Construction And Optimization Of Metabolic Pathways Of Unnatural Ginsenosides In Saccharomyces Cerevisiae

Ginsenosides are the dominant bioactive components of the traditional Chinese herb Panax ginseng.Ginsenosides can be divided into two types,dammarane type tetracyclic triterpenoid saponins and oleanane type pentacyclic triterpenoid saponins.Dammarane-type saponins account for the main active components of ginsenosides.The damarane-type tetracyclic triterpenoid saponins contain protopanaxadiol(PPD)-type saponins and protopanaxatriol(PPT)-type saponins.PPD-type saponins are usually produced through glycosylation at C3-OH and C20-OH of ginsenoside aglycone PPD.PPT-type saponins are produced through glycosylation at C6-OH and C20-OH of ginsenoside aglycone PPT.At present,ginsenosides,glycosylated at the C-12 hydroxyl group,haven’t been isolated from P ginseng.In our study,glycosyltransferase UGT109A1 indentified from bacillus subtilis,which can catalyze the glycosylation of ginsenoside aglycone PPD at both C3-OH and C 12-OH,were integrated into saccharomyces cerevisiac.Due to the low yield of 3β,12β-Di-O-Glc-PPD and massive accumulation of PPD in the recombinant strain,we speculated that the glycosyltransferase UGT109A1 may be a rate-limiting enzyme.It is necessary to modify UGT109A1 to improve the yield of 3β,12β-Di-O-Glc-PPD.The structure of glycosyltransferase UGT109A1 was predicted.UGT109A1 was docking with substrate molecule PPD.The hotspot of UGT109A1 were scanned with alanine.Half-saturated mutation of E69,K73 and L171 was conducted for improving the activity.By integrated the enzyme genes involved in the biosynthetic pathway of PPD and the mutated UGT109A1 into Saccharomyces cerevisiae,the yield of 3β,12β-Di-O-Glc-PPD in the recombinant strain was significantly improved.The specific results are as follows.(1)In order to enhance the catalytic activity of UGT109A1 to PPD,alanine scanning was conducted for the preliminary research of the relationship between structure and function of UGT109A1.The results showed,when H16,F231,M315 and E317 were mutated to alanine,the mutants completely lost their catalytic activities towards PPD.It suggested that these sites might be the key sites for maintaining glycosyltransferase catalytic activity.Mutations at site L80、V108、F136 and L138 decreased the catalytic activity of UGT109A1,suggesting that these sites might be essential for enzyme activity.The mutations at 111、E69、K73、T77、F107、S129 and L171 could enhance the catalytic activity.Among them,K73A was the highest activity,14.0 times higher than that of the wild type.The activity of E69A was 12.4 times higher than that of the wild type.The catalytic activity of L171A enhanced to 11.5 times compared with that of the wild type.(2)The half-saturated mutation method was used for further study.Among the half-saturated mutations at E69,E69A had the highest catalytic activity,which was 12.4 times higher than that of wild type.Among the half-saturated mutations at K73,the catalytic activity of K73A is the highest,which is 14.0 times higher than that of the wild type.Among the half-saturated mutations of L171,the catalytic activity of L171F enhanced to 11.7 times compared with that of the wild type.(3)In the study of optimal pH of K73A mutant,it was found that the optimal pH of the mutant protein was 11,and the enzyme was inactivated when the pH was higher than 11.5.In the study of optimal temperature,it was found that the optimal temperature of K73A mutant was 38℃.(4)In order to construct the biosynthetic pathway of 3β,12β-Di-O-Glc-PPD in S,cerevisiae,the codon-optimized genes of mutated UGT109A1 and DS,CYP1,CYP-Re genes were integrated into the chromosorme of Y-△HXK2 through the CRISPR/Cas9 system.Compared with the control strain,the yield of 3β,12β-Di-O-Glc-PPD was improved.The yield of 3β,12β-Di-O-Glc-PPD in YU-E69A is 1.70 times higher than that of control.The yield of 3β,12β-Di-O-Glc-PPD in YU-K73A was increased 1.84 times compared with control.The yield of 3β,12β-Di-O-Glc-PPD in YU-L-71F was 3.07 times higher than that of control.These results indicated that UGT109Al had been modified to exhibit better catalytic activity.The above research provides new ideas for improving the production of unnatural ginsenoside 3β,12β-Di-O-Glc-PPD in S cerevisiae,and lays a foundation for optimizing the engineering strain.It also lays the foundation for further research on pharmacological activity and new drug development of 3β,12β-Di-O-Glc-PPD.Previously,we have constructed engineered yeast to produce the anti-tumor ginsenoside 20S-O-D-glucopyranosyl-dammar-24-ene-3β,20S-diol(referred to as 20S-O-Glc-DM).The engineered yeast was optimized by optimizing chassis cells,overexpressing upstream pathway genes,suppressing expression of lanosterol synthase and overexpressing transcription factors.In this study,high-density fermentation was performed on the engineered strain Y7CSH,which had the highest yield at shake flask level,to further increase the yield of 20S-O-Glc-DM.The results are as follows.During the fed-batch fermentation,the ventilation rate was controlled to 3 L/min,the rotation speed and the dissolved oxygen were connected to maintain the DO value within 30±5%,the pH value was in the range of 5.5±0.2,and the glucose and ethanol contents were lower than 1.0 g/L and 5.0 g/L,respectively.The Y7CSH strain grew for 192 h,and the titer of 20S-O-Glc-DM reached 5.6 g/L,eventually.The above research provides an environmentally friendly and sustainable method for producing large amounts of unnatural ginsenoside 20S-O-Glc-DM,and also lays the foundation for research and development of innovative pharmaceutical drugs.