Abstract: A method to increase carotenoid production in carotenogenic microbial host cells is provided by down-regulating or disrupting glycogen synthesis. Disruption of glycogen synthase activity in a carotenogenic microbial host cell significantly increased carotenoid production. Carotenogenic microorganisms are also provided that have been optimized for the production of carotenoid compounds through the down-regulation and/or disruption of glycogen synthase activity.

Abstract: Provided is a method for stably expressing an introduced gene or genes in a methylotrophic microorganism host wherein the gene(s) are integrated into the hsdM region of the chromosome. This method provides stable, high-level expression of the integrated genes in which growth rate of the host strain is not highly affected and a selection marker is not required. The use of this method for expressing carotenoid biosynthetic genes and resulting production of astaxanthin is also described.

Abstract: Provided is a method for expressing an introduced gene or genes in a methylotrophic microorganism host wherein the gene(s) are integrated into the cytCP region of the chromosome. This method provides stable, high-level expression of the integrated genes in which growth rate of the host strain is not highly affected and a selection marker is not required. The use of this method for expressing carotenoid biosynthetic genes and resulting production of astaxanthin is also described.

Abstract: Provided is a method for stably expressing an introduced gene or genes in a methylotrophic microorganism host wherein the gene(s) are integrated into the cysH region of the chromosome. This method provides stable, high-level expression of the integrated genes in which growth rate and/or viability of the host strain is not significantly affected and a selection marker is not required. The use of this method for expressing carotenoid biosynthetic genes and resulting production of carotenoids is also described.

Abstract: Provided is a method for expressing an introduced gene or genes in a methylotrophic microorganism host wherein the gene(s) are integrated into the fliC region of the chromosome. This method provides stable, high-level expression of the integrated genes in which growth rate of the host strain is not adversely affected and a selection marker is not required. The use of this method for expressing carotenoid biosynthetic genes and resulting production of astaxanthin is also described.

Abstract: A method to increase carotenoid production in carotenogenic microbial host cells is provided by down-regulating or disrupting glycogen synthesis. Disruption of glycogen synthase activity in a carotenogenic microbial host cell significantly increased carotenoid production. Carotenogenic microorganisms are also provided that have been optimized for the production of carotenoid compounds through the down-regulation and/or disruption of glycogen synthase activity.

Abstract: A method for the positive selection of allelic exchange mutants is provided for C1 metabolizing bacteria. The chromosomal integration vectors, based on the pGP704 suicide vector, comprise at least one genetic selectable marker and the sacB gene, encoding levansucrase. A one- and two-step selection strategy is provided for the facile identification of double-crossover mutations in C1 metabolizing bacteria. This methodology enables production of “markerless” transformants, such that multiple rounds of mutation can be performed. Optimized conditions for conjugal transfer, homologous recombination, transformant purification, and screening are also presented.

Abstract: Methanotrophic bacterial strains are provided that have been optimized for the production of carotenoid compounds through the down-regulation of one or more of the crtN1, aid, crtN2 and crtN3 genes of the carotenoid biosynthetic pathway. The resulting strains lack pigmented C30 carotenoid compounds, and show an increase in the production of C40 carotenoids. The use of the optimized host strains for the production of the C40 carotenoids canthaxanthin and astaxanthin is also described.

Abstract: The present method is useful for the identification of genes, ORF's and other nucleic acid molecules which are essential for the expression of a specific phenotype in microorganisms. The method employs in vitro transposition in conjunction with an chromosomal integration vector containing a specific gene or genetic element whose function is unknown. Subsequent transformation of a recombination proficient host with the vector and growth first under non-integrating conditions and then under integrating conditions, followed by a selection screen for either single or double crossover events results in transformation that may be subjected to phenotypic screens to determine gene function.