Research On Strategy Of Modular Assembly Of Yeast Multigene Pathways

Development of synthetic biology has expanded from design of gene parts and devices to engineering of pathways and systems.During this process,synthesis of gene part comes to be standardized,and regulation of pathways and systems function comes to be fine and precise.This work has focused on developing strategy of modular assembly of yeast multigene pathways to break through the following key bottlenecks in synthetic biology.First,how to design standardized gene part to regulate and coordinate multigene expression? Second,how to construct functional gene combinations to remold artificial synthetic systems?For regulating multigene expression with standardized parts,we designed a new strategy of multigene assembly with regulatory linkers(M-PERL).The regulatory linkers contain two homologous ends for guiding assembly of gene parts,and have a central regulatory region for tuning gene expression levels.The design and synthesis of whole regulatory linkers can be operated by a standardized workflow.By using regulatory linkers,yeast transcriptional start site(TSS)and adjacent regions were rationally truncated,replaced and elongated,or endued with insertions of successive 5 to 40 random bases.As a consequence,the transcriptional strengths could be expanded to different magnitudes.For engineering the five-gene violacein synthesis pathway,cell libraries were constructed which presented varied product compositions.The fine-tuning of multigene expression was realized,increasing the violacein production by 80%.This strategy was also proved available for engineering industrial strain ?Angel Yeast?.For constructing functional gene combinations to remold artificial synthetic system,we designed standardized ?modular cassettes? to assist construction and assembly of functional gene modules.Based on seriate assembly of gene modules,alternative heterologous pathways for violacein,naringenin,?-carotene and lycopene synthesis were constructed in Saccharomyces cerevisiae or Yarrowia lypolytica.Further more,standardized modules of more than 250 genes were constructed and divided into 20 groups according to their biological functions.The introduction and assembly of modules affected yeast artificial system,making carotenoids production improved by 80% to 100% in S.cerevisiae.The modules of 12 groups in all groups were introduced into Y.lipolytica as totally heterologous gene modules,which greatly affected lycopene production.The obtained highest lycopene production was 89.73 mg/L,which was 52 times of initial production.To sum up,this work tried to explore principles and strategies of regulating artificial synthetic systems from several angles.Based on the M-PERL strategy,standardized design of artificial regulatory part was utilized to fine-tune multigene expression.Based on library construction and assembly of gene modules,artificial synthetic system could be remolded.The results of this work highly improved the ability of regulating artificial synthetic systems.