Reseach Of Pentose Transporter In Oleaginous Yeast

At present, bioenergy industry is transferred into the non-food era.However, the use of cellulose and hemicellulose is a technical problemfor decreasing the cost of raw materials. Specifically, the key of celluloseutilization is pentose utilization such as xylose in the hydrolyzate.Molecular biology techniques could expand the pentose utilizationcapacity of traditional oleaginous microbial, so the lignocellulosic wasregarded as raw materials for the production of microbial oil, which drewmuch concern in biodiesel technology.The main subject of this study is to find out the mechanism of theoleaginous yeast-Rhodotorula glutinis in the five-carbon sugar-specifictransmembrane transport. First, this study examined the reported Candidaintermedia xylose-specific transmembrane transporter protein expressionin the Rhodotorula glutinis. More specific, expression plasmid wasconstructed and then used in Rhodotorula glutinis added on the resistancegene for screening (shble). After that, adding the multiple cloning sitesfor expressing foreign genes in Rhodotorula glutinis. Finally, building free expression engineering carrier PRS424-Phxt7-GFP-Thxt7-shblewhich was applied in Rhodotorula glutinis genetic. It is a very valuablefor the Rhodotorula glutinis molecular biology subsequently.Then connection reported Candida intermedia specific xylosetransmembrane transporter gene GXS1(glucose/Xylose Symporter) withvector PRS424-Phxt1-GFP-Thxt1-shble was successfully conducted. Thexylose concentration was determined in the original strain and therecombinant strain respectively. It was found that xylose absorption andutilization efficiency in the recombinant strain were higher than theoriginal strain. Xylose was consumed totally in about80hours offermentation in recombinant bacteria while xylose was exhausted afterabout120h in the original strain. The results showed that pentosetransmembrane transporter protein expression in recombinant bacteria caneffectively work in xylose extracellular to intracellular transport.In this article, Rhodotorula glutinis specific xylosetransmembrane transporter gene was taken and obtained with the help ofreported homologous specificity of xylose transmembrane transporterprotein sequence and some technologies, such as RT-PCR techniques,RACE technique and chromosome walking techniques. Meanwhile,core sequence of1513bp Rhodotorula glutinis pentose transmembranetransporter protein had been cloned, and its similarity reached83%compared with reported Candida intermedia specific pentose transmembrane transporter gene by NCBI homology analysis, which laidthe foundation for cloning of the full-length cDNA and its functionalanalysis.