Engineering The Metabolism Of Tropane Alkaloids Based On AbPMT Promoter

A.belladonna is a commercial source plant of Tropane alkaloids(TAs)of the family Solanaceae designated by the Chinese Pharmacopoeia.And it is also the model plant for conducting research related to TAs.Tropane alkaloids(TA),including hyoscyamine,anisodamine and scopolamine,are widely used clinically as anticholinergic drugs to relieve pain and spasm,relieve Alzheimer’s disease,and treat pesticide poisoning.TAs is mainly dependent on the extraction from TAs medicinal plants such as Atropa belladonna.The content of TAs in wild Atropa belladonna is extremely low,such as scopolamine is only 0.02%-0.17% of the dry weight,resulting in a shortage of scopolamine drug resources.The shortage greatly restricts the industrial production and clinical application of scopolamine and its derivatives.Metabolic engineering is an effective way to increase the yield of TAs from medicinal plants and alleviate the shortage of drug resources.Elucidation of the biosynthetic pathway of TAs has laid an important foundation for metabolic engineering research.Phenyllactate UDP-glycosyltransferase(UGT1)and Littorine Synthase(LS)mediated conchine biosynthesis is an important rate-limiting step in the TAs synthesis pathway.In addition,the results of the enzymatic activities of Ab LS and DsLS showed that the catalytic activity of DsLS is higher than that of Ab LS;therefore,DsLS is more ideal molecular tool for TAs metabolic engineering than Ab LS.The Ca MV35 S promoter is a commonly engineered element for plant-related studies.Although it is defined as a strong promoter for constitutive expression,35 S varies across tissues and cell types.In addition the ubiquitous expression of target genes imposes unnecessary metabolic stress on plants.Therefore,for TAs specifically synthesized in root pericycle cells,metabolic engineering of TAs using pericycle cell-specific strong promoter-driven target genes is a theoretically worthwhile option.In A.belladonna and other medicinal plants of TAs,the pericycle cells of fibrous roots are the key site of TAs biosynthesis.Pathway genes such as PMT,UGT1 and LS are specifically/highly expressed in the pericycle cells of roots.The high expression abundance of PMT found in this study based on A.belladonna transcriptome analysis in the early stage suggests that the promoter of PMT(p PMT)has the strongest ability to drive the expression of downstream genes in the pericycle cells.Therefore p PMT can be used as a candidate molecular tool(promoter)to replace the 35 S promoter for metabolic engineering studies of TAs.Based on the literature analysis and the preliminary results of this study,this paper conducted a metabolic engineering study of tropane alkaloids in A.belladonna hairy roots,using the constitutive promoter 35 S and the pericycle-specific promoter p PMT to drive the target genes AbUGT1 and DsLS,respectively.The results are as follows:1.35S-driven overexpression of both AbUGT1 and DsLS significantly increased the content of TAs in A.belladonna hairy roots.Previous studies have shown that UGT1 and LS are rate-limiting enzymes for TAs biosynthesis.in the present study.In this study,we conducted a metabolic engineering study of TAs using A.belladonna germinating root system with 35 S driving target genes AbUGT1.1)Combined with positive g DNA identification and further q PCR results,the T-DNA regions of p1305.1-35S::AbUGT1 and p1305.1-35S::DsLS were successfully integrated into the genome of the belladella hair root,respectively.Moreover,AbUGT1 and DsLS were overexpressed in the corresponding transgenic roots,which could be used for subsequent metabolite analysis studies.2)The overexpression of 35S-driven target genes AbUGT1 and DsLS significantly promoted TAs biosynthesis in hair roots.Metabolite analysis showed that 35S-driven overexpression of either AbUGT1 or DsLS could promote the accumulation of hyoscyamine,anisodamine and scopolamine in the hair roots of A.belladonna.2.A.belladonna transcriptome based analysis reveals that PMT is expressed in the highest abundance in A.belladonna lateral rootsIn studies in metabolic engineering,the 35 S promoter is a commonly used constitutive promoter that drives the sustained and efficient expression of target genes in various tissues of the plant;Previous studies have found that the pan-expression of target genes may over-consume intracellular substances and energy,causing unnecessary metabolic stress to the plant.In this study.We analyzed the expression of13 TAs metabolic pathway genes in A.belladonna based on the A.belladonna transcriptome.It was found that the expression abundance of UGT1 and LS,the two hyoscyamine biosynthesis rate-limiting enzyme genes,was low in the lateral roots(TAs biosynthesis site).While the expression abundance of PMT was the highest,indicating that the promoter of AbPMT(p PMT)has the strongest ability to drive the expression of downstream genes in the pericycle cells cells.The above analysis results suggest that p PMT can be used as an ideal engineering element(promoter)to drive AbUGT1 or DsLS instead of 35 S promoter for metabolic engineering studies of TAs.3.AbPMT promoter-driven overexpression of AbUGT1 and DsLS was more significant than 35 S in promoting TAs in A.belladonna hairy roots development.Previous studies have shown that optimizing the expression localization of target genes can efficiently promote the biosynthesis of target products in natural product metabolic engineering studies.In this study,the root pericycle cell-specific promoter p PMT was used to drive overexpression of AbUGT1 and DsLS,respectively,in A.belladonna hairy roots.1)The results of g DNA based PCR detection and q PCR detection indicate that p PMT can efficiently drive the overexpression of downstream target genes in transgenic hair roots.2)The alkaloid analysis results of two transgenic hair roots showed that overexpression of p PMT driven target genes AbUGT1 and DsLS significantly promoted the biosynthesis of TAs in hair roots.3)Further comprehensive longitudinal comparison of alkaloids in transgenic hair roots driven by p PMT and 35 S promoters overexpressing AbUGT1 or DsLS indicates that p PMT has a more significant promoting effect on the biosynthesis of TAs.The above findings suggest that the pericycle cell-specific promoters in roots have important applications in the metabolic engineering of TAs.In summary,this study firstly conducted a metabolic engineering study of TAs in A.belladonna hair roots using the 35 S promoter to drive AbUGT1 and DsLS,and the results showed that overexpression of both AbUGT1 and DsLS could significantly promote the biosynthesis of TAs;The results of A.belladonna transcriptome analysis were used to screen and target the highly active promoter p PMT in lateral roots,and p PMT was used to drive the expression of AbUGT1 and DsLS in A.belladonna germinating roots.Molecular assays showed that it was able to drive the high level expression of target genes in germinating roots,and metabolite analysis showed that it drove the key enzyme genes of TAs biosynthesis,AbUGT1 and DsLS,to promote TAs accumulation.This study provides important engineering components for the genetic modification of TAs pharmacogenetic plants at a later stage,and also shows that by optimizing the expression localization of target genes,we can efficiently promote the efficient synthesis of target products,which provides an important idea for related research.