Construction Of Multigene Transcription Programs And RFN-based Biosensor In Bacillus Subtilis

Over-expression of key enzymes to relax the rate-limiting points in the biosynthetic processes and fine-tuning the expression of enzymes in shunt pathways to minimize their fluxes and maintain normal cell growth are the most commonly employed strategy to increase the productivity of target metabolites.In addition,construction of metabolite biosensor system for high-throughput screening of high-producing strains is also a valuable tool in metabolic engineering.In this study,high-efficiency synthetic expression elements were constructed and systematically evaluated to fine-tuning gene expression in Bacillus subtilis.Also,a multidirectional transcriptional program was developed for simultaneously activating and repressing of different genes.At last,a riboswitch-based target metabolite biosensor was constructed and optimized for further application.The main results of this study were shown as follows:1.Establishment of efficient genetic transformation system of Bacillus subtilis.The effects of media components,ComK expression level,host restriction-modification system,and antibiotics on competent cell development and DNA transformation were evaluated.The transformation efficiency of competent cells prepared by the optimized conditions was improved by 100-fold than that of the original.2.Construction of gene specific promoter library by a newly developed strategy named oligonucleotide annealing-based promoter shuffling.Firstly,four parent promoters with different strengths were selected to construct a small library using GFP as reporter.Library characterization showed that a total of 11 promoters with higher transcription activity than P43 were isolated,the strongest promoter exhibited 9-fold transcription activity than that of P43.Subsequently,promtoers with defferent strengthes wer used to express xylanase XynHB and pullulanase Pul703.The results showed that their expression levels were not correlated to the promoter strengths,suggesting that promoter strength is gene-dependent.To improve the expression of amylase BLA in B.subtilis,a BLA-specific library was constructed using the strategy mentioned above.As homologous recombination efficiency was significantly improved when DNA DSB happens,a CRISPR-assisted transformation system was developed by introducing DSB at the integration site by Cas9 during library transformation.Library characterization showed that 21 promoters were significantly stronger than P43,among which the transcription capacity of the strongest promoter P525 was 6-fold of the weakest promoter P232.3.Construction of CRISPR/Cas9 gene edit system and NHEJ pathway.The CRISPR/Cas9 system was constructed in B.subtilis,the gene edit efficiency in B.subtilis reaches 100%when using endogenous gene AmyE and exogenously integrated gene EmR as targets.Due to the lack of non-homologous end joining(NHEJ)pathway in B.subtilis vegetative cells,we constructed the B.subtilis spore-specific NHEJ pathway in vegetative cells,but it doesn't work.Then,the NHEJ pathways from Mycobacterium smegmatis and Mycobacterium tuberculosis were reconstructed to investigate whether they can repair DSB in B.subtilis.After introducing chromosome DSB via CRISPR/Cas9,about 20 transformants were obtained without providing homologous repair templates.However,the sequencing results showed that all the transformants have no mutation at the target gene.4.Constrcution of CRISPR-based multi-directional transcription program.The dCas9 was fused with ? or a subunit of RNA polymerase in B.subtilis,respectively.Using P43-GFP expression unit as a reporter system,when dCas9-?/? were guided to bind locations 305-459 bp upstream promtoer,GFP fluorescence were all significantly increased.A 3.3-fold increase of GFP fluorescence was observed when sgRNA targeting to 327 bp upstream of ATG.As the distance increases or decreases,the transcription activation decreases.When dCas9-? was guided to promoter(-35 motif),the GFP expression was not influenced.While the GFP expression were significantly inhibited when dCas9-?/? binds to the RBS or GFP coding region,which was able to completely repress GFP expression.Therefore,it is possible to simultaneously activate and repress the expression of different genes by designing guide RNAs targeting different regions.To understand whether dCas9-? can improve the expression of endogenous genes,a sgRNA target 224 bp upstream of AmyE initial codon was constructed,which improved AmyE expression by 3-fold.These resukts demonstrated that dCas9-?/? are capable of specificly improving gene expression in B.subtiis.In addition,guiding dCas9-? by two sgRNAs to the same gene or fusion dCas9 with a and ? simultaneously(dCas9-?-? or dCas9-?-?)could not further increase the expression of the target gene.5.Fine-tuning gene expression by regulating protein quality control system via dCas9-?.The effects of proteases degradation and protein folding on BLA production were evaluated by inhibiting extracellular proteases expression and activating chaperones expression via dCas9?.We found that inhibiting the expression of proteases nprB,bpr,and vpr can obviously improve BLA production,with a relative BLA activity of 200,145,and 160%,respectively,indicating that nprB,bpr and vpr have degradation activity toward BLA.When dCas9-? was targeted to the extracellular chaperone PrsA,the relative activity of BLA was increased by 73%,while sgRNAs target the intracellular chaperone groES and protease wprA,epr and mpr had no effect on the expression of BLA.6.Construction of RFN-based riboflavin biosensor.A FMN riboswitch-based biosensor was developed for high-throughput screening of high-producing riboflavin strains.Namely,the expression of lacI and GFP was controlled by FRN element and lacO,respectively,thus,the expression of GFP was correlated to the concentration of FMN.We analyzed the response potency of this biosensor by adding FMNs to culture medium and determining the GFP fluorescence.The results showed that GFP fluorescence was only increased by 35%after adding high concentration of FMN.Subsequently,the expression lacI was regulated by replacing its promoter with Pveg or dual promoter Plac-PribO,we found that the response efficiency of these constructs was deteriorated.Because the biosensor has a better response to riboflavin than FMN,we speculated that the low response of biosensor may attribute to the inefficient transport of FMN/riboflavin into the cytoplasm To analyze the effect of riboflavin transport on biosensor performance,ypaA was overexpressed with promoter P43 in an episomal vector.It was found that overexpression of ypaA can significantly increase the response efficiency of the biosensor.When exogenously added riboflavin reached 150 ?M,the relative GFP fluorescence was increased by 2-fold compared to the control(without riboflavin),and the GFP fluorescence was further increased to 3 times with 2 mM riboflavin.When the concentration of riboflavin reached 4 mM and 8 mM,cell growth was hampered and the GFP fluorescence intensity was greatly reduced.7.Coupling cell growth rate with riboflavin concentration.Because high-yield strain typically places heavy metabolic burdens on cell growth and reduce cellular fitness.To avoid displacement of high-yield strain by nonproductive mutants in subcultures,we attempted to couple riboflavin production with cell growth rate.Specifically,we first knocked out the ylaN gene of Bacillus subtilis so that the cells could not grow normally in the medium without FeCl3.Then,replace the GFP in the biosensor with ylaN,making ylaN expression correlated with riboflavin production.When the cellular riboflavin content is high,the expression of ylaN will increased,the strain can grow normally in the LB medium.