Engineering And Optimizing Saccharomyces Cerevisiae For The Production Of Protopanaxadiol From Xylose

Xylose is a kind of monosaccharide whose content is only inferior to glucose in lignocellulose hydrolysate.So far,xylose has been used to produce a variety of chemicals,such as bioethanol,butanol,acetoin,PHA and so on.The latest study found that xylose is more efficient to produce substances derived from acetyl-CoA than glucose.Acetyl-CoA is a precursor to the synthesis of isoprenoid compounds and it was found that isoprenoids production with xylose is more efficient than glucose.In this study,xylose was used to produce protopanoxadiol,an exogenous triterpene for Saccharomyces cerevisiae.The aim is to construct an engineering strain which utilizes xylose to produce protopanaxadiol.Simultaneously,the differences in fermentation of xylose and glucose are to be compared.First,using the original protopanaxdiol synthesis strain WLT as the starting strain,the XR-XDH xylose metabolism module is introduced to obtain WLT-2,which can metabolize xylose to produce protopanaxadiol;then,PP Pathway optimization module is introduced to obtain WLT-3,which was to accelerate the rate of xylose metabolism.The results showed that the yields of WLT-2 and WLT-3 were 72.32 mg/L and 93.65 mg/L,respectively,in the fermentation with xylose as a single carbon source,while the yields of PPD in glucose-based single carbon source were 17.94.mg/L and 18.11 mg/L.The PPD production of WLT-2 and WLT-3 with xylose was 3.03 and 4.17 times higher than that of glucose,respectively.Then,using strain Saccharomyces cerevisiae W303-1A as the starting strain,the xylose metabolism module,the PP Pathway optimization module,the Dammarenediol synthesis module and the protopanaxadiol synthesis module were constructed and W303-1A was transformed into an engineering strain GW6.The GW6 was fermented with mixed sugar and glucose as carbon sources respectively,the PPD titer was 11.16 mg/L and 8.75 mg/L.Although the titer of mixed sugar fermentation was lower than that of glucose,the yield of mixed sugar fermentation was 0.564 mg PPD/g xylose,which was 2.575 times the glucose yield 0.219 mg PPD/g glucose.Finally,the GW6 strain was optimized from the xylose metabolic pathway,MVA pathway,central carbon metabolism,and cofactor balance.It was found that the balance of cofactors regulated by NADH kinase POS5 increased the PPD yield of GW6 by 3.55 times to 50.78 mg/L,which was 1.91 times that of glucose fermentation.The strain was fermented in a 5L bioreactor and the final concentration of PPD reached 152.37 mg/L under mixed sugar conditions.which was 2.58 times that of glucose fermentation.It can be seen that xylose has a greater advantage and potential than glucose for the production of PPD.The strategies of xylose utilization coupled with metabolic engineering methods will lead to a higher level production of PPD.