manual of Deter to direct programmed, high-level gene expression in natural minative Bacteriology, pp. 217-289 (8th ed., 1974) (The environments, Abstracts of the 99th American Society for Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter Microbiology General Meeting (held May 30-Jun. 3, 1999 in “Bergey (1974)”). Table 1 presents the families and genera Chicago, Ill., USA). In a preferred embodiment of a secreted of organisms listed in this taxonomic “Part.” protein expression System, a constitutive promoter is used; in a preferred embodiment of a Secreted protein expression system, P, is used as the promoter for the extremozyme TABLE 3 encoding nucleic acid. Families and Genera Listed in the Part, “Gram-Negative Aerobic Rods and Cocci” (in Bergey (1974)) 0099) Other Elements and Methods Family I. Pseudomonadaceae Gluconobacter Pseudomonas 0100 Other elements may also be included within the Xanthomonas expression System according to the present invention. For Zoogloea example, a tag Sequence that facilitates identification, Sepa Family II. Azotobacteraceae Azomonas ration, purification, or isolation of an extremozyme Azotobacter expressed as a fusion protein there with can be encoded by a Beijerinckia Derxia coding Sequence attached to the coding Sequence of the Family III. Rhizobiaceae Agrobacterium extremozyme. In a preferred embodiment of the present Rhizobium invention, where use of a tag Sequence is desired, the tag Family IV. Methylomonadaceae Methylococcus Sequence is a hexa-histidine peptide and the extremozyme Methylomonas Family V. Halobacteriaceae Haiobacterium coding Sequence is fused to a hexa-histidine-encoding Halococcus Sequence. Similarly, the extremozyme may be expressed as Other Genera Acetobacter a fusion protein with a whole or partial viral Structural Alcaligenes protein, e.g., a viral (or phage) coat protein, by attaching all Bordetella Bruceiia or part of the viral coat protein coding Sequence to the Francisella coding Sequence of the extremozyme. Thermus 0101 Furthermore, one or more marker genes or reporter genes may be used in the expression System to verify expression of the extremozyme. Many Such useful marker or 0105 “Gram(-) Proteobacteria Subgroup 1” contains all reporter genes are known in the art. See, e.g., U.S. Pat. No. Proteobacteria classified thereunder, as well as all Proteo 4,753,876 to Hemming et al., and DL Day et al., in J. Bact. bacteria that would be classified thereunder according to the 157(3): 937-39 (March 1984). In a preferred embodiment, criteria used in forming that taxonomic "Part.” As a result, the marker gene is Selected from among the antibiotic “Gram(-) Proteobacteria Subgroup 1” excludes, e.g.: all resistance-conferring marker genes. In a preferred embodi Gram-positive bacteria; those Gram-negative bacteria, Such ment, the marker gene is Selected from among the tetracy as the Enterobacteriaceae, which fall under others of the 19 cline and kanamycin resistance genes. In a preferred “Parts” of this Bergey (1974) taxonomy; the entire “Family embodiment, a reporter gene is Selected from among those V. Halobacteriaceae "of this Bergey (1974) “Part,” which encoding: (1) fluorescent proteins (e.g., GFP); (2) colored family has since been recognized as being a non-bacterial proteins; and (3) fluorescence- or color-facilitating or -in family of Archaea; and the genus, Thermus, listed within this ducing proteins, the latter class (3) including, e.g., luminases Bergey (1974) “Part,” which genus which has since been and beta-galactoSideSe genes. Beta-galactosidases hydrolze recognized as being a non-Proteobacterial genus of bacteria. X-gal to create a blue-colored derivative. 0106 Also in accordance with this definition, “Gram(-) 0102). Further examples of methods, vectors, and trans Proteobacteria Subgroup 1 further includes those Proteo lation and transcription elements, and other elements useful bacteria belonging to (and previously called species of) the in the present invention are described in, e.g.: U.S. Pat. No. genera and families defined in this Bergey (1974) “Part,” 5,055,294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroy et and which have since been given other Proteobacterial al.; U.S. Pat. No. 5,281,532 to Rammler et al.; U.S. Pat. Nos. taxonomic names. In Some cases, these re-namings resulted 4,695,455 and 4,861,595 to Barnes et al.; U.S. Pat. No. in the creation of entirely new Proteobacterial genera. For 4,755,465 to Gray et al.; and U.S. Pat. No. 5,169,760 to example, the genera AcidovOrax, Brevundimonas, Burkhold Wilcox. eria, Hydrogenophaga, Oceanimonas, Ralstonia, and Stenotrophomonas, were created by regrouping organisms 01.03 Host Cells belonging to (and previously called species of) the genus 0104. Whether in native or altered form, the extrem Pseudomonas as defined in Bergey (1974). Likewise, e.g., Ozyme-encoding nucleic acids will be over-expressed, the genus Sphingomonas (and the genus Blastomonas, according to the present invention, in bacterial host cells derived therefrom) was created by regrouping organisms Selected from Pseudomonads and closely related bacteria. belonging to (and previously called species of) the genus The “Pseudomonads and closely related bacteria,” as used Xanthomonas as defined in Bergey (1974). Similarly, e.g., herein, is co-extensive with the group defined herein as the genus Acidomonas was created by regrouping organisms “Gram(-) Proteobacteria Subgroup 1.”“Gram(-) Proteobac belonging to (and previously called species of) the genus teria Subgroup 1 is more specifically defined as the group Acetobacter as defined in Bergey (1974). Such subsequently of Proteobacteria belonging to the families and/or genera reassigned species are also included within “Gram(-) Pro described as falling within that taxonomic “Part” named teobacteria Subgroup 1 as defined herein. US 2005/O13016.0 A1 Jun. 16, 2005

0107. In other cases, Proteobacterial species falling 0111 Exemplary host cell species of “Gram(-) Proteo within the genera and families defined in this Bergey (1974) bacteria Subgroup 2 include, but are not limited to the "Part” were simply reclassified under other, existing genera following bacteria (with the ATCC or other deposit numbers of Proteobacteria For example, in the case of the genus of exemplary Strain(s) thereof shown in parenthesis): Aci Pseudomonas, Pseudomonas enalia (ATCC 14393), domonas methanolica (ATCC 43581); Acetobacter aceti Pseudomonas nigrifaciens (ATCC 19375), and Pseudomo (ATCC 15973); Gluconobacter oxydans (ATCC 19357); nas putrefaciens (ATCC 8071) have since been reclassified Brevundimonas diminuta (ATCC 11568); Beijerinckia respectively as AlterOmonas haloplanktis, Alteromonas indica (ATCC 9039 and ATCC 19361); Derxia gummosa nigrifaciens, and AlterOmonas putrefaciens. Similarly, e.g., (ATCC 15994); Brucella melitensis (ATCC 23456), Bru Pseudomonas acidovorans (ATCC 15668) and Pseudomo cella abortus (ATCC 23448); Agrobacterium tumefaciens nas testosteroni (ATCC 11996) have since been reclassified (ATCC 23308), Agrobacterium radiobacter (ATCC 19358), as Comamonas acidovOranS and Comamonas testosteroni, Agrobacterium rhizogenes (ATCC 11325); Chelatobacter respectively; and Pseudomonas nigrifaciens (ATCC 19375) heintzii (ATCC 29600); Ensifer adhaerens (ATCC 33212); and Pseudomonas piscicida (ATCC 15057) have since been Rhizobium leguminosarum (ATCC 10004); Sinorhizobium reclassified respectively as PseudoalterOmonas nigrifacienS fredii (ATCC 35423); Blastomonas natatoria (ATCC and PseudoalterOmonas piscicida. Such Subsequently reas 35951); Sphingomonas paucimobilis (ATCC 29837); Alcali signed Proteobacterial Species are also included within genes faecalis (ATCC 8750); Bordetella pertussis (ATCC “Gram(-). Proteobacteria Subgroup 1” as defined herein. 