Systems Metabolic Engineering Of Candida Tropicalis For Terpenoid Production

Terpenoids are widespread in plants,animals,and higher fungi.Most of them have antitumoral,antiviral,and antibacterial activities.Terpenoids have tremendous applied value in foodstuff,medicament,energy and chemical industry.Due to their complex structures,most terpenoids in the market cannot be produced by chemical synthesis.Currently,the large-scale production of terpenoids is typically achieved via plant extraction.However,the terpenoids content in its original plants is low,and there are significant differences among varieties and regions.Considering the complicated process and depletion of natural resources,the plant extraction methods could not meet the market`s requirements.With the development of biotechnology,synthetic biology strategies transforming microbial cells to produce natural terpenoid products have high expectations.In this dissertation,Candida tropicalis ATCC 20336 was used as a chassis cell and a CRISPR-Cas9-based genome editing strategy for the strain was developed.Based on the CRISPR-Cas9 system,a series of C.tropicalis strains were constructed for producing the terpenoids including β-carotene,cembratriene-ol,miltiradiene,α-humulene and α-amyrin.The main results of this study are described as follows:1.A CRISPR-Cas9-based genome editing strategy for C.tropicalis was developed.A set of eight promoters from C.tropicalis were cloned and characterized based on genome data mining,in which the promoter activity spanned more than a 42-fold range.Then a CRISPR-Cas9-based genome editing strategy for C.tropicalis was developed using the screened promoters to control sg RNA and Cas9 expression.The system enables gene deleting,site-directed mutating,inserting,and assembling of multiple overlapping DNA fragments and integrating them into a target chromosomal locus in a single step.Based on the system,single-gene disruption(two loci,between 57% and 100%)and multi-gene deletions(four loci,32%)can be accomplished within nine days,saving the time for metabolic engineering and breeding.2.The β-carotene synthetic pathway in C.tropicalis was optimized using spatial combination of different subcellular compartments.Based on the bioinformatics analysis of the genomic data,three mitochondrial targeting signals and six peroxisome targeting signals were characterized.Using the signal peptide,a panel of engineering strains was constructed by localizing the β-carotene synthetic pathway to different subcellular compartments of the yeast.An optimized β-carotene synthetic pathway was obtained in which the pathway from acetyl-Co A to mevalonic acid located in cytoplasmic and mevalonic acid to β-carotene located in the peroxisome(generated strain 1C2P3P)The engineered strain 1C2P3 P can produce 203.6 mg/L β-carotene,which was about 1.6-fold than that of the strain(the entire β-carotene syntheic pathway was in the peroxisome),3.7-fold than that of the strain(in the cytoplasm),as well as 145.4-fold than that of the strain(in the mitochondria).Additionally,we analyzed the intermediates of terpenoid synthesis pathway permeability across the peroxisomal membrane using β-carotene as a reporter.The results showed that IPP and DMAPP could pass through the peroxisomal membrane,but not FPP and GGPP.3.A strain with high production of β-carotene was constructed through a modular engineering strategy(based on strain 1C2P3P).Moreover,this strategy was used to obtain the diterpenoidproducing strain.Through the overexpression of key enzymes,the β-carotene titer of the engineered strain was increased.To further improve β-carotene production,the ATP transport pathway in peroxisome was enhanced,and the engineered strain ANT06 was able to accumulate β-carotene with the titer of 585.2 mg /L,which was 187.4% higher than that of the control strain 1C2P3 P.The titer ofβ-carotene reached 6.5 g/L by fed-batch fermentation in a 5 L fermenter.This strategy was also used to engineer C.tropicalis for cembratriene-ol and miltiradiene production.The titer was 129.2 mg/L and 471.0 mg/L in the shaker,respectively.Finally,cembratriene-ol and miltiradiene titer reached 1.4g/L and 4.2 g/L in the 5 L fermenter,respectively.4.By compartmentalizing the entire syntheic pathway in the cytoplasm of C.tropicalis,α-humulene and α-amyrin production strains were constructed.An optimized α-humulene syntheic strain(DC-H01)was obtained by locating the entire α-humulene synthesis pathway in cytoplasmic.The engineered strain DC-H01 produced 12.9 mg/L α-humulene,about 5.3-fold more than that of the strain(the entire α-humulene synthesis pathway in the peroxisome).Furthermore,bottlenecks in theα-humulene synthase pathway were identified and relieved by overexpressing α-humulene synthase,acetoacetyl-Co A thiolase,and NADH-dependent HMG-Co A reductase.The engineered strain DCH21 D was able to produce 121.1 mg/L α-humulene,which was about 9.4-fold than that of the strain DC-H01.Combined with fermentation medium optimization,the engineered strain produced 195.31mg/L of α-humulene in shake flasks and 4.1 g/L in a bioreactor through fed-batch fermentation.This strategy was used to engineer C.tropicalis for α-amyrin production.The titer was 48.3 mg/L in shake flasks,which was about 68.1-fold than that of the strain DC-H01.