Metabolic Engineering Of Saccharomyces Cerevisiae And Fermentation Optimization For Lycopene Synthesis

Lycopene is a natural pigment with strong oxidation resistance.It has extensive biological activities for scavenging free radical,delaying cell senescence,preventing and inhibiting prostatic cancer,liver cancer and cardiovascular disease.Therefore,it has been widely used in food additives,functional food,and cosmetic industries.For the production of lycopene,the plant extraction method has problems such as limited extraction rate,low purity and high price,while chemical synthesis method suffers from uncontrollable chiral groups and low yield,which limit the efficient synthesis and wide application of lycopene.With the development of synthetic biology and metabolic engineering,constructing an efficient cell factory to biosynthesize lycopene has become a research focus.Current studies related to biosynthesis of lycopene focus on optimization of biosynthetic pathway and enhancement of precursor supply.In this study,the biosynthesis of lycopene in S.cerevisiae was achieved after the screening of lycopene biosynthesis genes and the optimization of metabolic pathways.The main results are summarized as follows:?1?A rapid and steady method for determining lycopene concentration was established.The n-hexane was used as the optimal solvent and 472 nm was defined as the detection wavelength after the absorption spectrum of lycopene and beta-carotene in five different solvents was analyzed.Finally,the standard curve was constructed:Y=0.0703X+0.0082(R²=0.9972,the liner interval was from 0.5 to 12 mg·L^-1).?2?Lycopene biosynthesis pathway genes were screened and then biosynthesis pathway was constructed and optimized.To further increase the production of lycopene,the metabolic pathway was engineered.Firstly,three kinds of Crt E,four kinds of Crt B and four kinds of Crt I were expressed in combination and then 48 different strains were constructed.One lycopene-overproducing strain was obtained?Crt E from Taxus x media,Crt B from Pantoea agglomerans and Crt I from Blakeslea trispora?through the above rapid determination method with the titer of 1.68 mg·L^-1(1.33 mg·g^-1 DCW).Secondly,ribosomal DNA multi-copy integration of Pa Crt B and Bt Crt I was implemented to obtain a mutant with higher production.Nine strains with r DNA multi-copy integration showed that there is a positive correlation between the titer of lycopene and gene copies.Recombinant strain R17 with 20 copies of Crt B and Crt I genes showed the highest lycopene titer,which reached 1.68 mg·L^-1(1.14 mg·g^-1DCW).Thirdly,to further increase the lycopene production,the strong promoter was used to express of Crt E.The resulting strain G2 produced 6.95 mg·L^-1(5.16 mg·g^-1 DCW)of lycopene,which was 4.04-fold than the control strain.Finally,to increase the supply of precursor acetyl-Co A,the YPL062W gene was knocked out.The resulting strain Q1 produced 12.32 mg·L^-1(5.16 mg·g^-1 DCW)of lycopene,which was 77.3%improvement than the strain G2.At the same time,An ACLa and An ACLb encoding ATP citrate lyase?ACL?from Aspergillus nidulans were introduced at YPL062W gene loci,the resulting strain Q2 could accumulate 11.04 mg·L^-1(6.48mg·g^-1 DCW)of lycopene.?3?The optimization of the culture medium and fermentation conditions for increasing the titer of lycopene.Firstly,the composition of culture media was optimized by the single-factor experiment and orthogonal experiment in shake flasks.Then the best composition of culture media was obtained:25 g·L^-1 sucrose,10 g·L^-1 yeast extract,25 g·L^-1 peptone,4 g·L^-1?NH₄?₂SO₄,0.05 g·L^-1 Mg SO₄,0.05 g·L^-1 K₂HPO₄·3H₂O,15 m L·L^-1 trace metal solution and 8m L·L^-1 vitamin solution.In this media,the titer of lycopene reached 26.17 mg·L^-1or 12.66mg·g^-1DCW.After that,we further optimized the culture conditions.According to the results of the single-factor experiment and orthogonal experiment,the best fermentation condition was obtained:the initial inoculation OD₆₀₀was 0.2,the initial p H of the media was 7.0 and the volume of the media was 50 m L?in 250-m L flask?.The titer of lycopene reached 26.25 mg·L^-1,which was 113.1%improvement than the initial culture condition.