9797); Burkholderia cepacia (ATCC 25416); Ralstonia 0108 Likewise in accordance with this definition, pickettii (ATCC 27511); Acidovorax facilis (ATCC 11228); “Gram(-) Proteobacteria Subgroup 1” further includes Pro Hydrogenophaga flava (ATCC 33.667); Zoogloea ramigera teobacterial Species that have Since been discovered, or that (ATCC 19544); Methylobacter luteus (ATCC 49878); have since been reclassified as belonging, within the Pro Methylocaldum gracile (NCIMB 11912); Methylococcus teobacterial families and/or genera of this Bergey (1974) capsulatus (ATCC 19069); Methylomicrobium agile (ATCC “Part.” In regard to Proteobacterial families, “Gram(-) Pro 35068); Methylomonas methanica (ATCC 35067); Methy teobacteria Subgroup 1 also includes Proteobacteria clas losarcina fibrata (ATCC 700909); Methylosphaera hansonii sified as belonging to any of the families: Pseudomona (ACAM 549); Azomonas agilis (ATCC 7494); Azorhizophi daceae, Azotobacteraceae (now often called by the lus paspali (ATCC 23833); Azotobacter chroococcum Synonym, the "Azotobacter group” of Pseudomonadaceae), (ATCC 9043); Cellvibrio mixtus (UQM 2601); Oligella Rhizobiaceae, and Methylomonadaceae (now often called urethralis (ATCC 17960); Pseudomonas aeruginosa (ATCC by the Synonym, "Methylococcaceae). Consequently, in 10145), Pseudomonas fluorescens (ATCC 35858); Fran addition to those genera otherwise described herein, further cisella tularensis (ATCC 6223); Stenotrophomonas malto Proteobacterial genera falling within “Gram(-) Proteobac philia (ATCC 13637); Xanthomonas campestris (ATCC teria Subgroup 1 include: 1) Azotobacter group bacteria of 33913); and Oceanimonas doudorofii (ATCC 27123). the genus Azorhizophilus, 2) Pseudomonadaceae family 0112 In a preferred embodiment, the host cell is selected bacteria of the genera Cellvibrio, Oligella, and Teredini from “Gram(-) Proteobacteria Subgroup 3.”“Gram(-) Pro bacter; 3) Rhizobiaceae family bacteria of the genera Che teobacteria Subgroup 3” is defined as the group of Proteo latobacter, Ensifer, Liberibacter (also called “Candidatus bacteria of the following genera: Brevundimonas, Agrobac Liberibacter”), and Sinorhizobium; and 4) Methylococ terium, Rhizobium, Sinorhizobium, Blastomonas, caceae family bacteria of the genera Methylobacter, Methy Sphingomonas; Alcaligenes, Burkholderia, Ralstonia, Aci localdum, Methylomicrobium, Methylosarcina, and Methy dovorax, Hydrogenophaga, Methylobacter, Methylocal losphaera. dum, Methylococcus, Methylomicrobium, Methylomonas; Methylosarcina, Methylosphaera, Azomonas, Azorhizophi 0109. In a preferred embodiment, the host cell is selected lus, Azotobacter, Cellvibrio, Oligella, Pseudomonas, Tere from “Gram(-) Proteobacteria Subgroup 1,” as defined dinibacter, Francisella, Stenotrophomonas, Xanthomonas; above. and Oceanimonas. 0110. In a preferred embodiment, the host cell is selected 0113. In a preferred embodiment, the host cell is selected from “Gram(-) Proteobacteria Subgroup 2.”“Gram(-) Pro from “Gram(-) Proteobacteria Subgroup 4.”“Gram(-) Pro teobacteria Subgroup 2 is defined as the group of Proteo teobacteria Subgroup 4” is defined as the group of Proteo bacteria of the following genera (with the total numbers of bacteria of the following genera: Brevundimonas, Blasto catalog-listed, publicly-available, deposited Strains thereof monas, Sphingomonas; Burkholderia, Ralstonia, indicated in parenthesis, all deposited at ATCC, except as Acidovorax, Hydrogenophaga, Methylobacter, Methylocal otherwise indicated): Acidomonas (2); Acetobacter (93); dum, Methylococcus, Methylomicrobium, Methylomonas; Gluconobacter (37); Brevundimonas (23); Beijerinckia (13); Methylosarcina, Methylosphaera, Azomonas, Azorhizophi Derxia (2); Brucella (4); Agrobacterium (79); Chelatobacter (2); Ensifer (3); Rhizobium (144); Sinorhizobium (24); Blas lus, Azotobacter, Cellvibrio, Oligella, Pseudomonas, Tere tomonas (1); Sphingomonas (27); Alcaligenes (88); Borde dinibacter, Francisella, Stenotrophomonas, Xanthomonas; tella (43); Burkholderia (73); Ralstonia (33); Acidovorax and Oceanimonas. (20); Hydrogenophaga (9); Zoogloea (9); Methylobacter 0114. In a preferred embodiment, the host cell is selected (2); Methylocaldum (1 at NCIMB); Methylococcus (2); from “Gram(-) Proteobacteria Subgroup 5.”“Gram(-) Pro Methylomicrobium (2); Methylomonas (9); Methylosarcina teobacteria Subgroup 5” is defined as the group of Proteo (1); Methylosphaera, Azomonas (9); Azorhizophilus (5); bacteria of the following genera: Methylobacter, Methylo Azotobacter (64); Cellvibrio (3); Oligella (5); Pseudomonas caldum, Methylococcus; Methylomicrobium, (1139); Francisella (4); Xanthomonas (229); Stenotroph Methylomonas; Methylosarcina, Methylosphaera, Azomo Omonas (50); and Oceanimonas (4). nas, Azorhizophilus, Azotobacter, Cellvibrio, Oligella, US 2005/O13016.0 A1 Jun. 16, 2005 10

Pseudomonas, Teredinibacter, Francisella, Stenotrophomo teobacteria Subgroup 15” is defined as the group of Proteo nas, Xanthomonas; and Oceanimonas. bacteria of the genus Pseudomonas. 0115) In a preferred embodiment, the host cell is selected 0.125. In a preferred embodiment, the host cell is selected from “Gram(-) Proteobacteria Subgroup 6.”“Gram(-) Pro from “Gram(-) Proteobacteria Subgroup 16.”“Gram(-) Pro teobacteria Subgroup 6' is defined as the group of Proteo teobacteria Subgroup 16' is defined as the group of Proteo bacteria of the following genera: Brevundimonas, Blasto bacteria of the following Pseudomonas species (with the monas, Sphingomonas; Burkholderia, Ralstonia, ATCC or other deposit numbers of exemplary strain(s) AcidovOrax, Hydrogenophaga, Azomonas, Azorhizophilus, shown in parenthesis): Pseudomonas abietaniphila (ATCC Azotobacter, Cellvibrio, Oligella, Pseudomonas, Teredini 700689); Pseudomonas aeruginosa (ATCC 10145); bacter, Stenotrophomonas, Xanthomonas; and Oceanimo Pseudomonas alcaligenes (ATCC 14909); Pseudomonas S. anguilliseptica (ATCC 33660); Pseudomonas citronellolis (ATCC 13674); Pseudomonas flavescens (ATCC 51555); 0116. In a preferred embodiment, the host cell is selected Pseudomonas mendocina (ATCC 25411); Pseudomonas from “Gram(-) Proteobacteria Subgroup 7..”“Gram(-) Pro nitroreducens (ATCC 33634); Pseudomonas oleovorans teobacteria Subgroup 7” is defined as the group of Proteo (ATCC 8062); Pseudomonas pseudoalcaligenes (ATCC bacteria of the following genera: Azomonas, Azorhizophilus, 17440); Pseudomonas resinovorans (ATCC 14235); Azotobacter, Cellvibrio, Oligella, Pseudomonas, Teredini Pseudomonas Straminea (ATCC33636); Pseudomonas aga bacter, Stenotrophomonas, Xanthomonas; and Oceanimo rici (ATCC 25941); Pseudomonas alcaliphila, Pseudomo S. naS alginovOra, Pseudomonas anderSOnii, Pseudomonas 0117. In a preferred embodiment, the host cell is selected asplenii (ATCC 23835); Pseudomonas azelaica (ATCC from “Gram(-) Proteobacteria Subgroup 8.”