Malaysian Applied Biology Journal

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47_05_30

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Malays. Appl. Biol. (2018) 47(5): 241–249

 

DEVELOPMENT OF Trichoderma virens ?pyrG AUXOTROPHIC

STRAIN VIA GENE DISRUPTION STRATEGY


ABDUL KARIM, N.A.1, AB WAHAB, A.F.F.1, ABU BAKAR, F.D.1, ILLIAS, R.2

and MURAD, A.M.A.1*


1School of Biosciences and Biotechnology, Faculty of Science and Technology,

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

2Department of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering,

Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

*E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it


Accepted 15 October 2018, Published online 30 November 2018


ABSTRACT

A dominant selection marker is an important prerequiste for gene transformation studies in most organisms. In fungi, two types or selection marker are usually used; the introduction of an antibiotic resistant gene or by using auxotrophic mutant. Due to the biosafety issues, auxotrophic genetic markers are widely used for the genetic manipulation, especially for downstream industrial applications. The aim of this work is to develop a uracil/uridine (?pyrG) auxotrophic mutant of Trichoderma virens UKM1, a locally isolated fungus capable of producing interesting industrial enzymes. The T. virens ?pyrG mutant was developed by the deletion of the gene encoding for orotidine-5-phosphate decarboxylase (pyrG), an enzyme involved in the biosynthesis of pyramidine, using a homologous recombination approach. Successful ?pyrG mutants exhibited a uracil auxotrophy phenotype and were able to grow on media containing 5-FOA (5-fluoroorotic acid) supplemented with uracil and uridine, that inhibit the growth of the wild-type strain. Transformation of the ?pyrG mutant with the pyrG from Aspergillus oryzae restored the pyrimidine biosynthesis pathway, transforming the ?pyrG mutant into a uracil prototroph. These results suggest uracil/uridine auxotrophic phenotype resulted from the deletion of the pyrG gene in T. virens UKM1. Hence, in this work we have successfully developed a Trichoderma virens UKM1 ?pyrG auxotrophic mutant that is amenable to further efforts in the genetic engineering of this interesting fungus.

Key words: Trichoderma virens, pyrG auxotroph, homologous recombination

 

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