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

Microbial Cell Factories

Research

Todd Vander Wall¹  Qi Xu¹  Hui Wei¹  Markus Alahuhta¹  John O. Baker¹  Wei Wang¹  Michael E. Himmel¹  Stephen R. Decker¹  Min Zhang²  Eric P. Knoshaug²  Shihui Yang³ 

[1] Biosciences Center, National Renewable Energy Laboratory, 80401, Golden, CO, USA;National Bioenergy Center, National Renewable Energy Laboratory, 80401, Golden, CO, USA;National Bioenergy Center, National Renewable Energy Laboratory, 80401, Golden, CO, USA;Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, 430062, Wuhan, People’s Republic of China;

关键词: Lipomyces starkeyi;    Oleaginous yeast;    Heterologous expression;    Cellulase;    Cellobiohydrolase I;    Endoglucanase II;    Advanced biofuels;    Consolidated bioprocessing;   

DOI  :  10.1186/s12934-017-0742-5

 received in 2016-12-23, accepted in 2017-07-13,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundLipomyces 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.

【 授权许可】

CC BY   
© The Author(s) 2017