“Gram(-) Pro 27162); Pseudomonas beijerinckii (ATCC 19372); teobacteria Subgroup 8” is defined as the group of Proteo Pseudomonas borealis, Pseudomonas boreopolis (ATCC bacteria of the following genera: Brevundimonas, Blasto 33662); Pseudomonas brassicacearum, Pseudomonas monas, Sphingomonas; Burkholderia, Ralstonia, butanovora (ATCC 43655); Pseudomonas cellulosa (ATCC AcidovOrax, Hydrogenophaga, Pseudomonas, Stenotroph 55703); Pseudomonas aurantiaca (ATCC 33663); Omonas, Xanthomonas, and Oceanimonas. Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, ATCC 17418, ATCC 17461); Pseudomonas fragi (ATCC 0118. In a preferred embodiment, the host cell is selected 4973); Pseudomonas lundensis (ATCC 49968); Pseudomo from “Gram(-) Proteobacteria Subgroup 9.”“Gram(-) Pro nas taetrolens (ATCC 4683); Pseudomonascissicola (ATCC teobacteria Subgroup 9” is defined as the group of Proteo 33616); Pseudomonas coronafaciens, Pseudomonas diter bacteria of the following genera: Brevundimonas; Burkhold peniphila, Pseudomonas elongata (ATCC 10144); eria, Ralstonia, Acidovorax, Hydrogenophaga, Pseudomonas flectens (ATCC 12775); Pseudomonas azoto Pseudomonas, Stenotrophomonas, and Oceanimonas. formans, Pseudomonas brenneri; Pseudomonas cedrella, Pseudomonas corrugata (ATCC 29736); Pseudomonas 0119). In a preferred embodiment, the host cell is selected extremorientalis, Pseudomonas fluorescens (ATCC 35858); from “Gram(-) Proteobacteria Subgroup 10.”“Gram(-) Pro Pseudomonas geSSardii, Pseudomonas libanensis, teobacteria Subgroup 10” is defined as the group of Proteo Pseudomonas mandelii (ATCC 700871); Pseudomonas bacteria of the following genera: Burkholderia, Ralstonia, marginalis (ATCC 10844); Pseudomonas migulae, Pseudomonas, Stenotrophomonas, and Xanthomonas. Pseudomonas mucidolens (ATCC 4685); Pseudomonas ori 0120 In a preferred embodiment, the host cell is selected entalis, Pseudomonas rhodesiae, Pseudomonas Synxantha from “Gram(-) Proteobacteria Subgroup 11.”“Gram(-) Pro (ATCC 9890); Pseudomonas tolaasi (ATCC 33618); teobacteria Subgroup 11” is defined as the group of Proteo Pseudomonas veronii (ATCC 700474); Pseudomonas fred bacteria of the genera: Pseudomonas, Stenotrophomonas; eriksbergensis, Pseudomonas geniculata (ATCC 19374); and Xanthomonas. Pseudomonas gingeri, Pseudomonas graminis, Pseudomo nas grimontii, Pseudomonas halodenitrificans, Pseudomo 0121. In a preferred embodiment, the host cell is selected nas halophila, Pseudomonas hibiscicola (ATCC 19867); from “Gram(-) Proteobacteria Subgroup 12.”“Gram(-) Pro Pseudomonas huttiensis (ATCC 14670); Pseudomonas teobacteria Subgroup 12” is defined as the group of Proteo hydrogenovora, Pseudomonas jessenii (ATCC 700870); bacteria of the following genera: Burkholderia, Ralstonia, Pseudomonas kilonensis, Pseudomonas lanceolata (ATCC Pseudomonas. 14669); Pseudomonas lini; Pseudomonas marginata (ATCC 25417); Pseudomonas mephitica (ATCC 33665); 0122) In a preferred embodiment, the host cell is selected Pseudomonas denitrificans (ATCC 19244); Pseudomonas from “Gram(-) Proteobacteria Subgroup 13.”“Gram(-) Pro pertucinogena (ATCC 190); Pseudomonas pictorum (ATCC teobacteria Subgroup 13” is defined as the group of Proteo 23328); Pseudomonas psychrophila, Pseudomonas fulva bacteria of the following genera: Burkholderia, Ralstonia, (ATCC 31418); Pseudomonas monteilii (ATCC 700476); Pseudomonas; and Xanthomonas. Pseudomonas mosselii, Pseudomonas Oryzihabitans (ATCC 0123. In a preferred embodiment, the host cell is selected 43272); Pseudomonas plecoglossicida (ATCC 700383); from “Gram(-) Proteobacteria Subgroup 14.”“Gram(-) Pro Pseudomonas putida (ATCC 12633); Pseudomonas reac teobacteria Subgroup 14” is defined as the group of Proteo tans, Pseudomonas Spinosa (ATCC 14606); Pseudomonas bacteria of the following genera: Pseudomonas and Xanth balearica, Pseudomonas luteola (ATCC 43273); Pseudomo OilotS. nas Stutzeri (ATCC 17588); Pseudomonas amygdali (ATCC 33614); Pseudomonas avellanae (ATCC 700331); 0.124. In a preferred embodiment, the host cell is selected Pseudomonas caricapapayae (ATCC 33615); Pseudomonas from “Gram(-) Proteobacteria Subgroup 15.”“Gram(-) Pro cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC US 2005/O13016.0 A1 Jun. 16, 2005 11

35104); Pseudomonas fuscovaginae, Pseudomonas meliae preferred embodiment, the host cell is selected from (ATCC 33050); Pseudomonas Syringae (ATCC 19310); “Gram(-) Proteobacteria Subgroup 19.” Pseudomonas viridiflava (ATCC 13223); Pseudomonas thermocarboxydovorans (ATCC 35961); Pseudomonas 0130 Transformation thermotolerans, Pseudomonas thivervalensis, Pseudomo 0131 Transformation of the host cells with the vector(s) nas vancouverensis (ATCC 700688); Pseudomonas wiscon may be performed using any transformation methodology Sinensis; and Pseudomonas Xiamenensis. known in the art, and the bacterial host cells may be transformed as intact cells or as protoplasts (i.e. including 0126. In a preferred embodiment, the host cell is selected cytoplasts). Exemplary transformation methodologies from “Gram(-) Proteobacteria Subgroup 17.”“Gram(-) Pro include poration methodologies, e.g., electroporation, pro teobacteria Subgroup 17” is defined as the group of Proteo toplast fusion, bacterial conjugation, and divalent cation bacteria known in the art as the “fluorescent Pseudomonads' treatment, e.g., calcium chloride treatment or CaCl/Mg" including those belonging, e.g., to the following Pseudomo treatment. naS Species: Pseudomonas azotoformans, Pseudomonas brenneri; Pseudomonas cedrella, Pseudomonas corrugata, 0132) Fermentation Pseudomonas extremorientalis, Pseudomonas fluorescens; 0.133 AS used herein, the term “fermentation” includes Pseudomonas geSSardii, Pseudomonas libanensis, both embodiments in which literal fermentation is employed Pseudomonas mandelii, Pseudomonas marginalis, and embodiments in which other, non-fermentative culture Pseudomonas migulae, Pseudomonas mucidolens, modes are employed. Fermentation may be performed at any Pseudomonas Orientalis, Pseudomonas rhodesiae, Scale. In a preferred embodiment, The fermentation medium Pseudomonas Synxantha, Pseudomonas tolaaSii, and may be Selected from among rich media, minimal media, Pseudomonas veronii. and mineral Salts media, a rich medium may be used, but is 0127. In a preferred embodiment, the host cell is selected preferably avoided. In a preferred embodiment either a from “Gram(-) Proteobacteria Subgroup 18.”“Gram(-) Pro minimal medium or a mineral Salts medium is Selected. In a teobacteria Subgroup 18' is defined as the group of all preferred embodiment, a minimal medium is Selected. In a Subspecies, varieties, Strains, and other Sub-Special units of preferred embodiment, a mineral Salts medium is Selected. the Species Pseudomonas fluorescens, including those Mineral Salts media are particularly preferred. belonging, e.g., to the following (with the ATCC or other 0.134 Mineral salts media consist of mineral salts and a deposit numbers of exemplary strain(s) shown in parenthe carbon Source Such as, e.g., glucose, Sucrose, or glycerol. Sis): Pseudomonas fluorescenS biotype A, also called biovar Examples of mineral Salts media include, e.g., M9 medium, 1 or biovar I (ATCC 13525); Pseudomonas fluorescens Pseudomonas medium (ATCC 179), Davis and Mingioli biotype B, also called biovar 2 or biovar II (ATCC 17816); medium (see, B D Davis & E S Mingioli, in J. Bact. 60: Pseudomonas fluorescens biotype C, also called biovar 3 or 17-28 (1950)). The mineral salts used to make mineral salts biovar III (ATCC 17400); Pseudomonas fluorescens biotype media include those Selected from among, e.g., potassium F, also called biovar 4 or biovar IV (ATCC 12983); phosphates, ammonium Sulfate or chloride, magnesium Sul Pseudomonas fluorescens biotype G, also called biovar 5 or fate or chloride, and trace minerals Such as calcium chloride, biovar V (ATCC 17518); and Pseudomonas fluorescens borate, and Sulfates of iron, copper, manganese, and zinc. No subsp. cellulosa (NCIMB 0.10462). organic nitrogen Source, Such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral Salts 0128. In a preferred embodiment, the host cell is selected medium. Instead, an inorganic nitrogen Source is used and from “Gram(-) Proteobacteria Subgroup 19.”