**Improved raw starch amylase production bySaccharomyces cerevisiaeusing codon optimisation strategies**

Cripwell, Rosemary A
Rose, Shaunita H
Viljoen-Bloom, Marinda
van Zyl, Willem H

¹Department of Microbiology, Stellenbosch University, JC Smuts Building, De Beer Street, Stellenbosch, 7600, South Africa

Corresponding author: Department of Microbiology, Stellenbosch University, JC Smuts Building, De Beer Street, Stellenbosch, 7600, South Africa. Tel: +27 21 808-5854; Fax: +27 21 808-5846; E-mail: [email protected]

[email protected]

Received October 23, 2018/Accepted December 07, 2018

FEMS Yeast Research (FEMSYR) 19(2):p foy127, March 2019. | DOI: 10.1093/femsyr/foy127

Abstract

Amylases are used in a variety of industries that have a specific need for alternative enzymes capable of hydrolysing raw starch. Five α-amylase and five glucoamylase-encoding genes were expressed in the Saccharomyces cerevisiae Y294 laboratory strain to select for recombinant strains that best hydrolysed raw corn starch. Gene variants of four amylases were designed using codon optimisation and different secretion signals. The significant difference in activity levels among the gene variants confirms that codon optimisation of fungal genes for expression in S. cerevisiae does not guarantee improved recombinant protein production. The codon-optimised glucoamylase variant from Talaromyces emersonii (temG_Opt) yielded 3.3-fold higher extracellular activity relative to the native temG, whereas the codon-optimised T. emersonii α-amylase (temA_Opt) yielded 1.6-fold more extracellular activity than the native temA. The effect of four terminator sequences was also investigated using temG and temG_Opt as reporter genes, with the ALY2_T terminator resulting in a 14% increase in glucoamylase activity relative to the gene cassettes containing the ENO1_T terminator. This is the first report of engineered S. cerevisiae strains to express T. emersonii amylase variants, and these enzymes may have potential applications in the industrial conversion of raw starch under fermentation conditions.