Over-expression of extremozyme genes in pseudomonads and closely related bacteria
An extremoyzme over-expression system in which Pseudomonads and closely related bacteria are used as host cells, and methods and kits for use thereof, extremozymes expressed therefrom.
1 . A recombinant bacterial host cell genetically engineered to contain an expression vector operative therein,
the expression vector containing a nucleic acid containing an exogenous extremozyme coding sequence operably linked to a control sequence,
said host cell being capable of overexpressing said coding sequence, so as to produce said extremozyme at a total productivity of at least 1 g/L, when grown on a medium under conditions permitting expression,
characterized in that the bacterial host cell is selected from the Pseudomonads and closely related bacteria.
2 . An extremozyme overexpression system having:
a recombinant bacterial host cell,
an expression vector operative in said host cell, the expression vector containing a nucleic acid containing an exogenous extremozyme coding sequence operably linked to a control sequence,
said overexpression system being capable of overexpressing said coding sequence so as to produce said extremozyme at a total productivity of at least 1 g/L when grown on a medium under conditions permitting expression,
characterized in that the bacterial host cell is selected from the 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:
(1) providing:
(a) a bacterial host cell selected from the Pseudomonads and closely related bacteria,
(b) an expression vector operative in said host cell and containing a nucleic acid containing an exogenous extremozyme coding sequence operably linked to a control sequence, and
(c) a medium;
(2) transforming said expression vector into said bacterial host cell to form a recombinant bacterial host cell; and
(3) growing said recombinant bacterial host cell on the medium under conditions permitting expression.
4 . A method for overexpressing an extremozyme, at a total productivity of at least 1 g/L, comprising:
(1) transforming an expression vector, containing a nucleic acid containing an exogenous extremozyme coding sequence operably linked to a control sequence, into a bacterial host cell selected from the Pseudomonads and closely related bacteria to produce a recombinant bacterial host cell; and
(2) growing said recombinant bacterial host cell on a medium under conditions permitting expression.
5 . Use, in a method for overexpressing an extremozyme at a total productivity of at least 1 g/L from a recombinant bacterial host cell grown on a medium under conditions permitting expression, of a recombinant bacterial host cell selected from the Pseudomonads and closely related bacteria.
6 . A commercial kit for overexpressing an extremozyme at a total productivity of at least 1 g/L, comprising:
(1) a quantity of a bacterial host cell selected from the Pseudomonads and closely related bacteria;
(2) a quantity of an expression vector operative in said bacterial host cell and containing a control sequence;
(3) instructions for inserting into said expression vector a nucleic acid containing an exogenous extremozyme coding sequence, so as to operably link the coding sequence to the control sequence, thereby preparing the expression vector;
(4) instructions for subsequently transforming said expression vector into said bacterial host cell to form a recombinant bacterial host cell; and
(5) instructions for growing said recombinant bacterial host cell on a medium under conditions permitting expression; and
(6) optionally, a quantity of said medium; and
(7) optionally, a quantity of an inducer for a regulated promoter where said control sequence utilizes said regulated promoter.
7 . A commercial kit for overexpressing an extremozyme at a total productivity of at least 1 g/L, comprising:
(1) a quantity of a bacterial host cell selected from the Pseudomonads and closely related bacteria,
(2) a quantity of an expression vector operative in said bacterial host cell and containing a control sequence and an exogenous extremozyme coding sequence operably linked thereto,
(3) instructions for transforming said expression vector into said bacterial host cell to form a recombinant bacterial host cell, and
(4) instructions for growing said recombinant bacterial host cell on a medium under conditions permitting expression; and
(5) optionally, a quantity of said medium; and
(6) optionally, a quantity of an inducer for a regulated promoter where said control sequence utilizes said regulated promoter.
8 . The extremozyme of any one of claims 1 - 7 wherein the extremozyme is selected from among any of the classes, IUBMB EC2-6.
9 . The extremozyme of claim 8 which is selected from among any of the extremophilic enzymes within any of the classes, IUBMB EC2-5.
10 . The extremozyme of claim 9 which is selected from among any of the extremophilic enzymes within any of the classes, IUBMB EC2-3.
11 . The extremozyme of claim 10 which is selected from among any of the extremophilic enzymes within the class IUBMB EC 3.
12 . The extremozyme of claim 11 which is selected from among any of the extremophilic enzymes within IUBMB EC 3.1-3.8.
13 . The extremozyme of claim 12 which is selected from among any of the extremophilic enzymes within IUBMB EC 3.1-3.2 or 3.4.
14 . The extremozyme of claim 13 which is selected from among any of the extremophilic enzymes within IUBMB EC 3.2 or 3.4.
15 . The extremozyme of claim 14 which is selected from among any of the extremophilic enzymes within IUBMB EC 3.2.1., 3.4.21, or 3.4.23.
16 . The extremozyme of claim 15 which is selected from the cellulases, amylases, serine endopeptidases, and aspartic endopeptidases.
17 . The extremozyme of claim 16 which is selected from the amylases, serine endopeptidases, and aspartic endopeptidases.
18 . The extremozyme of claim 17 which is selected from the alpha-amylases, pyrolysin, and thermopsin.
19 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 1.
20 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 2.
21 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 3.
22 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 5.
23 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 7.
24 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 12.
25 . The bacterial host cell of any one of claims 1 - 7 , 16 , and 18 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 15.
26 . The bacterial host cell of any one of claims 1 - 7 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 17.
27 . The bacterial host cell of any one of claims 1 - 7 , 16 , and 18 wherein the bacterial host cell is selected from Gram(−) Proteobacteria Subgroup 18.
28 . The expression vector of any one of claims 1 - 7 wherein the expression vector is selected from RSF1010 and derivatives thereof.
29 . The control sequence of any one of claims 1 - 7 wherein the control sequence contains a regulated promoter.
30 . The regulated promoter of claim 29 which is a negatively regulated promoter
31 . The negatively regulated promoter of claim 30 which is P tac .
32 . The growth of any one of claims 1 - 7 wherein said growth is done at or above a 10-Liter scale.
33 . The growth of any one of claims 1 - 7 wherein said growth under conditions permitting expression comprises growth of the recombinant bacterial host cells, said cells containing a regulated promoter operably linked to the extremozyme coding sequence, in the absence of an inducer therefor, followed by addition of such an inducer to the system.
34 . The medium of any one of claims 1 - 7 wherein said medium is selected from minimal media and carbon source-supplemented mineral salts media.
35 . The medium of claim 34 which is a carbon source-supplemented mineral salts medium.
36 . Any one of claims 3 - 4 further comprising separating, isolating, or purifying the extremozyme therefrom.
37 . The extremozyme expressed according to any one of claims 1 - 7 .
38 . Use in a biocatalytic process of an extremozyme expressed according to any one of claims 1 - 7 .
39 . The extremozyme of any one of claims 1 - 7 wherein the extremozyme is expressed in an inclusion body within the bacterial host cell and said inclusion body is thereafter solubilized.
40 . The extremozyme of any one of claims 1 - 7 and 39 wherein a refolding step is used to refold the extremozyme.