Cells engineered to contain genes of interest without expressed bacterial sequences and materials and methods therefor

A yeast integration plasmid, comprising:

• a selection gene marker;
• a targeting gene marker;
• a gene of interest;
• and
• a bacterial origin of DNA replication, wherein said plasmid contains neither any expressed bacterial sequence nor any yeast origin of replication.

A method of producing inositol, inositol-containing metabolites, phospholipids, phosphatidyl inositol, and other metabolites thereof, comprising the steps of:

• a) constructing haploid host yeasts of the genus Saccharomyces of each mating type which contain an Opi− phenotype which contains one or more target gene mutations in their genome which are non-lethal to said host and which are selectable;
• b) constructing one or more yeast integration plasmids such that the plasmids will be inserted to corresponding target gene mutation loci in said host, wherein each yeast integration plasmid comprises:
• a selection gene marker;
• a targeting gene marker;
• a gene of interest that is expressed in said host;
• and a bacterial origin of DNA replication, such that said integration plasmids neither contain any expressed bacterial sequence nor any yeast origin of replication;
• c) amplifying episomally each yeast integration plasmid in a bacterial plasmid amplification host microorganism;
• d) purifying the amplified yeast integration plasmids from step c);
• e) sequentially transforming said host yeast with each of the yeast integration plasmids such that each of the said integration plasmids is directed to a specific target gene mutation at each target loci in each of the haploid host cells;
• f) optionally mating the transformed host haploids of opposite mating types to form diploids;
• and
• g) fermenting the resulting genetically modified yeast from step e) or step f) to produce inositol, inositol-containing metabolites, phospholipids, phosphatidyl inositol and other metabolites.

A method of stably inserting at least one gene of interest into a specific locus in the genome of a host haploid yeast without adding any expressed bacterial sequences to said host to form a genetically modified haploid cell, comprising:

• constructing host haploid yeast strains to contain a non-lethal, selectable gene mutation, a target gene mutation, in at least one gene of said haploid;
• constructing at least one or a suite of a plurality of yeast integration plasmids, wherein each member plasmid comprises, a selection gene marker which allows for replication of said plasmid in a bacterial plasmid amplification host and for selection of the transformed colonies of said host;
• a targeting gene marker for directing the insertion of said plasmid into a specific mutated homologous locus, a target gene mutation, in said host yeast genome;
• a gene of interest that is desired to be expressed in said host yeast;
• and a bacterial origin of DNA replication functional in said bacterial plasmid amplification host, such that said yeast integration plasmids contain neither any expressed bacterial sequence nor any yeast origin of replication;
• amplifying each of said yeast integration plasmids in said plasmid amplification host wherein said plasmid amplification host has a selectable phenotype which is complemented by the selection gene marker of said yeast integration plasmid upon its transformation into said plasmid amplification host;
• purifying said amplified yeast integration plasmids;
• and
• transforming of said host yeast haploids of either or both mating types with yeast integration plasmids such that a targeting gene marker of a yeast integration plasmid directs the integration of said integration plasmid to said target gene mutation in said host to which it is homologous.