Production Of Astaxanthin By High-density Fermentation Of Engineered E. Coli

Astaxanthin is an important carotenoid,which has the strongest antioxidant effect among carotenoids.It has important economic value and broad application prospects in the fields of food,cosmetics and health products.Currently,most of the astaxanthin produced by fermentation are produced by Haematococcus pluvialis and Phaffia rhodozyma.The fermentation process is mature.However,it has the disadvantages of long fermentation cycle and difficult extraction,which is difficult to overcome fundamentally.Escherichia coli,as a mature cell factory,introduces heterologous genes to construct an astaxanthin synthesis pathway in E.coli engineering bacteria,and then produces astaxanthin through high-density fermentation.It is an excellent choice for astaxanthin industrialization production,with short cycle,low cost and easy extraction.Compared with the highest production of 432 mg/L?7.12mg/g DCW?to date for fed-batch fermentation by engineered E.coli reported by Robert.M.B.et al.,.the engineered E.coli-LY01 constructed and screened in our laboratory has a astaxanthin production of 10 mg/g DCW in shake flasks,which has great potential for astaxanthin fermentation production and the optimization of fermentation process on fermentor scale needs to be carried out.This study was based on E.coli-LY01,an engineering strain of E.coli with high astaxanthin production.This study successfully achieved its expanded culture from shake flask level to fermentor level.By optimizing the pH,temperature,the amount of inducer and the glucose feeding method during the fermentation,a set of high-density fermentation processes for E.coli were established:37?before induction,pH 7.0 culture,and when the OD₆₀₀ reached 60,L-Arabinose was induced by 0.5g/L inducer,and the culture conditions were adjusted to 32°C and pH 7.4.Glucose feeding uses variable speed feeding method.Astaxanthin volume production?mg/L?and dry weight production?mg/g DCW?reached a maximum of 958 mg/L and 17.2 mg/g DCW,and the bacterial biomass(OD₆₀₀)reached 165.Astaxanthin dry weight production?mg/g DCW?was increased by 63.1%compared to shake flask culture.The astaxanthin dry weight production?mg/g DCW?and volume production?mg/L?were increased by 28.6%and 26.8%.In terms of astaxanthin purity,the proportion of astaxanthin in the total pigment increased from 58.9%to 87.0%.The fermentation process basically realized the experimental purpose of obtaining high production and high purity astaxanthin by fermentation,which can provide a reference for the industrial production of astaxanthin.In this study,a batch of astaxanthin-synthesis-related genes enhanced engineering bacteria fermentation experiments were also constructed in the laboratory.The Isopentenyl diphosphate isomerase?IDI?gene-enhanced strain E.coli-IDI-3 achieves an astaxanthin dry weight production?mg/g DCW?of 18.7 mg/g DCW and an astaxanthin volume production?mg/L?was 964 mg/L,and astaxanthin accounted for 86.4%of the total pigment.The crt Z gene-enhanced strains E.coli-PanZ and E.coli-AauZ are mainly used to reduce the proportion of by-products.Among them,the production and purity of E.coli-AauZ are the best,and the astaxanthin volume production?mg/L?and dry weight production?mg/g DCW?are 18.6mg/g DCW and 944 mg/L,and the purity can reach 94.1%.The crtZ-deficient and crt W-deficient engineered strains E.coli-Can and E.coli-Zea target canthaxanthin and zeaxanthin as their target products.The dry weight and volume production of canthaxanthin reached 28.5 mg/g DCW and 2423 mg/L,dry weight and volume production of zeaxanthin reached 27.8 mg/g DCW and 1445 mg/L.The productions of canthaxanthin and zeaxanthin were both high.The preliminary trials of high-density fermentation production of canthaxanthin and zeaxanthin were completed.