Microbial proteins hold great promise as sustainable alternatives for future protein sources,and oleaginous yeast Yarrowia lipolytica has emerged as a recognized platform for heterologous protein expression and secret...Microbial proteins hold great promise as sustainable alternatives for future protein sources,and oleaginous yeast Yarrowia lipolytica has emerged as a recognized platform for heterologous protein expression and secretion.N-terminal signal peptides(SPs)are crucial for directing proteins to the secretion pathway,which offers advantages in both academic and industrial protein production.Although some of the innate SPs of Y.lipolytica have been reported,there is a growing need to expand the genetic toolkit of SPs to support the increasing use of Y.lipolytica as a cell factory for overproduction of various secretory proteins.In this study,we employed an efficient evolutionary approach to rapidly evolve the innate SP XPR2-pre by leveraging Gibson assembly with two syn-thetic overlapping oligos containing high portion of degenerate nucleotides.Using Nanoluc(Nluc)luciferase as a robust reporter,we characterized the intracellular and extracellular enzymatic activity of 447 SP mutants and identified previously undescribed SPs exhibiting superior performance compared to XPR2-pre in Nluc luciferase secretion,with improvements of up to 2.91-fold of enzymatic activity in the supernatant.The generalizability of the top-performing SPs was evaluated using three additional heterologous enzymes(β-galactosidase,α-amylase,and PET hydrolase).Our results confirmed their versatility across different proteins with protein-specific effi-ciency.Additionally,based on our screening,we also evaluated the performance of different feature engineering strategies and machine learning models in the design and prediction of SP mutants.This study integrated rational design,directed evolution and machine learning to identify novel SPs,expanding the repertoire of signal peptides and benefiting secretory protein overexpression in Y.lipolytica.展开更多
Cycloastragenol is a bioactive,high-value triterpenoid derived from Astragalus membranaceus.Conventional plant-based extraction and chemical synthesis methods are expensive.To our knowledge,this is the first report on...Cycloastragenol is a bioactive,high-value triterpenoid derived from Astragalus membranaceus.Conventional plant-based extraction and chemical synthesis methods are expensive.To our knowledge,this is the first report on the de novo biosynthesis of cycloastragenol in yeast.The mevalonate pathway was reconstituted in yeast peroxisomes,and the engineered yeast produced 656.55 mg/L squalene.Further introduction of heterologous enzymes led the engineered yeast to produce 1.04 mg/L cycloastragenol,which demonstrated the yeast production of value-added medicinal molecules.展开更多
基金supported by the National Natural Science Foundation of China(no.22378083)Mengniu group with project number EN2400037.
文摘Microbial proteins hold great promise as sustainable alternatives for future protein sources,and oleaginous yeast Yarrowia lipolytica has emerged as a recognized platform for heterologous protein expression and secretion.N-terminal signal peptides(SPs)are crucial for directing proteins to the secretion pathway,which offers advantages in both academic and industrial protein production.Although some of the innate SPs of Y.lipolytica have been reported,there is a growing need to expand the genetic toolkit of SPs to support the increasing use of Y.lipolytica as a cell factory for overproduction of various secretory proteins.In this study,we employed an efficient evolutionary approach to rapidly evolve the innate SP XPR2-pre by leveraging Gibson assembly with two syn-thetic overlapping oligos containing high portion of degenerate nucleotides.Using Nanoluc(Nluc)luciferase as a robust reporter,we characterized the intracellular and extracellular enzymatic activity of 447 SP mutants and identified previously undescribed SPs exhibiting superior performance compared to XPR2-pre in Nluc luciferase secretion,with improvements of up to 2.91-fold of enzymatic activity in the supernatant.The generalizability of the top-performing SPs was evaluated using three additional heterologous enzymes(β-galactosidase,α-amylase,and PET hydrolase).Our results confirmed their versatility across different proteins with protein-specific effi-ciency.Additionally,based on our screening,we also evaluated the performance of different feature engineering strategies and machine learning models in the design and prediction of SP mutants.This study integrated rational design,directed evolution and machine learning to identify novel SPs,expanding the repertoire of signal peptides and benefiting secretory protein overexpression in Y.lipolytica.
基金supported by the Program for Scientific and Techno-logical Innovation Talents in Universities of Henan Provincial Education Department(25HASTIT057)the Henan Province Outstanding Youth Foundation(242300421097)National Natural Science Foundation of China(32371485 and 32111530179).
文摘Cycloastragenol is a bioactive,high-value triterpenoid derived from Astragalus membranaceus.Conventional plant-based extraction and chemical synthesis methods are expensive.To our knowledge,this is the first report on the de novo biosynthesis of cycloastragenol in yeast.The mevalonate pathway was reconstituted in yeast peroxisomes,and the engineered yeast produced 656.55 mg/L squalene.Further introduction of heterologous enzymes led the engineered yeast to produce 1.04 mg/L cycloastragenol,which demonstrated the yeast production of value-added medicinal molecules.