“Gram(-) Pro this may be Selected from among, e.g., ammonium Salts, teobacteria Subgroup 19” is defined as the group of all aqueous ammonia, and gaseous ammonia. A preferred min Strains of Pseudomonas fluorescenS biotype A. Aparticularly eral Salts medium will contain glucose as the carbon Source. preferred strain of this biotype is P. fluorescens strain In comparison to mineral Salts media, minimal media also MB101 (see U.S. Pat. No. 5,169,760 to Wilcox), and deriva contain mineral Salts and a carbon Source, but are further tives thereof. Supplemented with, e.g., low levels of amino acids, Vita 0129. In a particularly preferred embodiment, the host mins, peptones, or other ingredients, though these are added cell is selected from “Gram(-) Proteobacteria Subgroup 1.” at very minimal levels. In a particularly preferred embodiment, the host cell is 0.135 The extremozyme expression system according to selected from “Gram(-) Proteobacteria Subgroup 2.” In a particularly preferred embodiment, the host cell is Selected the present invention can be cultured in any fermentation from “Gram(-) Proteobacteria Subgroup 3.” In a particu format. For example, batch, fed-batch, Semi-continuous, and larly preferred embodiment, the host cell is selected from continuous fermentation modes may be employed herein. “Gram(-) Proteobacteria Subgroup 5.” In a particularly 0.136 The expression systems according to the present preferred embodiment, the host cell is selected from invention are useful for extremozyme expression at any “Gram(-) Proteobacteria Subgroup 7..” In a particularly Scale (i.e. volume) of fermentation. Thus, e.g., microliter preferred embodiment, the host cell is selected from Scale, centiliter Scale, and deciliter Scale fermentation Vol “Gram(-) Proteobacteria Subgroup 12.” In a particularly umes may be used; and 1 Liter Scale and larger fermentation preferred embodiment, the host cell is selected from Volumes can be used. In a preferred embodiment, the “Gram(-) Proteobacteria Subgroup 15.” In a particularly fermentation volume will be at or above 1 Liter. In a preferred embodiment, the host cell is selected from preferred embodiment, the fermentation volume will be at or “Gram(-) Proteobacteria Subgroup 17.” In a particularly above 5 Liters. In a preferred embodiment, the fermentation preferred embodiment, the host cell is selected from volume will be at or above 10 Liters. In a preferred embodi “Gram(-) Proteobacteria Subgroup 18.” In a particularly ment, the fermentation volume will be at or above 15 Liters. US 2005/O13016.0 A1 Jun. 16, 2005

In a preferred embodiment, the fermentation volume will be chemical, or enzymatic means, See, e.g., P. Prave et al. (eds.), at or above 20 Liters. In a preferred embodiment, the Fundamentals of Biotechnology (1987) (VCH Publishers, fermentation volume will be at or above 25 Liters. In a New York) (especially Section 8.3), following by separation preferred embodiment, the fermentation volume will be at or of the proteins, e.g., by any one or more of microfiltration, above 50 Liters. In a preferred embodiment, the fermenta ultrafiltration, gel filtration, gel purification (e.g., by PAGE), tion volume will be at or above 75 Liters. In a preferred affinity purification, chromatography (e.g., LC, HPLC, embodiment, the fermentation volume will be at or above FPLC), and the like. Alternatively, variations of these com 500 Liters. In a preferred embodiment, the fermentation monly known protein recovery and protein purification volume will be at or above 150 Liters. In a preferred methods can be used which capitalize on the Specific prop embodiment, the fermentation volume will be at or above erties of these enzymes. For example, it has been reported 200 Liters. In a preferred embodiment, the fermentation that hyperthermophilic enzymes can be easily Separated volume will be at or above 250 Liters. In a preferred from cellular materials by heating which resuspends the embodiment, the fermentation volume will be at or above extremozymes while causing precipitation of the other cel 500 Liters. In a preferred embodiment, the fermentation lular proteins and materials; this method is particularly volume will be at or above 750 Liters. In a preferred preferred for use with hyperthermophilic enzymes herein. embodiment, the fermentation volume will be at or above 0141 Where the extremozyme is secreted from the host 1,000 Liters. In a preferred embodiment, the fermentation cell, it can be directly Separated, isolated, and/or purified volume will be at or above 2,000 Liters. In a preferred from the medium. Where the extremozyme is expressed in embodiment, the fermentation volume will be at or above the host cell as, or as part of, an insoluble inclusion body, the 2,500 Liters. In a preferred embodiment, the fermentation inclusion body will be solubilized to permit recovery of volume will be at or above 5,000 Liters. In a preferred functional enzymes. For example, the host cells can be lysed embodiment, the fermentation volume will be at or above to obtain Such inclusion bodies therefrom, and then solubi 10,000 Liters. In a preferred embodiment, the fermentation lized; alternatively, Some extremozyme inclusion bodies can volume will be at or above 50,000 Liters. In a particularly be directly extracted from the host cell by solubilization in preferred embodiment, the fermentation volume will be at or cyto without use of a cell lysis Step. In either embodiment, above 10 Liters. Such Solubilization may result in Some degree of unfolding 0.137 In the present invention, growth, culturing, and/or of the expressed extremozyme. Where solubilization results fermentation of the host cells is performed within a tem in unfolding of the expressed extremozyme, a refolding Step perature range of about 4 C. to about 55 C., inclusive. will preferably follow the Solubilization step. Thus, e.g., the terms “growth' (and “grow,”“growing”), 0.142 Various techniques for solubilizing and refolding “culturing” (and “culture”), and “fermentation” (and “fer the enzymes and other proteins expressed in inclusion ment,”“fermenting”), as used herein in regard to the host bodies are known in the art. See, for example: E De cells of the present invention, inherently and necessarily BernardeZ Clark, Protein refolding for industrial processes, means “growth,”“culturing,” and “fermentation,” within a Curr. Opin. in Biotechnol. 12(2): 202-07 (Apr. 1, 2001); M temperature range of about 4 C. to about 55 C., inclusive. M Carrio & A Villaverde, Protein aggregation as bacterial In addition, “growth” is used to indicate both biological inclusion bodies is reversible, FEBS Lett. 489(1): 29-33 States of active cell division and/or enlargement, as well as (Jan. 26, 2001); R Rudolph & H Lilie, In vitro folding of biological States in which a non-dividing and/or non-enlarg inclusion body proteins, FASEB J. 10: 49-56 (1996); B ing cell is being metabolically Sustained, the latter use of the Fischer et al., in Biotechnol. Bioeng. 