Genetically Engineered Kluyveromyces Marxianus For Mix-sugar Utilization

Kluyveromyces marxianus is thermo-tolerant yeast,which can use a wide range of sugar,such as glucose,xylose,galactose,raffinose and so on.These properties make the yeast an attractive candidate to produce thermo-tolerant enzymes and bioethanol.Although K.marxianus keeps these promising characteristics,the glucose repression in mix-sugar is still a big problem preventing it from being widely used in industrial production.As a crabtree negative yeast,when metabolizing mixed sugar which consisted of glucose and other non-glucose sugar as carbon source,the non-glucose sugar will not be utilized until the glucose was used up or at low concentration.In this study we aimed to construct glucose-derepressed thermo-tolerant yeast for mixed sugar fermentation.The genes which have high sequence similarity with the ScHXK1,ScHXK2,ScGLK,ScMIGl or ScMIG2 were cloned and named as KmHXK,KmGLK and KmMIGl.These three genes were disrupted respectively in K.marxianus to evaluate their function in glucose repression.ScURA3 expression cassette was inserted into the three genes to construct the disruption cassettes.The three genes in YHJ010,a K.marxianus with three auxotrophic selectable tags(?KmURA3,?KmLEU2,?KmTRP1),were disrupted respectively,and resultant strains were named as YLM001,YLM002 and YLM012.Then the 2-DG(2-Deoxy-D-glucose)plate was used to evaluate the glucose repression.Glucose repression of YLM001 in which KmHXK was disrupted was released,while the growth on glucose was hindered.Then KmGLK coupled with ScTRPl as selection marker was expressed in YLM001 and the resultant strain was named as YLM005.The growth ability,sugar consumption characteristics,transcription level and enzyme activity of xylose metabolism were investigated in glucose/xylose mix culture.YLM005 with glucose de-repression like its parent strain had a rather high growth rate.Different kinds of mix sugars were also used to evaluate the de-repression of YLM005.The result showed that it can co-metabolism other sugar with glucose.Furthermore,the effect of KmHXK was evaluated in a xylitol producting yeast,which was constructed in our lab previously.The resultant strain was named as YLM011.With OD600 of 10 YLM011 produced 67.9 g/1 xylitol from 79.5 g/1 xylose and 27.2 g/1 glucose with productivity of 1.85 g/1/h at 42?,and the xylitol yield was 1.04g/g.The productivity and xylitol yield are considerably high among currently reported mix-sugar fermentation strains at elevated temperature.