Phylogenetic and morphological analyses of the mycoparasitic genus Piptocephalis.

The Piptocephalidaceae (Zoopagales, Zoopagomycota) contains three genera of mycoparasitic, haustoria-forming fungi: Kuzuhaea, Piptocephalis, and Syncephalis. Although the species in this family are diverse and ubiquitous in soil and dung, they are among the least studied fungi. Co-cultures of Piptocephalis and their hosts are relatively easy to isolate from soil and dung samples across the globe, making them a good model taxon for the order Zoopagales. This study focuses on the systematics of the genus Piptocephalis. Despite the fact that there are approximately 40 described Piptocephalis species, there are no modern taxonomic or molecular phylogenetic treatments of this group. Minimal sequence data are available, and relatively little is known about the true diversity or biogeography of the genus. Our study addresses two aspects: Piptocephalis systematics and analyses of the length and inter- and infraspecific variation of the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. First, we generated a large subunit (28S) nuc rDNA phylogeny and evaluated several morphological characters by testing their correlation with the phylogeny using Bayesian Tip-association Significance testing (BaTS). We found monophyly of Piptocephalis species identified based on morphological traits, but morphological character states were not conserved across clades, suggesting that there have been multiple gains and losses of morphological characters. We also found that Kuzhuaea is nested within Piptocephalis. Second, we amplified the ITS from many Piptocephalis isolates, created a sequence alignment, and measured the lengths using the software ITSx. Piptocephalis species had ITS regions that were longer than the average for most Dikarya but were similar in length to those of the related genus Syncephalis.

Constructing a cellulosic yeast host with an efficient cellulase cocktail.

Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.

Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production.

BACKGROUND: Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed. RESULTS: To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called "promoter-based gene assembly and simultaneous overexpression" (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-ß-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the 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 convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.