41: 3-13 (1993) (refold term “growth' being synonymous with the term “mainte ing of eukaryotic proteins expressed in E. coli); G. Dong et nance.” al., in Appl. Envir. Microbiol. 63(9): 3569-3576 (September 0.138. In addition, growth “under conditions permitting 1997) (refolding of an extremophilic amylase enzyme); A expression' when used in regard to the recombinant bacte Yamagata et al., in Nucl. Acids Res., 29(22): 4617-24 (Nov. rial host cells and expression Systems of the present inven 15, 2001) (urea denaturation to Solubilize a heterologous, tion, is defined herein to mean: (1) growth of the recombi thermophilic Rec.J exonuclease enzyme, followed by refold nant bacterial host cells per se, where the promoter used in ing to obtain an active enzyme); and C Pire et al., in FEMS the control Sequence operably linked to the extremozyme Microbiol. Lett. 200(2) 221-27 (Jun. 25, 2001) (refolding of coding sequence is a constitutive promoter, and (2) where an archaeal halophilic glucose dehydrogenase expressed in the promoter used in the control Sequence operably linked to E. coli). the extremozyme coding Sequence is a regulated promoter, 0143. The extremozyme expressed according to the (a) growth of the recombinant bacterial host cells in the present invention can be used in a biocatalytic process, Such presence of (i.e. in contact with) an inducer therefor, and (b) as described above. Preferred biocatalytic processes are growth of the recombinant bacterial host cells in the absence industrial biocatalytic processes. Once Separated, isolated, of an inducer therfor, followed by addition of such an or purified, the extremozymes can then be used to perform inducer to the System, thereby causing contact between the biocatalysis, e.g., in free-enzyme or immobilized-enzyme cell and the inducer. bioreactors, e.g. in place of current industrial enzymes. 0139 Biocatalyst Preparation Alternatively, once the extremozyme has been expressed (or while it is being expressed) by the host cell, it can be used 0140. Once expressed, the extremozymes can then be in cyto for biocatalysis. For example, the cell can be used as Separated, isolated, and/or purified using any protein recov a biocatalytic unit, e.g., in a whole-cell bioreactor, whether ery and/or protein purification methods known in the art. For a free-cell or immobilized-cell bioreactor; in this format, the example, where the extremozyme is expressed intracellu extremozyme can be expressed intracellularly or on the cell larly, the host cell can be lysed by Standard physical, Surface or can be Secreted or otherwise exported from the US 2005/O13016.0 A1 Jun. 16, 2005

cell. In a preferred embodiment using this format, the least 110 g/L. In a preferred embodiment, the cell density extremozyme is expressed either intracellularly or on the will be at least 120 g/L. In a preferred embodiment, the cell cell Surface. The resulting enzyme or whole-cell bioreactor density will be at least 130 g/L. In a preferred embodiment, can itself be a batch, fed-batch, Semi-continuous, or con the cell density will be at least 140 g/L. In a preferred tinuous bioreactor. embodiment, the cell density will be at least 150 g/L. 0144) Expression Levels 0151. In a preferred embodiment, the cell density will be 0145 The expression systems according to the present at or below 150 g/L. In a preferred embodiment, the cell invention express extremozymes at a level at or above 5% density will be at or below 140 g/L. In a preferred embodi tcp. In a preferred embodiment, the expression level will be ment, the cell density will be at or below 130 g/L. In a at or above 8% tep. In a preferred embodiment, the expres preferred embodiment, the cell density will be at or below sion level will be at or above 10% tep. In a preferred 120 g/L. In a preferred embodiment, the cell density will be embodiment, the expression level will be at or above 15% at or below 110 g/L. In a preferred embodiment, the cell tcp. In a preferred embodiment, the expression level will be density will be at or below 100 g/L. In a preferred embodi at or above 20% tep. In a preferred embodiment, the ment, the cell density will be at or below 90 g/L. In a expression level will be at or above 25% tep. In a preferred preferred embodiment, the cell density will be at or below 80 embodiment, the expression level will be at or above 30% g/L. In a preferred embodiment, the cell density will be at or tcp. In a preferred embodiment, the expression level will be below 75 g/L. In a preferred embodiment, the cell density at or above 40% tep. In a preferred embodiment, the will be at or below 70 g/L. expression level will be at or above 50% tep. 0152. In a preferred embodiment, the cell density will be between 20 g/L and 150 g/L, inclusive. In a preferred 0146 In a preferred embodiment, the expression level embodiment, the cell density will be between 20 g/L and 120 will be at or below 35% tep. In a preferred embodiment, the g/L, inclusive. In a preferred embodiment, the cell density expression level will be at or below 40% tep. In a preferred will be between 20 g/L and 80 g/L, inclusive. In a preferred embodiment, the expression level will be at or below 45% embodiment, the cell density will be between 25 g/L and 80 tcp. In a preferred embodiment, the expression level will be g/L, inclusive. In a preferred embodiment, the cell density at or below 50% tep. In a preferred embodiment, the will be between 30 g/L and 80 g/L, inclusive. In a preferred expression level will be at or below 60% tep. In a preferred embodiment, the cell density will be between 35 g/L and 80 embodiment, the expression level will be at or below 70% g/L, inclusive. In a preferred embodiment, the cell density tcp. In a preferred embodiment, the expression level will be will be between 40 g/L and 80 g/L, inclusive. In a preferred at or below 80% tep. embodiment, the cell density will be between 45 g/L and 80 0147 In a preferred embodiment, the expression level g/L, inclusive. In a preferred embodiment, the cell density will be between 5% top and 80% tep. In a preferred will be between 50 g/L and 80 g/L, inclusive. In a preferred embodiment, the expression level will be between 8% tep embodiment, the cell density will be between 50 g/L and 75 and 70% tep, inclusive. In a preferred embodiment, the g/L, inclusive. In a preferred embodiment, the cell density expression level will be between 10% tep and 70% tep, will be between 50 g/L and 70 g/L, inclusive. In a particu inclusive. In a preferred embodiment, the expression level larly preferred embodiment, the cell density will be at least will be between 15% tep and 70% tep, inclusive. In a 40 g/L. In a particularly preferred embodiment, the cell particularly preferred embodiment, the expression level will density will be between 40 g/L and 80 g/L. be between 20% tep and 70% tep, inclusive. 