Xylitol Dehydrogrnase As A Selective Marker For Yarrowia Lipolytica And Its Application In Synthesis Of Threitol

Yarrowia lipolytica is used in synthetic biology chassis microorganisms due to its unique physicochemical properties,mature molecular operation system and safety.With the development of synthetic biology,molecular biology methods can be used to accurately edit the Yarrowia lipolytica genome and develop an advanced version of yeast chassis microbes.However,the selective markers available for this yeast are very limited,which increases the difficulty of gene editing such as gene knock-in and knock-out.Therefore,it is very important to develop more yeast selective markers to improve the efficiency of molecular manipulation.In this study,Yarrowia lipolytica was used as a chassis organism,and a method using xylitol dehydrogenase as a selective marker was successfully developed.Different xylitol dehydrogenase genes were expressed in Yarrowia lipolytica,respectively,so that the yeast could grow well on minimal medium with xylitol as the sole carbon source.Xylitol is a safe and inexpensive advantage as a selective agent relative to other screening markers such as the antibiotic hygromycin and geneticin.By studying the enzymatic properties of different xylitol dehydrogenases,it was found that xylitol dehydrogenase derived from Scheffersomyces stipitis CBS 6054 can oxidize erythritol to erythrulose and also erythritol.The sugar is reduced to threitol.This enzyme is predicted to convert erythritol to threitol.Then we heterologous expression the Scheffersomyces stipitis CBS 6054 xylitol dehydrogenase gene in Y.lipolytica CGMCC7326 and successfully obtained the yeast engineered strain HC110.It was found that the yeast overexpressing the xylitol dehydrogenase can be glucose-based.The synthesis of threitol by fermentation of the substance achieved the first direct fermentation of glucose to produce threitol with a yield of 37.3%.In order to increase the purity of threitol in the fermentation broth,the fermentation broth was biologically purified using the yeast Candida parapsilosis CGMCC2.431 mediated biological purification method,so that the purity of threitol in the fermentation broth was over 98%.This study not only developed an effective Y.lipolytica screening marker,but also developed a one-step engineering of Yarrowia lipolytica engineering strain that synthesizes threitol by glucose fermentation,which simplifies the traditional synthesis of threitol method.