Construction Of Recombinant E.Coli Strain For Erythritol Production

Erythritol (butane-1,2,3,4-tetraol) widely exists in fungi, lichens and fruits such as grapes. As its sweetness is0.65, it is generally used as low caloric sweetener in food, pharmaceuticals, health care products etc. Production methods of erythritol divided into:chemical synthesis、Biological extraction and microorganism fermentation. Because of polluting and high cost of the chemical synthesis, it is difficult to industrialize. Therefore, microorganism fermentation is used as the main method so far. Candida lipolytica and Candida magnoliae are the main producing strain. To obtain higher yield rate of erythritol with yeast, some problems have yet to be solved such as low sugar resistance, low conversion rate, long fermentation period and low fermentation temperature.Based on study of the metabolic pathways for erythritol in the yeast, Genes encoding yidA and erythrose reductase (ER) were cloned from the genome of Escherichia coli BW25113and Candida magnolia, respectively. We chose pZE12-luc vector of E.coli to construct gene engineering bacteria. For the problem that the gene encoding ER was existing in inclusion bodies after inducible expression, co-expression of molecular chaperones and ER were implemented, followed by research of inducing fermentation. Main work has been done are listed below:We successfully constructed recombinant bacteria pZE12-luc-yidA-ER and pZE12-luc-ER-yidA in the Escherichia coli expression system. After inducing fermentation of the two kinds of constructed recombinant bacteria, the fermentation broths were analyzed by HPLC. The results showed that the fermentation broth contained erythritol with low yield. The SDS-PAGE revealed that the gene encoding ER is existing in inclusion bodies after inducible expression.We constructed gene engineering bacteria pCS-27-IbpA in the E.coli expression system, which could express heat shock protein (HSP). After co-expression of HSP with ER and fermentation, the SDS-PAGE results showed the expression quantity of ER was improved, which means HSP help protein folding.Co-expression of pCS-27-IbpA with pZE12-luc-yidA-ER or pZE12-luc-ER-yidA enhanced the production of erythritol by271.22%and308.42%, respectively. Order to further improve the yield of erythritol, we used the orthogonal method to obtain the desired inducing conditions to further increase the yield of7.4%.