0153. Total Productivity 0148 Cell Density 0154) In the expression systems according to the present 014.9 The expressions systems according to the present invention, the total productivity, i.e. the total extremozyme invention provide a cell density, i.e. a maximum cell density, productivity, is at least 1 g/L. The factors of cell density and of at least about 20 g/L (even when grown in mineral Salts expression level are Selected accordingly. In a preferred media); the expressions Systems according to the present embodiment, the total productivity will be at least 2 g/L. In invention likewise provide a cell density of at least about 70 a preferred embodiment, the total productivity will be at g/L, as Stated in terms of biomass per Volume, the biomass least 3 g/L. In a preferred embodiment, the total productivity being measured as dry cell weight. will be at least 4 g/L. In a preferred embodiment, the total 0150. In a preferred embodiment, the cell density will be productivity will be at least 5 g/L. In a preferred embodi at least 20 g/L. In a preferred embodiment, the cell density ment, the total productivity will be at least 6 g/L. In a will be at least 25 g/L. In a preferred embodiment, the cell preferred embodiment, the total productivity will be at least density will be at least 30 g/L. In a preferred embodiment, 7 g/L. In a preferred embodiment, the total productivity will the cell density will be at least 35 g/L. In a preferred be at least 8 g/L. In a preferred embodiment, the total embodiment, the cell density will be at least 40 g/L. In a productivity will be at least 9 g/L. In a preferred embodi preferred embodiment, the cell density will be at least 45 ment, the total productivity will be at least 10 g/L. g/L. In a preferred embodiment, the cell density will be at O155 In a particularly preferred embodiment, the expres least 50 g/L. In a preferred embodiment, the cell density will Sion System will have an extremozyme expression level of at be at least 60 g/L. In a preferred embodiment, the cell least 5% top and a cell density of at least 40 g/L, when grown density will be at least 70 g/L. In a preferred embodiment, (i.e. within a temperature range of about 4 C. to about 55 the cell density will be at least 80 g/L. In a preferred C., inclusive) in a mineral Salts medium. In a particularly embodiment, the cell density will be at least 90 g/L. In a preferred embodiment, the expression System will have an preferred embodiment, the cell density will be at least 100 extremozyme expression level of at least 5% top and a cell g/L. In a preferred embodiment, the cell density will be at density of at least 40 g/L, when grown (i.e. within a US 2005/O13016.0 A1 Jun. 16, 2005 temperature range of about 4 C. to about 55 C., inclusive) Luria-Bertani Broth (“LB'), supplemented with 15 lug/mL in a mineral Salts medium at a fermentation Scale of at least tetracycline HCl, in 15 ml Falcon tubes and growth for 10 Liters. 16-20h, at 32° C., 300 rpm. 1 mL of the seed culture (in LB) was placed into 50 mL of the Terrific Broth (TB) medium EXAMPLES (see Table 4), Supplemented with 15 lug/mL tetracycline Example 1 HCl, in 250 ml bottom baffled shake-flasks, and incubated for 5 h at 32 C., 300 rpm. Induction was performed by the Extremophilic Cellulase addition of IPTG to a final concentration of 0.5 mM. Samples were taken at 16-24 hours post-induction. Example 1A Construction of Pseudomonas fluorescens Strains TABLE 3 Expressing Thermotoga maritima and PyrococcuS TB Medium Recipe furiosus Cellulases Bacto tryptone 12 g/L 0156 Methods Bacto yeast extract 24 Glycerol 1O O157 Molecular Biology techniques were as described in KHPO 2.3 Ausubel et al. (eds.), Current Protocols in Molecular Biol KHPO, 12.5 ogy (1995) (John Wiley & Sons); Sambrook, Fritsch, & Maniatis (eds.), Molecular Cloning (1989) (Cold Spring Harbor Laboratory Press, NY). 0.164 Results for shake-flask scale results are presented 0158 Expression Cassettes in Table 5. 0159. The parent plasmid pMYC1803 is a derivative of TABLE 5 pTJS260 (see U.S. Pat. No. 5,169,760 to Wilcox), carrying a regulated tetracycline resistance marker and, the replica List of strains constructed and their performance in shake-flasks tion and mobilization loci from RSF1010 plasmid. Cellulase Yield (pMYC1803 is a source for many derivative plasmids useful Expression by SDS-PAGE in expression extremozymes according to the present inven Cassette Strain Isolates # (g/L) tion. Most such derivatives differ from pMYC1803 prima PTs MB214pMYC1951 5 O.6 rily around the ORF in order to introduce convenient restric 18 O.6 tion sites for cloning different exogenous genes.). Plac MB214pMYC1954 3.1 0.5 3.2 0.5 0160 The Thermotoga maritima cellulase gene (0.94 kb 3.3 0.4 encoding the 314 aa, 38 kD cellulase) and the Pyrococcus 2.2 0.4 furiosus endoglucanase gene (0.90 kb encoding the 300aa, 2.5 O.3 34 kD endoglucanase) were PCR-amplified using primers designed to introduce a Spel Site at the N-terminal end, along with the translational start site of the ORF in 0.165 Results for 10-Liter scale results are presented in pMYC1803, and a XhoI site at the C-terminus of the coding Table 6. Sequences of the genes. The Spe-XhoI fragment of the respective PCR products were independently inserted into TABLE 6 pMYC1803 at the corresponding sites such that the enzyme Performance of representative strains in 10 liter fermentations genes replaced an exogenous gene already present therein hence, their expression was initiated from the tac promoter. Cellulase Yield The resulting constructs, pMYC1954 and plDOW2408, in E. Expression by SDS-PAGE coli JM109 was screened by restriction digests and qualita Cassette Strain Isolates # (g/L) tive enzyme assays and then, alkaline lysis miniprep plasmid Plac MB214pMYC1954 3.1 1O DNA's of the correct constructs were electroporated into P. MB214pMYC1954 2.2 7 fluorescens MB214. MB214pDOW2408 1.2 0161) Host Strain Pseudomonas fluorescens MB214 0162 MB214 is a derivative of MB101 (a wild-type 0166 The cellulases were expressed at levels above 8% prototrophic P. fluorescens Strain), derived by a procedure tcp in both Shake-flask and high cell density fermentor wherein the lacIZYA operon (deleted of the lacZ promoter cultures. The cellulases were separated and tested for activ region) had been integrated into the chromosome to provide ity and were found to be active. a host background where derivatives of the lac promoter can Example 2 be regulated by lactose or IPTG. MB101 is Lac whereas MB214 is Lac". However, MB101 can be rendered Lac+ by Extremophilic Amylases introducing an E. coli lad gene on a plasmid into the Strain. 0.167 Alpha-amylase genes from a Thermococcal and a Example 1B Sulfolobus Solfataricus source were PCR amplified and cloned onto pMYC1803 as in Example 1, so that they Expression of Extremophilic Cellulases became operably linked to a control Sequence including the 0163) Seed cultures were produced as follows. Pfluore P promoter in, an RSF1010-based vector also carrying a Scens MB214 transformants were inoculated into 2-5 mL of tetracycline resistance marker, as shown in FIG. 1. The US 2005/O13016.0 A1 Jun. 16, 2005 resulting constructs were transformed into LacI P. fluore 0173 5. R. Leemans et al., Abroad-host-range expres Scens MB101. The resulting recombinant host cells were Sion vector based on the p promoter of coliphage W. cultured in 10 L fermentors by growth in a mineral salts regulated Synthesis of human interleukin 2 in Erwinia medium (Supplemented with tetracycline and fed with glu and Serratia species, J. Bact. 169: 1899-1904 (1987). cose or glycerol). The transformants were grown in fed batch fermentation cultures, ultimately to cell densities 0.174 6. J. L. Ramos et al., Broad-host-range expres providing biomasses within the range of about 20 g/L to Sion vectors containing manipulated meta-cleavage more than 70 g/L (dry cell weight). The gratuitous inducer pathway regulatory elements of the TOL plasmid, of the P promoter, IPTG, was added to induce expression. FEBS Lett. 226: 241-246 (1988). Thereupon, the amylases were expressed (i.e. over-ex 0.175 7. N. T. Keen et al., Improvedp broad-host-range9. pressed) to a level within the range of about 5% tep to more plasmids for DNA cloning in Gram-negative bacteria, than 30% tep. Thus, total productivity ranged from about 2 Gene 70:191-197 (1988). g/L to over 10 g/L, offering a yield above 100 g of extrem 0176 8. J. M. Blatny et al., Construction and use of a ozyme from a single 0.10 L batch. After host cell lysis, the Versatile Set of broad-host-range cloning and expres extremozymes were purified by microfiltration followed by sion vectors based on the RK2 replicon. Appl. Env. ultrafiltration. The resulting enzymes were characterized and Microbiol. 