Natalia Sedkova

Diversity of Carotenoid Synthesis Gene Clusters from Environmental Enterobacteriaceae Strains

ABSTRACT Eight Enterobacteriaceae strains that produce zeaxanthin and derivatives of this compound were isolated from a variety of environmental samples. Phylogenetic analysis showed that these strains grouped with different clusters of Erwinia type strains. Four strains representing the phylogenetic diversity were chosen for further characterization, which revealed their genetic diversity as well as their biochemical diversity. The carotenoid synthesis gene clusters cloned from the four strains had three different gene organizations. Two of the gene clusters, those from strains DC416 and DC260, had the classical organization crtEXYIBZ; the gene cluster from DC413 had the rare organization crtE-idi-XYIBZ; and the gene cluster from DC404 had the unique organization crtE-idi-YIBZ. Besides the diversity in genetic organization, these genes also exhibited considerable sequence diversity. On average, they exhibited 60 to 70% identity with each other, as well as with the corresponding genes of the Pantoea type strains. The four different clusters were individually expressed in Escherichia coli, and the two idi-containing clusters gave more than fivefold-higher carotenoid titers than the two clusters lacking idi. Expression of the crtEYIB genes with and without idi confirmed the effect of increasing carotenoid titer by the type II idi gene linked with the carotenoid synthesis gene clusters.

Expression of bacterial hemoglobin genes to improve astaxanthin production in a methanotrophic bacterium Methylomonas sp.

Astaxanthin has been widely used as a feed supplement in poultry and aquaculture industries. One challenge for astaxanthin production in bacteria is the low percentage of astaxanthin in the total carotenoids. An obligate methanotrophic bacterium Methylomonas sp. 16a was engineered to produce astaxanthin. Astaxanthin production appeared to be dramatically affected by oxygen availability. We examined whether astaxanthin production in Methylomonas could be improved by metabolic engineering through expression of bacterial hemoglobins. Three hemoglobin genes were identified in the genome of Methylomonas sp. 16a. Two of them, thbN1 and thbN2, belong to the family of group I truncated hemoglobins. The third one, thbO, belongs to the group II truncated hemoglobins. Heterologous expression of the truncated hemoglobins in Escherichia coli improved cell growth under microaerobic conditions by increasing final cell densities. Co-expression of the hemoglobin genes along with the crtWZ genes encoding astaxanthin synthesis enzymes in Methylomonas showed higher astaxanthin production than expression of the crtWZ genes alone on multicopy plasmids. The hemoglobins likely improved the activity of the oxygen-requiring CrtWZ enzymes for astaxanthin conversion. A plasmid-free production strain was constructed by integrating the thbN1–crtWZ cassette into the chromosome of an astaxanthin-producing Methylomonas strain. It showed higher astaxanthin production than the parent strain.