Establishment Of Homology-Independent Genome Integration In Yarrowia Lipolytica And Its Application For Succinic Acid Biosynthesis

Non-conventional yeasts such as Yarrowia lipolytica,Pichia pastoris,Hansenula polymorpha and Kluyveromyces lactis usually have unique physiological and metabolic characteristics which make it as microbial hosts for the production of important compounds including biodiesel,recombinant protein and ethanol,and are widely used in industrial fermentation.Due to the low efficiency of genetic manipulation,metabolic pathway modification of unconventional yeasts and the production of non-natural compounds are still difficult.Y.lipolytica is an important industrial microorganism with high safety,strong acid resistance,secretion of various metabolites and the ability to utilize a variety of substrates.It has been received increasing attention and considered a potential biotechnological strain.Unlike Saccharomyces cerevisiae,Y.lipolytica is more prone to utilize non-homologous end joining(NHEJ)rather than homologous recombination(HR)during DNA damage repair.This feature has led to a lack of the genetic editing tools,and the time-consuming and labor-intensive for genome integration of large DNA fragments.In addition,strategies for constructing expression libraries to optimize multigene biosynthetic pathways are also absent in Y.lipolytica.In order to detect the transformation rate of linear DNA fragments in Y.lipolytica,we first introduced a complete LEU2 expression cassette into yeast and calculated the number of transformants in the corresponding selection medium.Up to 1.6×104 colonies can be obtained from 1 ?g of DNA,indicating that Y.lipolytica can efficiently absorb foreign linear DNA.Interestingly,the LEU2 was cleaved into two fragments using the restriction endonuclease HinC?,which can be precisely joined and integrated into the genome after transformation.Knockout ofthe key gene ku 70 in the NHEJ repair pathway resulted in a dramatic decrease in the number of transformants.The genomes of integrated strains were extracted and verified by PCR.It was found that the foreign DNA was efficiently and randomly inserted into the Y.lipolytica genome in homology-independent manner.With the help of selection markers,homology-independent genomic integration allows for efficient transformation of DNA fragments up to 12.5 kb with an integration rate of 1.7 ×103 colonies/?g DNA.As the number of integrated fragments increases,integration rate declines,and up to three DNA fragments can be integrated into genome via one-step transformation.To investigate the protein expression profile of the integrated genes mediated by homology-independent integration,the reporter gene hrGFP was fused with the LEU2 selection marker and transformed into strain Po1g.Compared with the fluorescence intensity of the strain expressing hrGFP from an episomal plasmid,integrated strains showed a wide variation in hrGFP expression,which indicated that the integration location may affect the expression.We therefore analyzed the integration location of six randomly selected strains by Genome walking method.The hrGFP gene insertion sites are distributed on different chromosomes and there is no correlation,which further confirms the randomness of homology-independent integration.Subsequently,three concentration gradients of hygromycin(400 mg/L,800 mg/L and 1600 mg/L)were used to screen for hygromycin resistance and hrGFP gene high expression strains.The relative fluorescence intensity and gene copy number were positively correlated with the screening pressure,while the integration rate was negatively correlated.Most integrated strains contained a single copy of the hrGFP gene at low screening pressure,while up to 8 copies of hrGFP were inserted into the integrated strain 25 obtained from the higher hygromycin concentration.These results indicate that homology-independent random integration results in differences in protein expression levels that are affected by a combination of gene insertion sites and copy numbers.To expand the application field of NHEJ-mediated random genome integration method,this study takes the optimized synthesis of lipase and ?-carotene as an example.Firstly,an overexpression library of Y.lipolytica endogenous lipase LIP2 was constructed under high concentration hygromycin,and a series of lipase production strains were obtained by enzyme activity screening.The lipase activity of the engineered strain Polf LIP2-2 was 1967 U/mL,which was 5.3 times that of the control strain.In view of the ?