Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

Abstract

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.

Evaluation of heterologous α‐amylase production in two expression platforms dedicated for Yarrowia lipolytica: commercial Po1g–pYLSC (php4d) and custom‐made A18–pYLTEF (pTEF)

“…Nevertheless, recent studies by Duquesne et al . () and Boonvitthya et al . () showed pTEF promoter to be useful in efficient heterologous expression of xylanases and cellulases in Y. lipolytica .…”

“…XPR2 and Lip2 pre‐ and prepro‐signal peptides are the most commonly used in heterologous protein production in Y. lipolytica . These sequences were found to be highly efficient in directing the protein to the extracellular space (Boonvitthya et al ., ; Duquesne et al ., ; Madzak et al ., ). On the other hand, it has been reported that some signal peptides, native for the heterologous protein itself, may lead the protein to the secretory pathway of Y. lipolytica cells.…”

“…Furthermore, it has been observed that, by driving selection of prototrophs by defective markers (with truncated promoter region; ura3d or leu2d ), one can increase the copy number of an integrated heterologous sequence (Le Dall et al ., ; Juretzek et al ., ; Nicaud et al ., ), which offers a relatively straightforward technique for amplification of gene expression. Currently, a significant workload is being devoted to developing potent episomal plasmids (primary studies by Fournier et al ., ; Nicaud et al ., ), which earlier used to have low copy number and cause stability problems (Liu et al ., ), improved native promoters (Blazeck et al ., , ), or surface‐display vectors (Bulani et al ., ; Duquesne et al ., ; Yue et al ., ), altogether making Y. lipolytica a competitive expression platform compared to conventional yeasts. Several advantageous traits distinguish Y. lipolytica from the other expression systems available, such as a highly efficient co‐translational secretion mechanism, high expression and secretion capacity (Tobe et al ., ), regularity of performance, lower glycosylation in comparison with S. cerevisiae (Jolivet et al ., ), with probable lack of an immunogenic α 1,3‐mannose linkage (Song et al ., ).…”

Metabolic Engineering for Expanding the Substrate Range of Yarrowia lipolytica

“…It is one of the most abundant hemicelluloses in lignocellulosic materials and therefore its use as a carbon source is desirable for biotechnology due to its low costs. Xylanases, the enzymes that break xylan down into xylose, have been expressed in Y. lipolytica; they have been either secreted into growth media [43] or displayed on cell surfaces [56]. However, a discrepancy in the engineered strain's ability to grow on xylose has been seen in the literature.…”

“…However, a discrepancy in the engineered strain's ability to grow on xylose has been seen in the literature. On the one hand, although xylanase originating from Thermobacillus xylanilyticus and displayed on cell surfaces was used to determine the efficiency of three different anchoring systems, yeast growth on xylan was not observed [56]. On the other hand, Trichoderma harzianum endo-1,4-b-xylanase (XynII) and A. niger exo-1,4-b-xylosidase xlnD were successfully independently secreted and together allowed strains to grow on xylan [43].…”