Expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi

Abstract

Background Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates.ResultsTo evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method.ConclusionsOur results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel production. The effectiveness of the tested secretion signals will also benefit future secretion of other heterologous proteins in L. starkeyi and, given the effectiveness of the cross-genus secretion signal, possibly other oleaginous yeasts as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0742-5) contains supplementary material, which is available to authorized users.

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

“…Yeast strains were grown in YPD at 30 °C (with shaking at 220 rpm) for general growth and transformation unless noted. The Gibson Assembly Cloning Kit (NEB, Ipswich, MA, USA) was used to insert target genes into vectors [ 16 ] and then followed by standard gene cloning protocols [ 30 ]. The primers and plasmids used in this study are listed in Additional files 1 and 2 , respectively.…”

“…Lipomyces starkeyi NRRL Y-11557 was acquired from the ARS Culture Collection (NRRL) and was transformed following the optimized transformation protocol as described [ 31 ] and modified by Xu et al [ 16 ]. Briefly, cells were grown to an OD 600 of approximately 10.…”

“…Of particular importance is cellobiohydrolase I (CBHI), an essential cellulase in fungal cellulase systems [ 9 , 10 ]. Previous research in developing yeast CBP organisms has focused on S. cerevisiae [ 1 , 11 , 12 ], Y. lipolytica, [ 13 – 15 ], and L. starkeyi [ 16 ]. However, these previous studies of the expression of fungal CBHIs in yeast have encountered major challenges, such as low secretion, yield, and activity of the recombinant proteins which directly discourages the application of these yeast to CBP [ 17 , 18 ].…”

“…Clearly, low secretion levels of heterologous CBHI are one of the key obstacles for yeast CBP that must be resolved. Some fungal cellulases, such as T. reesei endoglucanase II ( Tr EGII), have been reported to have high secretion levels (compared to CBHI enzymes) in L. starkeyi [ 16 ]. Specifically, we reported previously that the secretion level of Tr EGII in L. starkeyi is much higher than that of a chimeric CBHI generated by the fusion of the catalytic module from Talaromyces emersonii CBHI with the linker peptide and cellulose-binding module from T. reesei CBHI ( TeTr CBHI) [ 16 ].…”

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

“…The enzyme displayed its maximum activity at a temperature of 50 °C and a pH value of 5.0. This enzyme is highly stable in a pH range from 4.0 to 7.0 and at NaCl concentrations up to 3.0 M. Xu et al 21 reported the expression and secretion of fungal endoglucanase II and chimeric cellobiohydrolase I in the oleaginous yeast Lipomyces starkeyi and the authors demonstrated that the engineered L. starkeyi was capable of expressing and secreting both enzymes with high activity toward cellulose.…”