Constructing a cellulosic yeast host with an efficient cellulase cocktail

Abstract

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.

Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous β-carotene and cellulase production

“…Three types of cellulases ( CBHI , CBHII , EgIII , EgI , EglA and BGS ) were transformed into the R . glutinis genome according to our previous studies with other hosts 18 , 42 , 43 . The genomic integration of each gene was confirmed by PCR using gene specific primer pairs (Supplementary Table S2 , 9 – 20 ).…”

“… 43 engineered a cellulolytic K . marxianus with the similar cellulase genes as in this study 43 . The total cellulase activity of R .…”

“…glutinis , three types of fungal cellulase genes were selected and integrated into the R . glutinis genome 18 , 43 . The cellulase genes included two cellobiohydrolases ( CBHI and CBHII , from Trichoderma reesei ), three endo-β-1,4-glucanases ( EgIII , from T .…”

“…The amplification of six cellulase gene cassettes used the same method as in the carotenogenic gene cassettes. Each cassette was in the pUC18 vector as in previous studies 18 , 43 . The gene cassettes were integrated using three selection marker genes: G418 resistance gene (neomycin phosphotransferase gene, KanMx ), Zeocin resistance gene ( Sh ble ) and Hygromycin phosphotransferase gene ( hph ).…”

Kluyveromyces marxianus: Current State of Omics Studies, Strain Improvement Strategy and Potential Industrial Implementation

“…Since the anchoring protein OlpB has seven type II cohesins, the interaction between CipA-OlpB can accommodate up to 9 × 7 = 63 cellolosomal enzymes in a single cellulosome complex. Up to now, several research groups have been sought to design cellulosome microbes that can express a full size of cellulosome structure instead of some individual cellulosomal genes called mini-cellulosomes [31][32][33][34][35][36]. Recently, the group of Anandharaj et al [37] succeeded in developing an engineered K. marxianus host that can express a full size of H. thermocellum cellulosome on its cell surface.…”