Engineering ?-carotene Producers Of Escherichia Coli Using Gene Elements From Zymomonas Mobilis

Beta-carotene is widely used as a natural colorant and nutrition enhancer in the food industry.Currently,metabolic engineering enables the high yield of ?-carotene,being promising as an alternative technology to natural extraction and chemical synthesis.Since ?-carotene's biosynthesis involves with multiple genes,it is crucial to construct an efficient metabolic pathway for the high yield of ?-carotene.In this thesis,we metabolically engineer the Escherichia coli strains producing ?-carotene,and establish a feasible strategy to increase the yield of ?-carotene in the engineered strains by combining rational engineering with random mutagenesis and the optimization of fermentation processing.The contents and results are summarized as follows:1.The assembly of ?-carotene's metabolic pathways and engineering the E.coli strains producing ?-carotene.Firstly,we analyzed native promoters' activity of Zymomonas mobilis ZM4 strain,and build libraries using representative promoters and terminators,respectively.Using the Golden Gate Assembly(GG)technology,then we assembled the gene cluster for ?-carotene synthesis,crtEXYIB,from Pantoea agglomerans CGMCC 1.2244 with the promoters and terminators libraries,and obtained 43 engineered strains,we screened a strain with high yield,Crt1-29,in which the ?-carotene yield reached 0.68 mg/g dry cell weight(DCW).2.Optimization of the biosynthesis of ?-carotene by rational engineering strategy.We enhanced the yield of ?-carotene using a two-round assembly strategy.In the first round,the ?-carotene gene cluster was divided into three parts: crtE,crtXYI and crtB,and then assembled them with the promoters and terminators libraries into transcriptional units(TUs),respectively.In the second,the TUs were assembled into entire metabolic pathways for carotene's biosynthesis.Among forty engineered strains,the ?-carotene yield of Crt2-20 reached 0.83 mg/g DCW,which is 21.5% higher than that of Crt1-29,respectively.The results demonstrate that the optimization of TUs is an effective strategy to improve biosynthesis.In addition,we sequenced the DNA elements used in the ?-carotene metabolic pathways and calculated the usage rate and activity of biological elements,confirming that the optimization of promoters and terminators helps improve the biosynthesis of ?-carotene.3.Mutagenesis by Atmospheric and room temperature plasma(ARTP).To further increase the yield of ?-carotene,we conducted ARTP mutagenesis and screened 65 mutants in total.Significant increases in ?-carotene's yield were found in six mutants,ranging from 3-9% higher than that before mutagenesis,respectively.4.The optimization of fermentation processing.We carried out the single-factor experiments to determine fermentation parameters such as medium type,carbon source,nitrogen source,dissolved oxygen and temperature.The results showed that culturing in 2YT+1% sucrose with 30% of the dissolved oxygen at 37? is optimal to accumulate ?-carotene.Under such conditions,the mutants A7 and B8 produced higher ?-carotene yield in flask fermentation than did the control.In batch fermentation with 3 L fermentation working volume,the yield of B8 reached 1.50 mg/g DCW,19.2% higher than that of the parent strain Crt2-20.In summary,we established a strategy combining rationally metabolic engineering,ARTP random mutagenesis with fermentation optimization in this study,which can efficiently increase the yield of ?-carotene biosynthesis.