Use Of The Optimized Membrane Engineering To Improve The Ability Of Escherichia Coli To Accumulate And Transport Carotenoids

Carotenoids are natural lipid-soluble pigments with good physiological activity and nutritional function.The species Escherichia coli is industrially used as cell factory to produce carotenoids because of its clear genetic background,simple genetic manipulation and easy cultivation.In this study,metabolically engineered ?-carotene or lycopene producing E.coli strains were employed to further boost their final production titers via modification of cell membrane.The storage and transport capacity of carotenoids were optimized by raising the content of cell membrane and constructing the excretion system,and the accumulation level of the carotenoids was improved.The main results are as follows:1.Enhance cell membrane content contributes to carotenoids storage.Firstly,overexpression of lipoglycosyltransferase Almgs gene resulted in morphological changes of E.coli cell membrane,which led to a fonnation of more cell membrane analogues.The change of cell membrane morphology resulted in a 28.3%increase in ?-carotene production in Almgs expression mutant than in the parental strain CAR015.We further fine-tuning of the transcriptional intensity of Almgs gene,and found that promoter M1-37,which has moderate expression intensity,was more conducive to Almgs biological function.After transcriptional regulation,the specific production of ?-carotene increased by more than 50%in mutant compared with that of the original strain.Secondly,by overexpressing two acyltransferase(PlsB and PlsC)in CAR015,the synthesis of phospholipid in cell membrane was enhanced,and the specificproduction of ?-carotene in the transformed cells was increased by 43.8%compared with the control.Moreover,the combination of the above two strategies had a synergistic effect,which caused a 2.9-fold increase of ?-carotene specific production(from 6.7 mg/g to 19.6 mg/g DCW).Further transmission electron microscopy observation of sub-cellular morphology and cell membrane composition analysis showed that the main storage area of ?-carotene was cell membrane.Subsequently,the above cell membrane modification strategy was extended to the(3-carotene hyper-producing strain CAR025,and the specific production of ?-carotene could also be increased significantly.The specific production of ?-carotene increased from 31.8 to 44.2 mg/g DCW,raising about 39%.2.Build up excretion system to enhance carotenoid transport capacity.By knocking out genes related to the outer membrane structure of E.coli,such as tolA,tolR and nlpI.It's found that the secretory capacity of the outer membrane vesicles can be significantly increased and the specific production of extracellular(3-carotene was elevated.Secondly,by characterizing double deletion of all pairwise combinations of tolA,tolR and nlpl genes,it was found that ?tolA ?nlpl or ?tolR ?nlpI could significantly superimpose the release effect of outer membrane vesicles.In addition,we combined the cell membrane synthesis module and the excretion system,and also co-expressed cell membrane component AccABCD and PlsBC.The results made the number of the outer membrane vesicles of strain TW-012and strain TW-013 increased,and the specific production of the excreted ?-carotene in the extracellular was significantly higher than that of the control strain which was 48 fold and 71.5 fold respectively.Finally,after extending this outer membrane modification strategy in CAR025,the?-carotene excretion ability was improved which led to a marked enhancing of ?-carotene final production3.The application of membrane strategy in the production of other terpenoids.The membrane modification strategy for E.coli is also suitable for improving the ability of the cell membrane to store the lycopene.Compared with the control strain LYC101,the lycopene content of the unit cell of the mutant LYC101-37Almgs(pPlsBC)after the cell membrane modification is 1.32-fold that of the starting strain,indicating that cell membrane modification is general and universal strategy for terpenoids productionIn conclusion,the storage and transport capacity of carotenoids can be effectively improved by enhancing the content of cell membrane and constructing the excretion system.This work provides a novel engineering mode and practical route for the future use of E.coli as host to produce carotenoids and other terpenoids...