Engineering

Background

Pectin is a kind of high molecular carbohydrate existing in the cell wall of plants. The main chain is polymerized by galacturonic acid (PGA), while the side chain is composed of various polysaccharides such as galactose, rhamnose, and xylose. In fruit wine production, Saccharomyces cerevisiae uses the sugars in fruit juice to ferment to produce alcohol. It is necessary to add pectinase to destroy the pectin in the cell wall to increase the juice yield and increase the dissolution of aromatic substances such as pigments or terpenes. In addition, pectinase can also eliminate the turbidity of fruit wine caused by pectin colloids, so as to ensure the light transmittance and stability of fruit wine to the greatest extent. Most of the pectinase currently on the market is a composite enzyme obtained from the fermentation of Aspergillus. This undoubtedly increases the cost of fruit wine production.

There have been successful cases of exogenous expression of pectinase. All these achievements aimed to obtain high-yielding pectinase production strains, but yeast strains that can both decompose pectin and complete alcohol fermentation have never been obtained.

Design

This project uses the CRISPR-Cas technology to realize the heterologous expression of the cohesive galacturonase gene endo-pgaA of Aspergillus niger SC323 in the fruit wine yeast, and obtain a yeast strain that can degrade pectin. This will help to reduce the cost of fruit juice and fruit wine production process.

A double-plasmid expression system of CRISPR-Cas9 was designed to edit genome of Saccharomyces cerevisiae (Figure 1). One plasmid is pHCas9-Nours plasmid expressing Cas protein and another is pYES2-gRNA-hyg-MCS plasmid containing sgRNA. First, pHCas9-Nours plasmid was introduced yeast cells, and then the pYES2–gRNA-hyg-MCS plasmid and the homology arm for repair into the wine yeast. After obtaining the transformant, verify endo-pgaA Whether to replace to the HXK1 gene locus.

Figure 1. Design of integrating endo-pgaA gene in wine yeast using CRISPR-Cas9 technology. P is the promoter (promotor); T is the terminator (terminator); CEP is the Cas effector protein; HR is the homology arm; endo-pgaA is the pectinase gene.

Build

Plasmids pHCas9-Nourse and pYES2-gRNA-hyg-MCS were successfully constructed (Figure 2). The integration of endo-pgaA gene in wine yeast genome was verified by DNA sequencing.

Figure 2. Schematic maps of plasmids pHCas9-Nourse and pYES2-gRNA-hyg-MCS.

Test

Yeast transformants were picked pectinase production. The pectinase activity was determined using DNS method (inducer is β-galactose) (Table 1). The specific activity was calculated from a reduce sugar standard curve.

Table 1. Pectinase activity in yeast transformants.

Learn

In this project, we successfully prepared genetically engineered wine yeast strain which contains pectinase in its genome. The pectinase produced from yeast can well degrade pectin into small sugars.