Engineering

Background

China is one of the oldest countries in the world to cultivate wheat, and wheat is also one of China's three major food crops (rice, wheat, and corn). There are usually two types of industrially produced wheat starch, A-type and B-type. A starch is a refined starch and B starch is a by-product of the production of A starch. The non-starch polysaccharides (pentosans, β-glucans) in B starch cannot be decomposed by the endogenous digestive enzymes of monogastric animals. It enters the large intestine directly and is decomposed and fermented by microorganisms in the large intestine, which usually causes nutritional disorders in monogastric animals, especially poultry.

Alcohol (ethanol) is an important chemical in the fields of food, medicine, and biofuels. The traditional fermentation method uses corn or tapioca starch as raw material to produce alcohol.

Wheat B starch is a by-product of deep processing of wheat starch. It is often used directly as feed, and the added value of the industry is not high. If wheat B starch is used as a raw material to produce alcohol, some of the disadvantages of wheat starch alcohol can be avoided and the utilization value of wheat B starch can be improved. After wheat B starch is pretreated by liquid saccharification to produce sugar, it can be fermented with conventional Saccharomyces cerevisiae to produce alcohol. However, the pentosan component of wheat B starch is not used in this process. Therefore, the ideal approach is to develop Saccharomyces cerevisiae strains which can secret pentosanase.

Design

We selected codon-optimized xylanase gene AnXlnB, β-xylosidase gene AnXlnD (from Aspergillus niger) and acetyl xylan esterase CcXynA (from Clostridium cellulophilum). To express these gens in Saccharomyces cerevisiae strain, generating a genetically modified S. cerevisiae with the ability to decompose and utilize pentosan. The ability of the strain to ferment pentosan to produce alcohol was tested in a specific medium. The overall design is shown in Figure 1.

Figure 1. Genetically modified S. cerevisiae with the ability to decompose and utilize pentosan, and to ferment pentosan to produce alcohol.

Build

AnXlnB, AnXlnD, and CcXynA were inserted into yeast expression vector (Figure 2).

Figure 2. Plasmid maps in the project.

Test

The xylan utilization S. cerevisiae strain construction and fermentation test (Figure 3).

Figure 3. Fermentation performance of the plasmids transformed S. cerevisiae strains in the simulated wheat B starch medium. A: OD value. B: Sugar concentration.

The plasmids pXylan-B and pXylan-BD were transformed into the S. cerevisiae strain, respectively. The resulting positive transformants were undergo the fermentation test. In the simulated wheat B starch medium (YPD20Xylan20), all the strains showed almost the same growth performance during the first 8 h, this is due to the strains preferentially utilized the glucose present in the media. This was verified again in Figure 7B, all the strains showed the comparable sugar utilization capacity, the xylan utilization ability may be covered by the glucose. Therefore, to verify the strains’ xylan utilization capacity, a xylan as the sole carbon source medium was essential in further study.

The sugar consumption data showed that starting from 2 hours, the WXA/pXylan-B and WXA/pXylan-BD strain was slightly higher than the WXA control, which could be interpreted as decomposing xylan and producing reducing sugars.

Learn

The engineered bacteria we constructed can decompose the xylan successfully.