Corynebacterium Glutamicum Chassis Construction For Biosynthsis Of Geraniol

Geraniol is an acyclic monoterpene alcohol,with a mild rose fragrance,often used in food additives,as the main agent for the fragrance of rose.As a pharmaceutical raw material,it is used for antibacterial and insect repellent.It is good for clinical treatment of chronic bronchitis,and it is also helpful for improving the immune function of the body.In this paper,the metabolic engineering of the synthesis of geraniol was carried out by using the wild type Corynebacterium glutamicum ATCC13032 as the starting strain.The research on the synthesis and yield improvement of geraniol was carried out in the following aspects:1.In wild type C.glutamicum ATCC13032,expression plasmid pEC-XK99E as the carrier,Catharanthus roseus or source of Valeriana officinalis geraniol synthase gene CrGES or VoGES was introduced,and determined VoGES geraniol for optimal synthesis genes.2.To improve the intracellular geranyl pyrophosphate GPP,expression plasmid pEC-XK99E as the carrier,four different geranyl pyrophosphate synthase gene(the source of Saccharomyces cerevisiae ERG20^ww,the source of Catharanthus roseus CrGPPS,the source of Arabidopsis thaliana GGPPS-11-12,the source of Arabidopsis thaliana AtGPS1)respectively with VoGES express in wild type C.glutamicum ATCC13032.The sources of Saccharomyces cerevisiae ERG20^ww was best,The yield of Cg-ERG20^WW-VoGES was 5.38±0.38 mg/L.3.In order to improve the main metabolic fluence,pXMJ19 was used as the carrier in the strain Cg-ERG20^WW-VoGES to express the key genes dxs and idi in the synthesis pathway of terpenes,and the yield of geraniol was further increased to12.18±0.44 mg/L.4.Study the effect of the fusion protein yield of geraniol,ERG20^ww with VoGES was linked together by two different linkers,express plasmid pEC-XK99E as the carrier in the wild type C.glutamicum ATCC13032 expressed fusion protein,the results showed that the fusion protein strategy did not the same as the synthesis of geraniol in Saccharomyces cerevisiae,geraniol yields down to 1.60±0.07 mg/L and2.13±0.08 mg/L.5.The genes involved in the synthesis of monoterpenes in the pathway were studied,and the gene with the highest impact on the yield of geraniol was idsA,and the yield of the isolates from idsA was significantly decreased.The knockout of crtE,crtB and crtB2 had little effect on the yield.6.Based on the preliminary optimization of geraniol shaking flask fermentation conditions,determined the best shaking flask fermentation process,the initial glucose concentration20 g/L in CGXII medium,fermentation temperature 30?,initial pH6.0,dodecanoic 10%,180 rpm,Cg-dxs-idi-ERG20^WW-VoGES geraniol production increased to 15.19±0.54 mg/L.