Research On Synthetic Biology Technologies And Central Metabolism Reprogramming In Saccharomyces Cerevisiae

Saccharomyces cerevisiae is currently the most thoroughly studied model eukaryote.In recent decades,it has greatly promoted the development of molecular biology,metabolic engineering,genomics,and synthetic biology.S.cerevisiae is also an ideal microbial cell factory and widely used for the large-scale production of a variety of high value-added compounds.This dissertation focused on the development of novel genome editing and regulation tools and the identification of metabolic engineering targets related to glucose repression in yeast.Firstly,by optimizing the CRISPR/Cas9 system,a multi-functional CRISPR system was developed for combinatorial metabolic engineering applications that enables simultaneous gene activation,repression,and editing(CRISPR-ARE)in S.cerevisiae.In the proof-of-concept study,CRISPR-ARE was demonstrated for activating the expression of mCherry for 3.8-fold,repressing the expression of m Venus by 64%,and editing an endogenous ADE2 gene with nearly 100%efficiency.In addition,CRISPR-ARE was applied for combinatorial optimization of ?-santalene biosynthetic pathway(simultaneous HMG1 activation,ERG9 repression,and UPC2 editing),which improved the production of ?-santalene for 2.66-fold in a single step.Subsequently,a pyruvate decarboxylase deficient Pdc strain and a derivative PDH strain were constructed by knocking out the pyruvate decarboxylation pathway and further integrating the cytosol-localized pyruvate dehydrogenase genes.Based on these two strains,genome-scale engineering was performed to identify glucose repression related targets and construct pyruvate and acetyl-CoA overproducing platform strains.Since the MAGIC genome evolution system contains the most comprehensive and diversified genomic library for activation,interference,and deletion in yeast,it was applied in genome-wide screening.Over 70 targets were identified that relieved the Crabtree effect and recovered the growth of Pdc strains in glucose-containing medium.With the next two rounds of high-throughput screening,a large number of novel targets were identified.33 and 34 screened targets of the following two rounds were further verified via reverse metabolic engineering,respectively.The glucose repression effect of the resultant strains was gradually relieved,and the glucose utilization capacity was gradually increased.The final engineered strains Pdc-R3 and PDH-R3 were capable of high pyruvate accumulation(over 3 g/L),efficient R-BDO production(over 5 g/L),and co-utilization of mixed carbon sources.These established strains promise to be further engineered to be platform cell factories for high level production of value-added compounds.Finally,to take advantage of the increased carbon metabolic flux in the mitochondria of Pdc and its derivative strains,mitochondrial compartmentalization of the whole metabolic pathway is an effective metabolic engineering strategy with practical applications.Considering that mitochondrial localization signal peptides were required for compartmentalizing metabolic pathways into the mitochondria,a series of S.cerevisiae mitochondrial localization signal peptides were constructed and characterized,and its application in Pdc platform strains was further explored.For the first time,18 mitochondrial signal peptides with good mitochondrial localization ability were systematically characterized through growth complementation and fluorescence confocal methods.After systematic characterization,seven of the well-performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria.As a proof of concept,the full ?-santalene biosynthetic pathway consisting of 10 genes capable of converting acetyl-CoA to ?-santalene was compartmentalized into the mitochondria,leading to a 3.7-fold improvement in the production of ?-santalene.When applied to the PDH-R3 strain,the production of ?-santalene was also increased by more than 50%compared to the wild-type strain.In this thesis,a novel metabolic engineering tool was established that enables convenient and simultaneous regulation of multiple targets in S.cerevisiae.On the other hand,novel glucose repression targets were identified via genome-scale engineering and the resultant pyruvate and acetyl-CoA overproducing platform strain were constructed.This research has developed new metabolic regulation tools and laid the foundation for elucidating the mechanism of glucose repression and the development of yeast platform cell factories.