Deana J. Dicosimo

Use of Transposon Promoter-Probe Vectors in the Metabolic Engineering of the Obligate Methanotroph Methylomonas sp. Strain 16a for Enhanced C40 Carotenoid Synthesis

ABSTRACT The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C40 carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations—the fliCS, hsdM, ccp-3, cysH, and nirS regions—were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.

Construction and Utilization of Carotenoid Reporter Systems: Identification of Chromosomal Integration Sites That Support Suitable Expression of Biosynthetic Genes and Pathways

In order to metabolically engineer microorganisms to produce compounds of interest, it is often desirable to integrate foreign genes into the chromosome of the host. However, the consequences of these genetic alterations are not always predictable. The use of a reporter system can often assist in determining chromosomal locations for optimal expression of foreign biosynthetic genes. The method described here involves the construction and utilization of promoterless carotenoid transposons, which provides a colorimetric screen for identifying the best chromosomal integration sites for the expression of the genes of interest. The transposons (pUTmTn5::392W and pUTmTn5::392) contain the carotenoid genes required for the production of canthaxanthin and astaxanthin, respectively. Thus, when promoterless transposons insert into the hosts genome, the color of the colonies will vary based on their chromosomal location. There is a correlation between the color intensity of the colonies and the expression of the carotenoid transposon. The transposon insertion site can be determined via direct chromosomal sequencing. This sequence information is used to guide the site-specific integration of biosynthetic genes and pathways of interest.