-carotene biosynthesis pathway involving multiple genes,we modularized and constructed three integrated fragments based on metabolic characteristics.Module 3 was initially integrated into the Y.lipolytica genome to evaluate the possibility of modular integration and optimization.We found that most of the colonies carrying randomly integrated M3 showed the expected orange or red color to different degrees.Subsequently,three modular fragments were transformed into Y.lipolytica wild strain in one step to construct a P-carotene producing library.By detecting the production of ?-carotene,the library generated a nearly 27-fold range of 6-carotene titer,with the highest titer reaching 12.1 mg/g DCW.The transcription level of genes in the ?-carotene pathway was also measured,and transcription levels of the genes of integrated strains were greatly different.Most pathway associated genes in the optimum ?-carotene producing strain showed high levels of transcription.By comparing high titer strains(strains 30,34 and 40)with low titer strains(strains 1 and 12),it was found that high level expression of module 3 may be critical for ?-carotene overexpression.Succinic acid has wide range of applications in many fields like food,chemical and agricultural,and was selected as the first of the twelve most commercially valuable platform compounds by the US Department of Energy.The microbial synthesis of succinic acid relies mainly on a variety of bacteria,while their fermentation process normally requires anaerobic and neutral pH condition.The addition of alkali increases the probability of bacterial contamination during the cultivation and it leads to more complex downstream industrial processing(DSP)steps.In contrast to prokaryotes,yeasts are highly tolerant of low pH,makes it an attractive industrial host.In the long run,yeasts are more suitable for the biosynthesis of succinic acid than bacteria if it can achieve high titer and high productivity.This study firstly explored the acetic acid anabolic pathway of Y.lipolytica.It was found that the inactivation of pyruvate decarboxylase PDC had no significant effect on the accumulation of acetic acid.Overexpression of Salmonella enteric derived SeACS16411P reduced the acetic acid concentration to 4.7 g/L.By excavating the literature and strain modification,it was found for the first time that knockout of the CoA transferase encoding gene Ylach can effectively relieve the acetic acid overflow in SDH-deficient strain.The succinic acid production capacity was further improved by combining optimization to enhance the metabolic flux of the reductive carboxylation,oxidized TCA and glyoxylate pathways associated with succinic acid biosynthesis.When the Saccharomyces cerevisiae derived ScPCK was overexpressed,the succinic acid production in the shake flask reached 30.2 g/L,which was 150.2%higher than the control.Synergistic overexpression of the succinyl-CoA synthase subunits YlSCS2 and ScPCK can further increase succinic acid production by 24%to 37.0 g/L.In the fed-batch fermentation,we demonstrated that the addition of high concentrations of initial glycerol resulted in the accumulation of reducing substances such as erythritol and mannitol,which affected the efficiency of succinic acid synthesis.By adjusting the initial glycerol concentration,the succinic acid titer,yield and productivity of final engineering strain PGC202 can reach 110.7 g/L,0.8 g/h/L and 0.5 g/g glycerol,respectively,without adjusting the pH.To verify the industrial application potentiality of strain PGC202,small and pilot plant fermentations were carried out.The succinic acid production reached 45.4 g/L during the small-scale fermentation with glucose as the sole carbon source.The results of the pilot fermentation were basically the same as those of the 2.5 L and 50 L fermenters,succinic acid titer was 123.9 g/L after 56 h fed-batch cultivation,while the yield(0.76 g/g glycerol)and productivity(2.2 g/h/L)of succinic acid has been greatly improved.This indicates that succinic acid producing strain Y.lipolytica PGC202 possesses great application prospects,and its fermentation process is relatively stable.This study systematically explores the homology-independent genome integration in Y.lipolytica,and develops a rapid and efficient expression library construction method for the microbial production of high value compounds.Rationally reconstructing the TCA pathway of Y.lipolytica can solve the problem of by-product overflow during succinic acid fermentation,and finally achieve high-efficiency biosynthesis of succinic acid under low pH.