ENGINEERED LIGHT-EMITTING REPORTER GENES AND METHODS OF USE
Compositions and methods are provided for enhanced expression of light emitting reporters. Such reporters are used in methods for monitoring cultures for production of target compounds. Additionally, methods are provided for the use of light emitting reporter systems and other reporter systems for selecting desired traits in an organism and for identifying or optimizing culture conditions.
1 - 33 . (canceled)
34 . A method for selecting mutants with a desired characteristic that produce an intermediate or end product of a fermentative, synthetic, or metabolic pathway at a higher titer, higher yield, or at a higher ratio over a less desired product, comprising:
a) mutagenizing a plurality of recombinant cells that comprise a recombinant nucleic acid molecule comprising an expression control sequence operatively linked with a coding nucleotide sequence encoding a reporter;
b) isolating pure cultures derived from individual mutants;
c) measuring the expression of the reporter in the cultures; and
d) selecting mutants that have a different expression level of the reporter compared to unmutagenized recombinant cells, wherein the different expression level indicates that the intermediate or end product is produced at the higher titer, yield, or ratio.
35 . The method of claim 34 , wherein the reporter is a light-emitting reporter.
36 . The method of claim 34 , wherein the reporter is codon biased for expression in Gram positive bacteria.
37 . The method of claim 34 , wherein the coding nucleotide sequence of the reporter is not naturally occurring.
38 . The method of claim 34 , wherein the recombinant cells are Gram positive bacteria.
39 . The method of claim 38 , wherein the Gram positive bacteria are a Clostridium species.
40 . The method of claim 34 , wherein the different expression level of the reporter is a higher expression level.
41 . The method of claim 40 , wherein the higher expression level correlates with a desired characteristic.
42 . (canceled)
43 . The method of claim 34 , wherein the end product of the fermentative, synthetic, or metabolic pathway is a solvent.
44 . The method of claim 43 , wherein the solvent is an alcohol, an aldehyde, or a ketone.
45 . The method of claim 43 , wherein the solvent is butanol.
46 . The method of claim 34 , wherein the different expression level correlates with higher expression of a gene.
47 . The method of claim 34 , wherein the different expression level of the reporter is a lower expression level.
48 . The method of claim 47 , wherein the lower expression level correlates with a desired characteristic.
49 . The method of claim 34 , wherein the expression control sequence is from a gene that a protein.
50 . The method of claim 49 , wherein the protein is an enzyme in the fermentative, synthetic, or metabolic pathway that produces the intermediate or end product.
51 . A mutant recombinant cell selected by the method of claim 34 .
52 . A culture of mutant recombinant cells selected by the method of claim 34 .
53 . A product produced by culturing the mutant recombinant cell of claim 51 .
54 . A product according to claim 53 , wherein the product is the intermediate of end product of the fermentative, synthetic, or metabolic pathway.
55 . A product according to claim 54 , wherein the product is the end product of the fermentative, synthetic, or metabolic pathway, and wherein the end product is a solvent.
56 . The method of claim 34 , wherein the expression control sequence is from a gene in the pathway of interest and the different expression level of the reporter in the selected mutants is a higher level of expression.
57 . The method of claim 34 , wherein the expression control sequence is from a gene in a competing pathway and the different expression level of the reporter in the selected mutants is a lower level of expression.
58 . The method of claim 35 , wherein the light-emitting reporter is luciferase.
59 . The method of claim 34 , wherein the coding nucleotide sequence encoding the reporter comprises an A/T content of at least 65%.
60 . A method according to claim 34 , further comprising applying selection pressure to the cultures of recombinant mutants to select mutants with a selectable characteristic.
61 . A method according to claim 60 , wherein the selectable characteristic comprises growth at a particular temperature range, increased tolerance to a solvent, ability to grow and adhere to a solid support, increased substrate degradation, increased resistance to low pH, growth at high osmolarity, or increased assimilation of organic acids.