63: 370-379 (1997); and J. M. Blatney et al., further tested for starch liquefaction activity and found to be Improved broad-host-range RK2 vectors for high and active, hyperthermophilic, and acidophilic. low regulated gene expression levels in Gram-negative Example 3 bacteria, Plasmid 38: 35-51 (1997). 0177) 9. A. A. Watson et al., Construction of improved Extremophilic Proteases Vectors for protein production in Pseudomonas aerugi 0168 Pyrococcus furiosus and Sulfolobus acidocaldarius nosa, Gene 172: 163-164 (1996). protease genes respectively encode pyrolysin (IUBMB EC 0178 10. H. P. Schweizer et al., Vector design and host 3.4.21.-), a serine protease active at 115° C. and pH 6.5-10.5, systems for Pseudomonas, in J. K. Setlow (ed.), and thermopsin (IUBMB EC 3.4.23.42), an acid protease Genetic Engineering, vol. 23 (2001) (Kluwer Plenum operating optimally at 90° C. and pH 2.0, respectively. These Press, New York). genes were PCR amplified and cloned onto pMYC1803 as in Example 1, So that they became operably linked to a 0179 11. J. Eichler, Biotechnological uses of archaeal control Sequence including the P promoter in an extremozymes, Biotech. Adv. 19:261-78 (2001). RSF1010-based vector also carrying a tetracycline resis 0180 12. M. C. Srinivasan et al., Production and tance marker, as shown in FIG. 1. The resulting constructs application of enzymes Stable to and active under were transformed into LacI P. fluorescens MB214. The extreme environments: an overview, Proc. Indian Natl resulting recombinant host cells were cultured in 10 L fermentors by growth in a mineral salts medium (Supple Sci. Acad. 65(Pt. B): 143-62. (1999). mented with tetracycline and fed with glucose or glycerol). 0181. 13. G. A. Sellek & J. B. Chaudhuri, Biocatalysis The transformants were grown in fed-batch fermentation in organic media using enzymes from extremophiles, cultures, ultimately to cell densities providing biomasses Enz. Microb. Technol. 25: 471-82 (1999). within the range of about 20 g/L to more than 70 g/L (dry cell weight). Upon induction with IPTG, the proteases were 0182 14. K. O. Stetter, Extremophiles and their adap expressed to levels within the range of about 5% top to more tation to hot environments, FEBS Lett. 452: 22-25 than 30% tep. Thus, total productivity ranged from about 1 (1999). g/L to over 10 g/L, offering a yield above 100 g of extrem 0183) 15. M. W. W. Adams et al., Extremozymes: Ozyme from a single 10 L batch. Expanding the limits of biocatalysis, Bio/technology 13:662-68. (1995). REFERENCES 0.184 16. D. C. Demirian et al., Enzymes from extre 0169. 1. C. Nieto et al., Cloning vectors, derived from mophiles, Curr. Opin. Chem. Biol. 5: 144-51 (2001). a naturally occurring plasmid of Pseudomonas SavaS tanoi, Specifically tailored for genetic manipulations in 0185. 17. E. Leveque et al., Thermophilic archaeal Pseudomonas, Gene 87: 145-149 (1990). amylolytic enzymes, Enz. Microb. Technol. 26: 3-14. (2000). 0170 2. M. Bagdasarian et al., Molecular and func tional analysis of the broad host range plasmid 0186 18. F. Niehaus et al., Extremophiles as a source RSF1010 and construction of vectors for gene cloning of novel enzymes for industrial application, Appl. in Gram-negative bacteria, in Microbial Drug Resis Microbiol. Biotech. 51: 711-29 (1999). tance. Proceedings of the Third Int'l Symp., pp. 183-97 0187. 19. C. Vieille & G. J. Zeikus, Hyperthermophilic (Tokyo, 1982). enzymes: Sources, uses, and molecular mechanisms for 0171 3. J. P. Fuerste et al., Molecular cloning of the thermostability, Microbiol. Mol. Biol. Rev. 65: 1-43 plasmid RP4 primase region in a multi-host-range tacP (2001). expression vector, Gene 48: 119-131 (1986). 0188 20. D. W. Hough & M. J. Danson, Extrem 0172 4. E. Brunschwig & A. Darzins, A two-compo ozymes, Curr. Opin. Chem. Biol. 3: 39-46 (1999). nent T7 System for the overexpression of genes in 0189 It is to be understood that the preferred embodi Pseudomonas aeruginosa, Gene I11: 35-41 (1992). ments described above are merely exemplary of the present US 2005/O13016.0 A1 Jun. 16, 2005 invention and that the terminology used therein is employed 4. A method for overexpressing an extremozyme, at a total Solely for the purpose of illustrating these preferred embodi productivity of at least 1 g/L, comprising: ments; thus, the preferred embodiments Selected for the above description are not intended to limit the Scope of the (1) transforming an expression vector, containing a present invention. The invention being thus described, other nucleic acid containing an exogenous extremozyme embodiments, alternatives, variations, and obvious alter coding Sequence operably linked to a control Sequence, ations will be apparent to those skilled in the art, using no into a bacterial host cell selected from the more than routine experimentation, as equivalents to those Pseudomonads and closely related bacteria to produce preferred embodiments, methodologies, protocols, vectors, a recombinant bacterial host cell; and reagents, elements, and combinations particularly described (2) growing said recombinant bacterial host cell on a herein. Such equivalents are to be considered within the medium under conditions permitting expression. Scope of the present invention and are not to be regarded as 5. Use, in a method for overexpressing an extremozyme a departure from the Spirit and Scope of the present inven at a total productivity of at least 1 g/L from a recombinant tion. All Such equivalents are intended to be included within bacterial host cell grown on a medium under conditions the Scope of the following claims, the true Scope of the permitting expression, of a recombinant bacterial host cell invention thus being defined by the following claims, as selected from the Pseudomonads and closely related bacte construed under the doctrine of equivalents or like doc ria. trine(s) applicable in the present jurisdiction. 