The development of a Trichoderma virens expression system for heterologous protein production

Abstract

Numerous eukaryotic expression systems have been developed for use in filamentous fungi since these microorganisms have a high capacity for protein production and secretion.In order to fulfill the necessity of exploring new fungal expression systems as an alternative to those covered by patents, a locally isolated fungus, Trichoderma virens, is currently being developed as an expression host. Expression vectors comprising of several important elements that includes an expression cassette, hygromycin cassette as dominant selection marker and a rDNA sequence for chromosomal integration were constructed. For this construction, three separate expression cassettes were made. basic elements employed for the construction of the expression cassettes of this study the promoter region, signal secretion sequence, Glomerella cingulata cutinase a heterologous gene reporter and Aspergillus nidulans TrpC as terminator. Using plasmid pUC19 as a backbone vector, the expression cassettes were constructed by fusing the cutinase gene to the constitutive A. niger glucoamylase gla-promoter (PglaScutTrp) and inducible T virens endochitinase ech-promoter (PSechMcutTrp and PechscutTrp0.Expression vectors transformed into the T virens protoplasts yielded about 0.34 transformants/ug, The cutinase production by positive transformants containing cut expression cassettes was analyzed via shake jlask induction experiments. The ac measurements using p-nitrophenyllauerate (pNPL) as a substrate showed that the cut produced and secreted into the culture medium had esterase activity with the highest specific activity of 33.29 U/ug derived from the gla-promoter driven expression all U/ug for the inducible gla-promoter driven expression. Although the amount of the enzyme secreted into the medium is low as compared to the cutinase produced by other expression systems commercially available, it can be concluded that the heterologous express the G. cingulata cutinase and the subsequent secretion of the recombinant enzyme were achieved in the T. virens expression system.