Among the current approaches to cellulosic ethanol production, consolidated bioprocessing (CBP) is most preferred because of its simplicity and potential low cost . To achieve CBP, a microbe that can carry out cellulase production, hydrolysis, and fermentation in a single process is needed. Currently, however, there is no single microbe available for doing CBP efficiently. Although Saccharomyces cerevisiae has been considered the best ethanol producer from hexose sugars, its genome lacks genes for cellulolytic enzymes. Thus, there have been efforts to introduce cellulase genes into S. cerevisiae[2, 3]. Previously, we made attempts to improve the signal peptide for secretion or to reduce the glycosylation strength of S. cerevisiae, so that we could over-express cellulase genes of other fungi in S. cerevisiae. Unfortunately, most of the expressed proteins were either non-functional or could not be efficiently secreted out of the cell (data not shown). Recently, we isolated a kefir yeast, Kluyveromyces marxianus KY3 (data not shown), that has the potential to serve as a host for bioethanol production and a biorefinery platform, because the strain has broad substrate spectrum, including both hexose and pentose sugars, and produce valuable flavor byproducts such as 2-phenylethanol. The strain also shows resistant to inhibitors generated from chemical pretreatment of lignocellulose and is heat-tolerant [4, 5]. Moreover, many genetic and genomic tools such as those developed for K. lactis[6, 7] are applicable to KY3.
Recently, synthetic biology has been recognized as a powerful approach for the design and construction of new biological systems. Although cloning tools such as the Univector plasmid-fusion system , ligase-free  or ligation-independent cloning (LIC) , and the Gateway cloning system [11, 12] have been developed and widely adopted, a technique that can assemble multiple genes in a desired order in a single step and integrate the large DNA piece into a genome is highly desirable. To achieve this goal, various techniques have been developed to enable the assembly of several genes or DNA modules into a larger construct, including chain reaction cloning , the OGAB method , DNA assembler in vivo, USER cloning , MAGIC , SLIC , In-Fusion Clontech , Illegitimate recombination , Circular polymerase extension cloning , and one-step assembly in yeast [22, 23]. These methodologies are based mainly on historically well-characterized hosts, such as Escherichia coli, Bacillus subtilis and S. cerevisiae, whereas newly discovered organisms with great characteristics for bioprocessing are still in need of synthetic biology tools.
The synthetic biology technique we developed in this study relies on homologous recombination, which is responsible for a number of important transformation processes of microorganisms and is very useful for the generation of host cells for both cloning and expression of heterologous genes. Several transformation systems have been developed for use with S. cerevisiae by episomal plasmids and integrating plasmids with foreign DNA fragments [15, 24, 25]. In a previous study, an extending homologous recombination approach was demonstrated by assembling a megaplasmid from multiple overlapping fragments in a single step in S. cerevisiae. Moreover, episomal plasmid and integrating plasmid transformation studies on other yeast species, such as K. lactis, have also been reported [6, 7, 26]. Although non-homologous end-joining (NHEJ) is a common phenomenon in fungi and serves as a transformation method in K. marxianus[20, 27], the homologous recombination strategy has several advantages for genetic engineering in fungi, such as ordered multiple gene assembly in one step [22, 23]. Compared to NHEJ, this strategy is simpler for controlling the copy numbers of interested genes in a genome, and for targeting specific genes for insertion or disruption.
The technique we developed is called “Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO)”. PGASO has four advantages for genome engineering: (1) Multiple genes can be transformed into a genome in a single step; (2) specific upstream promoter sequences can be used in the gene assembly in a predesignated order without linker sequence; (3) each gene cassette has a unique promoter, so that its expression level can be adjusted; and (4) PGASO can be applied to a host that can undergo homologous recombination. As an example of application, we applied PGASO to integrate five gene cassettes in a predesignated order into a specific site in the genome of K. marxianus KY3 in a single step. The five genes include one reporter gene, one selectable marker gene, and three different essential cellulase genes for cellulose saccharification. The purpose of this construct is to engineer K. marxianus KY3 into a host for cellulose saccharification and ethanol fermentation in one step. This is the first example of multi-gene assembly in a yeast species other than S. cerevisiae.