Background: Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but donot have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform suchenzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for aconsolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that cantransform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interactsynergistically is needed.Results: To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-basedgene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiplegenes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulasecocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study andused to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and onebeta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen totransport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembledinto the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could expressthe five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.Conclusion: Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker,were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convertcellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktailformulation protocol and a synthetic biology technique to develop a designer yeast host
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