6. A commercial kit for overexpressing an extremozyme What is claimed is: at a total productivity of at least 1 g/L, comprising: 1. A recombinant bacterial host cell genetically engi (1) a quantity of a bacterial host cell Selected from the neered to contain an expression vector operative therein, Pseudomonads and closely related bacteria; the expression vector containing a nucleic acid containing (2) a quantity of an expression vector operative in Said an exogenous extremozyme coding Sequence operably bacterial host cell and containing a control Sequence; linked to a control Sequence, (3) instructions for inserting into said expression vector a Said host cell being capable of overexpressing Said coding nucleic acid containing an exogenous extremozyme Sequence, So as to produce Said extremozyme at a total coding Sequence, So as to operably link the coding productivity of at least 1 g/L, when grown on a medium Sequence to the control Sequence, thereby preparing the under conditions permitting expression, expression vector; characterized in that the bacterial host cell is Selected from the Pseudomonads and closely related bacteria. (4) instructions for Subsequently transforming said 2. An extremozyme overexpression System having: expression vector into Said bacterial host cell to form a recombinant bacterial host cell; and a recombinant bacterial host cell, (5) instructions for growing said recombinant bacterial an expression vector operative in Said host cell, the host cell on a medium under conditions permitting expression vector containing a nucleic acid containing expression; and an exogenous extremozyme coding Sequence operably linked to a control Sequence, (6) optionally, a quantity of said medium; and Said overexpression System being capable of overexpress (7) optionally, a quantity of an inducer for a regulated ing Said coding Sequence So as to produce Said extrem promoter where said control Sequence utilizes Said Ozyme at a total productivity of at least 1 g/L when regulated promoter. grown on a medium under conditions permitting 7. A commercial kit for overexpressing an extremozyme expression, at a total productivity of at least 1 g/L, comprising: characterized in that the bacterial host cell is Selected (1) a quantity of a bacterial host cell Selected from the from the Pseudomonads and closely related bacteria. Pseudomonads and closely related bacteria, 3. A process for overexpressing an extremozyme at a total productivity of at least 1 g/L, comprising the Steps of (2) a quantity of an expression vector operative in Said bacterial host cell and containing a control Sequence (1) providing: and an exogenous extremozyme coding Sequence oper (a) a bacterial host cell selected from the ably linked thereto, Pseudomonads and closely related bacteria, (3) instructions for transforming said expression vector (b) an expression vector operative in said host cell and into Said bacterial host cell to form a recombinant containing a nucleic acid containing an exogenous bacterial host cell, and eXtremozyme coding Sequence operably linked to a (4) instructions for growing said recombinant bacterial control Sequence, and host cell on a medium under conditions permitting (c) a medium; expression; and (2) transforming said expression vector into said bacterial (5) optionally, a quantity of said medium; and host cell to form a recombinant bacterial host cell; and (6) optionally, a quantity of an inducer for a regulated (3) growing said recombinant bacterial host cell on the promoter where said control Sequence utilizes Said medium under conditions permitting expression. regulated promoter. US 2005/O13016.0 A1 Jun. 16, 2005

8. The extremozyme of any one of claims 1-7 wherein the 25. The bacterial host cell of any one of claims 1-7, 16, extremozyme is Selected from among any of the classes, and 18 wherein the bacterial host cell is selected from IUBMB EC2-6. Gram(-) Proteobacteria Subgroup 15. 9. The extremozyme of claim 8 which is selected from among any of the extremophilic enzymes within any of the 26. The bacterial host cell of any one of claims 1-7 classes, IUBMB EC2-5. wherein the bacterial host cell is selected from Gram(-) 10. The extremozyme of claim 9 which is selected from Proteobacteria Subgroup 17. among any of the extremophilic enzymes within any of the 27. The bacterial host cell of any one of claims 1-7, 16, classes, IUBMB EC2-3. and 18 wherein the bacterial host cell is selected from 11. The extremozyme of claim 10 which is selected from Gram(-) Proteobacteria Subgroup 18. among any of the extremophilic enzymes within the class 28. The expression vector of any one of claims 1-7 IUBMB EC 3. wherein the expression vector is selected from RSF1010 and 12. The extremozyme of claim 11 which is selected from derivatives thereof. among any of the extremophilic enzymes within IUBMB EC 31-38. 29. The control sequence of any one of claims 1-7 wherein 13. The extremozyme of claim 12 which is selected from the control Sequence contains a regulated promoter. among any of the extremophilic enzymes within IUBMB EC 30. The regulated promoter of claim 29 which is a 3.1-3.2 or 3.4. negatively regulated promoter 14. The extremozyme of claim 13 which is selected from 31. The negatively regulated promoter of claim 30 which among any of the extremophilic enzymes within IUBMB EC is P tact 3.2 or 3.4. 32. The growth of any one of claims 1-7 wherein said 15. The extremozyme of claim 14 which is selected from growth is done at or above a 10-Liter Scale. among any of the extremophilic enzymes within IUBMB EC 33. The growth of any one of claims 1-7 wherein said 3.2.1.3.4.21, or 3.4.23. growth under conditions permitting expression comprises 16. The extremozyme of claim 15 which is selected from growth of the recombinant bacterial host cells, Said cells the cellulases, amylases, Serine endopeptidases, and aspartic containing a regulated promoter operably linked to the endopeptidases. extremozyme coding Sequence, in the absence of an inducer 17. The extremozyme of claim 16 which is selected from therefor, followed by addition of such an inducer to the the amylases, Serine endopeptidases, and aspartic endopep System. tidases. 18. The extremozyme of claim 17 which is selected from 34. The medium of any one of claims 1-7 wherein said the alpha-amylases, pyrolysin, and thermopsin. medium is Selected from minimal media and carbon Source 19. The bacterial host cell of any one of claims 1-7 Supplemented mineral Salts media. wherein the bacterial host cell is selected from Gram(-) 35. The medium of claim 34 which is a carbon Source Proteobacteria Subgroup 1. Supplemented mineral Salts medium. 20. The bacterial host cell of any one of claims 1-7 36. Any one of claims 3-4 further comprising Separating, wherein the bacterial host cell is selected from Gram(-) isolating, or purifying the extremozyme therefrom. Proteobacteria Subgroup 2. 37. The extremozyme expressed according to any one of 21. The bacterial host cell of any one of claims 1-7 claims 1-7. wherein the bacterial host cell is selected from Gram(-) 38. Use in a biocatalytic process of an extremozyme Proteobacteria Subgroup 3. expressed according to any one of claims 1-7. 22. The bacterial host cell of any one of claims 1-7 wherein the bacterial host cell is selected from Gram(-) 39. The extremozyme of any one of claims 1-7 wherein Proteobacteria Subgroup 5. the extremozyme is expressed in an inclusion body within 23. The bacterial host cell of any one of claims 1-7 the bacterial host cell and said inclusion body is thereafter wherein the bacterial host cell is selected from Gram(-) Solubilized. Proteobacteria Subgroup 7. 40. The extremozyme of any one of claims 1-7 and 39 24. The bacterial host cell of any one of claims 1-7 wherein a refolding Step is used to refold the extremozyme. wherein the bacterial host cell is selected from Gram(-) Proteobacteria Subgroup 12.