Heterologous Biosynthesis Of Lycopene In Lactic Acid Bacteria

Lycopene,a C₄₀ terpenoid compound,has strong anti-oxidation and immunity enhancement effects,which has been widely used in the food and cosmetics industries.At present,the industrial production cost of lycopene is high and the yield is low,which is far from meeting the growing demand.With the rapid development of synthetic biology,the use of microbial heterologous synthesis of lycopene has become a research hotspot.As food-grade microorganisms,lactic acid bacteria are potential excellent hosts for heterologous synthesis of lycopene,but there are few related research reports.Therefore,in this article,lactic acid bacteria are used as chassis cells to synthesize lycopene heterologous,and metabolic engineering methods and fermentation process are used to optimize the synthesis efficiency of lycopene,which lays the foundation for the production of lycopene by lactic acid bacteria.the main researches are as follows:（1）Since lactic acid bacteria lack the ability to synthesize lycopene,this study firstly constructed a module for heterologous synthesis of lycopene by lactic acid bacteria.Three enzyme genes,including geranylgeranyl diphosphate synthase（crt E）,phytoene synthase（crt B）and phytoene dehydrogenase（crt I）were cloned into the backbone of shuttle vectors for lactic acid bacteria p NZ44,respectively.Thus,the lycopene expression plasmid p GZQ01 was constructed.Electroporated into three kinds of lactic acid bacteria,using high performance liquid chromatography（HPLC）determination found that only Lactococcus lactis NZ9000 can synthesize lycopene,the yield is 0.46 mg/L.The fermentation extract of recombinant L.lactis NZ9000 was further analyzed by ultra-performance convergence chromatography（UPC²）,and the pigment was determined to be lycopene.（2）In order to improve the efficiency of lycopene synthesis by recombinant L.lactis NZ9000,this study used metabolic engineering methods to knock out lactate dehydrogenase（ldh）using CRISPR-Cas9 technology to obtain recombinant strain L.lactis NZ9000-△ldh/p GZQ01,the yield of lycopene increased to 0.92 mg/L,which was1 time higher than that of the control strain.The MVA pathway genes from Streptococcus thermophilus,Lactobacillus plantarum and Lactococcus lactis were overexpressed,respectively,and the results showed that the overexpression of Streptococcus thermophilus 3-hydroxy-3-methylglutaryl-Co A reductase（mva A）The highest concentration of lycopene in the recombinant strain GZQ12 was 1.213 mg/L,and the yield was increased by 31.7%.（3）In order to further improve the efficiency of lycopene synthesis by recombinant GZQ12,the fermentation conditions were optimized by single factor experiment.The optimized culture conditions were GM17 medium supplemented with glucose 7.5 g/L,tryptone 7.5 g/L,yeast powder 5 g/L,initial fermentation p H 7.0,fermentation temperature 30°C and culture time 12 h.After optimization,the lycopene yield of GZQ12 reached 1.448 mg/L,which was 20%higher than that before optimization.In addition,compared with the control L.lactis NZ9000/p NZ44,the DPPH,ABTS and·OH free radical scavenging abilities of GZQ12 itself and in skim milk were significantly enhanced,antioxidant activity increased by 46%～103%.