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Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome

Paper ID Volume ID Publish Year Pages File Format Full-Text
23511 43444 2013 3 PDF Available
Title
Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome
Abstract

•A method was developed to efficiently integrate many genes into the K. marxianus chromosome.•Sequential integration resulted in extremely low chance of obtaining a desired colony.•Already-integrated gene was excised out during sequential integration procedures.•Many genes could be simultaneously integrated into K. marxianus with a high efficiency.•Over 10% colonies of many strains contained all genes after simultaneous transformation.

While Kluyveromyces marxianus is a promising yeast strain for biotechnological applications, genetic engineering of this strain is still challenging, especially when multiple genes are to be transformed. Sequential gene integration, which takes advantage of repetitive insertion/excision of the URA3 gene as a marker, has been the best option until now, because the URA3-deletion mutant is the only precondition for this method. However, we found that the introduced gene is co-excised during the URA3 excision step for next gene introduction, resulting in a very low cumulative probability (<1.57 × 10−6% for 4 genes) of integrating all genes of interest. To overcome this extremely low probability, and to reduce labor and time, all 4 genes were simultaneously transformed. Surprisingly, the infamously high ‘non-homologous end joining’ activity of K. marxianus enabled simultaneous integration of all 4 genes in a single step, with a probability of 7.9%. Various K. marxianus strains could also be similarly transformed. Our finding not only reduces the labor and time required for such procedures, but also removes a number of preconditions, such as pre-made vectors, selection markers and knockout mutants, which are needed to introduce many genes into K. marxianus.

Keywords
Kluyveromyces marxianus; Integration; Ura-blaster; URA3
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Simultaneous integration of multiple genes into the Kluyveromyces marxianus chromosome
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 167, Issue 3, 10 September 2013, Pages 323–325
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us