Method for the Identification of a Metabolic Pathway Family by Means of Positive Selection

Abstract

The invention relates to the direct selection of metabolic pathways having a determined function in the transformation of a substrate {Ai} into a target product {B}, which is of interest in the industrial, pharmaceutical or agri-food sectors. More specifically, the invention relates to the detection, within metagenomic libraries, of novel biosynthesis pathways involved in a biochemical reaction having a known product {B}. The selection and characterisation of said novel metabolic pathways enables {B} to be produced enzymatically. The invention provides an alternative to the chemical synthesis of the molecule in question {B}. Moreover, and above all, the invention can be used specifically to target and exploit the only metabolic pathways enabling the transformation of {Ai} into {B}, while eliminating the associated metabolic pathways that can catabolise the target product {B}.

1 . A method for selecting or preparing cells comprising at least one metabolic pathway or metabolic pathway family enabling the transformation of one or more substrate(s) {Ai} into a desired product {B}, comprising the following steps:
 a) providing a population of host cells (Ai−; B−) incapable of metabolizing said substrate or substrates {Ai} and said product {B};   b) transforming said population of host cells with a library of sequences of nucleic acid;   c) testing in parallel said population of transformed host cells on minimum media containing either one of the substrates {Ai}, or said product {B} as the only source of an element essential to growth; and   d) selecting said host cell(s) capable of growth on a minimum medium containing one of the substrates {Ai} and on a minimum medium containing said product {B} (Ai+; B+).   
     
     
         2 . The method according to  claim 1 , comprising, before step c), a step consisting of testing said population of transformed host cells on a minimum medium containing the substrate(s) {Ai} and said product {B} as the only source of an element essential to growth and selecting said host cell(s) capable of growth on said minimum medium containing the substrate(s) {Ai} and said product {B}; said selected host cell(s) then being subjected to step c) and the subsequent steps. 
     
     
         3 . The method according to  claim 1 , comprising, after step d), the following steps:
 e) implementing in vitro mutagenesis of the molecule of nucleic acid isolated from said transformed host cell(s) (Ai+; B+) in step d);   f) re-transforming the population of host cells (Ai−; B−) described in step a) with the population of nucleic acids mutated in vitro in step e) and testing the host cell(s) thus transformed on minimum media containing either one of the substrate(s) {Ai}, or said product {B} as the only source of an element essential to growth; and   g) selecting said transformed host cell(s) incapable of growth on a minimum medium containing one of the substrate(s) {Ai} and capable of growth on a minimum medium containing said product {B} (A ; B+), and optionally isolating the mutated molecule of nucleic acid.   
     
     
         4 . The method according to  claim 3 , comprising the characterization of the gene or genes encoding the enzyme or enzymes involved in the conversion of the substrate {Ai} into product {B} in said host cell(s) (Ai−; B+) selected in step g). 
     
     
         5 . The method according to  claim 3 , comprising, after step f), in parallel to step g):
 h) selecting said transformed host cell(s) which has (have) become incapable of growth on a minimum medium containing one of the substrate {Ai} and on a minimum medium containing said product {B} (Ai−; B−);   i) implementing a quantitative analysis of the accumulation of the product {B} of said transformed host cells(s) (Ai−; B−) on a rich medium supplemented by {Ai}; and   j) selecting said transformed host cell(s) (Ai−; B−) accumulating the product {B} on a rich medium and optionally isolating in parallel the mutated molecule of nucleic acid introduced during the transformation of step f).   
     
     
         6 . The method according to  claim 5 , comprising the characterization of the gene or genes encoding the enzyme or enzymes involved in the conversion of the substrate {Ai} into product {B} in said host cell(s) (Ai−; B−) selected in step j). 
     
