Transcriptome And Visualized Red Fluorescence Screening Based Promoter Library Construction And Application In Rhodobacteria Sphaeroides

The emergence of synthetic biology field led to the use of a variety of microorganisms,such as Escherichia coli,Saccharomyces cerevisiae,Strepomyces,etc.,as classic chassis cells in various research fields.Due to their special metabolic and physiological characteristics,some special microorganisms such as halophilic bacteria,thermophilic bacteria,acidophilic bacteria,and some photosynthetic bacteria,also have great potential for microbial chassis development for industrial application.Rhodobacter sphaeroides is a GRAS(Generally Regarded As Safe)purple non-sulfur bacteria,that has been used for the synthesis of various products such as Coenzyme Q10,5-aminolevulinic acid and hydrogen gas.As a new chassis strain,Rhodobacter sphaeroides has attracted a growing attention,and also been developed for the synthesis of many other products.However,the lack of available synthetic biology elements,including the most critical transcriptional control elements:promoters,in Rhodobacter sphaeroides has greatly restricted the metabolic engineering of the bacteria and synthetic biology research.This study aims to develop a set of strong promoter library in Rhodobacter sphaeroides HY01(HY01)to promote the development and application of the photosynthetic chassis cells.In order to obtain the natural strong promoter of Rhodobacter sphaeroides,we first analyzed the transcriptomes of different strains(HY01 and the wild-type Rhodobacter sphaeroides 2.4.1)at different growth phases(logarithmic phase and stationary phase)through comparative transcriptomics.Ten strong promoter candidates were selected.Then,the activity of the candidate promoters were evaluated by ?-glucuronidase(GusA).Two strong promotors were obtained,Prsp-7571 and Prsp-6124.Compared to the activity of the commonly used promoter Ptac,the activity of Prsp-7571 and Prsp-6124 were increased by 6.4 and 8 times respectively(p<0.05),however,the latter affected the growth of the cells.Next,Prsp-7571 was selected as a natural constitutive strong promoter.Then,a promoter library was further obtained by genetic modification.Through the analysis of the promoter structure,the truncated three promoters Prep-7571-1,Prsp-7571-2 and Prsp-7571-3 were obtained with sizes of 334 bp,167 bp and 84 bp respectively.The activity of truncated promoter was lower than that of prsp-7571.In order to further screen and obtain more active promoter,error-prone PCR was conducted on the fragments of the promoters Prsp-7571-1 and Prsp-7571-2,and the mutant library of the promoter were introduced into HY01 through transformation.Then,the portable red fluorescence(RFP)visualization equipment was used for the evaluation of promoters activities using direct screening of fluorescence intensity of RFP protein expressed under the control of a total of 29 promoters obtained in this study,and activity ranging from 54%to 3000%(p<0.05)compared with Ptac was obtained.In order to evaluate the efficiency of our screening in applications,the strong promoter Prsp-7571 was selected to express the key genes of the MEP pathway and the ubiquinone synthesis pathway dxs and ubi,respectively.As a result,the unit cell yield of coenzyme Q10 was increased by 27.82%(p<0.05),and the transcription level of dxs and ubiE was increased by 28 and 20 times compared with the original strain HY01.Then,we used Prsp-7571 to construct a CRISPR-dCas12a-based transcriptional inhibition system,which showed inhibition of target genes(e.g.hemB or fnrL)reached more than 95%(p<0.05).Finally,the Prsp-7571 promoter was used to express the synthetic pathway of ?-carotene and lycopene in HY01,in order to realize the efficient synthesis in industrial chassis.In summary,based on transcriptome analysis,activity of GusA and fluorescence screening system,a set of highly efficient strong promoter library was established successfully in Rhodobacter sphaeroides.As a result,we achieved improvement of the coenzyme Q10 synthesis efficiency,establishment of a transcriptional inhibition system using CRISPR-dCas12a and efficient production of the carotenoids ?-carotene and lycopene.Our study,shows a great potential of the use of these promoters in efficient regulation of target genes and pathways to facilitate the development of the photosynthetic microbial chassis Rhodobacter sphaeroides for synthetic biology research and applications.