Proline auxotrophs

A method for selecting transformed methanotrophic cells, the method comprising:

• (a) culturing, in a proline-free culture medium and in the presence of a C1 substrate, a population of methanotrophic proline auxotrophs transformed with a nucleic acid molecule construct having:
• (1) a first recombinant polynucleotide encoding a desired protein;
• and (2) a second recombinant polynucleotide encoding a pyrroline-5-carboxylate reductase, wherein the culturing is under conditions and for a time sufficient to allow expression of the first and second recombinant polynucleotides;
• and
• (b) selecting cells that grow in the proline-free culture medium, whereby cells that grow in the proline-free culture medium correspond to transformed cells.

A method of making a proline auxotroph, the method comprising:

• (a) introducing a chromosomal mutation into a parental methanotrophic microorganism to generate a population of mutagenized methanotrophic microorganisms, wherein the chromosomal mutation is selected from the group consisting of a deletion of an endogenous proC gene and a mutation in an endogenous proC gene;
• (b) culturing the population of mutagenized methanotrophic microorganisms in the presence of methane under two independent sets of culture conditions, wherein the first condition is culturing in a proline-containing liquid culture medium and wherein the second condition is culturing in a proline-free liquid culture medium;
• and
• (c) identifying a mutagenized methanotrophic microorganism from the population of mutagenized methanotrophic microorganisms which grows in a proline-containing culture medium and which does not grow in a proline-free culture medium.