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Biotechnol Lett (2011) 33:721725 DOI 10.1007/s10529-010-0479-2
ORIGINAL RESEARCH PAPER
Expression and secretion of a single-chain sweet protein, monellin, in Saccharomyces cerevisiae by an a-factor signal peptide
Zhongjun Chen Zhengying Li Na Yu
Lili Yan
Received: 7 October 2010 / Accepted: 12 November 2010 / Published online: 24 November 2010 Springer Science+Business Media B.V. 2010
Abstract The sweet protein monellin gene was expressed in Saccharomyces cerevisiae under the control of the GAL1 promoter and a-factor signal peptide sequence of S. cerevisiae. The gene, which was obtained through mutation of the synthesized single-chain monellin gene, was cloned into an E. coli-yeast shuttle vector pYES2.0 which carries the galactose-inducible promoter GAL1. Then the a-factor signal peptide of
S. cerevisiae was linked also, resulting in the secreting expression vector pYESMTA. The recombinant plasmid was subsequently transformed into strain S. cerevisiae INVsc1. The peptide efciently directed the secretion of monellin from the recombinant yeast cell. A maximum yield of active monellin was 0.41 g l-1 of
the supernatant from INVsc1 harboring pYESMTA.
Keywords Monellin Saccharomyces cerevisiae
Secretion Signal peptide
Introduction
Monellin is a sweet protein from the berries of the African plant Dioscoreophyllum cumminsii. On a molar basis, it is many thousands of times sweeter than sucrose (Morris and Cagan 1972) and can be used
as a highly potent sweetener as it is readily soluble in water. Monellin is of potential use as a non-carbohydrate sweetener and could be particularly benecial to diabetics who must control their sugar intake. However, monellin is not stable to heat or pH (Kim et al. 1989). A single-chain monellin with 97 amino acids and a molecular weight of 10.7 kDa, in which the two polypeptides are connected by a glycine residue was designed and had greatly improved thermal stability without changing its sweetness (Kim et al. 1989). In addition, monellin is costly to extract from the fruit and the plant is difcult to grow. Many attempts have been made to produce the single-chain monellin via recombinant technology. Monellin has been expressed in different systems (Zhang et al. 2002; Kondo et al. 1997). We have expressed monellin in E. coli (Chen et al. 2005) and B. subtilis (Chen et al. 2007).
In this study, the synthetic gene encoding single-chain monellin (Chen et al. 2005) was...