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Applied and Environmental Microbiology, March 2007, p. 1721-1728, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.01332-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Pamela L. Sharpe,* Deana DiCosimo, Melissa D. Bosak, Kyle Knoke, Luan Tao, Qiong Cheng, and Rick W. Ye

Biochemical Sciences and Engineering, Central Research and Development, E. I. DuPont de Nemours Inc., Wilmington, Delaware 19880-0328

Received 9 June 2006/ Accepted 10 January 2007

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.

* Corresponding author. Mailing address: E. I. DuPont de Nemours Inc. Experimental Station, E328/B49, Wilmington, DE 19880-0328. Phone: (302) 695-6692. Fax: (302) 695-1829. E-mail: pamela.l.sharpe{at}usa.dupont.com.

{triangledown} Published ahead of print on 19 January 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, March 2007, p. 1721-1728, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.01332-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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