     
         7 . The method according to  claim 1 , comprising, after step c), in parallel to step d) and the subsequent steps, the following steps:
 k) selecting said transformed host cell(s), incapable of growth on a minimum medium containing one of the substrates {Ai} and capable of growth on a minimum medium containing said product {B}, called receiving cell(s) (Ai−; B+);   l) transforming said receiving cell(s) (Ai−; B+) with a library of sequences of nucleic acid;   m) testing in parallel said transformed receiving cell(s) (Ai−; B+) on a minimum medium containing one of the substrate(s) {Ai};   n) selecting said transformed receiving cell(s) capable of growth on a minimum medium containing one of the substrates {Ai}; and   o) characterizing the gene or genes encoding the enzyme or enzymes involved in the conversion of the substrate {Ai} into product {B} in said transformed receiving cell(s) (Ai+; B+) selected in step n).   
     
     
         8 . The method according to  claim 7 , comprising, before step m), testing said host cell(s) (Ai−; B+) transformed on a minimum medium containing several substrates {Ai} as the only source of an element essential to growth and selecting said host cell(s) capable of growth on said minimum medium containing several substrates {Ai}; said selected host cell(s) then being subjected to step m) and the subsequent steps. 
     
     
         9 . The method according to  claim 7 , wherein:
 between steps k) and l), said host cell(s) (Ai−; B+) is/are modified by replacing the first selection marker present in the vector containing the sequence of nucleic acid introduced in step b) with a new selection marker;   said library of sequences of nucleic acid from step l) includes a selection marker different to that carried by said host cell(s) (Ai−; B+); and   the method further includes the following steps:
 kk) the extraction and purification of the vectors contained in said host cell(s) selected in step k); 
 kkk) the in vitro mutagenesis of said vector purified in step kk), advantageously by transposition with a transposable element carrying a functional resistance to an antibiotic different to that previously existing on this vector; 
 kkkk) the transformation of said host cell(s) (Ai−; B−) incapable of metabolising said substrate(s) {Ai} and said product {B} by the mutated nucleic acids obtained in the previous step; and 
 kkkkk) the selection of transformed host cells containing just said second selection marker; these transformed cells, of phenotype (Ai−B+), called receiving cells, are then the object of the transformation described in step l). 
   
     
     
         10 . The method according to  claim 1 , wherein said host cells are eukaryotic or prokaryotic cells. 
     
     
         11 . The method according to  claim 10 , wherein said host cells are:
 cultivable under standard conditions known by the man skilled in the art,   transformable, and   capable of stably maintaining the transforming exogenous DNA.   
     
     
         12 . The method according to  claim 10 , wherein said host cells are bacteria. 
     
     
         13 . The method according to  claim 1 , wherein said library of sequences of nucleic acid is a metagenomic library. 
     
     
         14 . The method according to  claim 1 , in which said library of nucleic acid sequences originates from cultivatable prokaryotic or eukaryotic organisms. 
     
     
         15 . The method according to  claim 1 , in which said library of nucleic acid sequences originates from non-cultivatable prokaryotic or eukaryotic organisms. 
     
     
         16 . A process for preparing the product {B} from the substrate {Ai} comprising:
 cultivating a host cell incapable of growth on a minimum medium containing one of the substrate(s) {Ai} and capable of growth on a minimum medium containing said product {B} (Ai−; B+); or   cultivating a host cell that has been selected on the basis that it has become: incapable of growth on a minimum medium containing one of the substrate {Ai} and on a minimum medium containing said product {B} (Ai−; B−); and accumulates the product {B} when grown on a rich medium containing the substrate {Ai}; and   recovering the product {B}.   
     
     
         17 . A process for preparing the product {B} from the substrate {Ai} comprising cultivating a host cell transformed with a gene or genes encoding the enzyme or enzymes involved in the conversion of the substrate {Ai} into product {B} on a medium containing the substrate {Ai} and recovering the product {B}. 
     
     
         18 . A method for selecting or preparing a host cell (Ai−; B−) incapable of metabolising said substrate(s) {Ai} and said product {B} comprising the following steps:
 testing a population of host cells, cultivatable under standard laboratory conditions and under industrial production conditions, transformable, and capable of stably maintaining the transforming exogenous DNA, on a minimum medium containing the substrate(s) {Ai} and said product {B} as the only source of an element essential to growth; and, 
 selecting the host cell(s) incapable of growth on said minimum medium containing the substrate(s) {Ai} and said product {B}.