| Organisms often need to regulate their own gene expression to adapt to the complex environment.There are many approaches for regulation of gene expression,including regulation at the transcription level or translation level,posttranslational modification and so on.In addition to the biological significance of regulation of gene expression,artificial manipulation of gene expression also plays fundamental roles in determining and optimizing functions of genes and metabolic pathways.Development of methods for manipulating gene expression at the transcriptional level has immense value,for design and construction of biological systems for efficient biosynthesis of useful chemicals.The most widely applied approach to improve the transcription level of the target gene by using strong promoters.For this purpose,researchers have identified a number of inducible and constitutive strong promoters in different species to modify the target metabolic pathway.Researchers can use strong promoters to overexpress structural genes,which can achieve increased production of natural products.There is at least one regulatory gene in these biosynthetic gene clusters,and the positive regulatory gene is often favored by researchers.The traditional method for increasing the yield of natural products is using strong promoters to overexpress positive transcriptional regulatory genes which can activate the expression of some or all structural genes in corresponding biosynthetic gene clusters.Some strong promoters could not be shared because of interspecific differences among different organisms.Limited by the strength of the promoters,the traditional method usually has limited yield gains for natural products.Therefore,it is very important to develop a new way to activate gene transcription to improve the synthesis of target products.The purpose of this research is to find a new method of overexpressing target gene to improve the problem of the limitation of species diversity and promoter strength.In this study,we firstly focused on Pseudomonas stutzeri DSM 4166 which has the ability of biological nitrogen fixation(nif).Twenty-six candidate promoters were identified from DSM 4166 by RNA-seq analysis,and the strength of these 26 promoters was characterized using firefly luciferase reporter genes.Using the strong promoter P12445 to overexpress nifA gene,a pathway-specific positive regulatory gene of biological nitrogen fixation,the transcription level of nif genes in the engineered strain DSM4166-P12445-nifA was significantly higher than that in the wild-type strain.The nitrogenase activity and the concentration of extracellular ammonium of DSM4166-P12445-nifA strain increased by 4.1 and 25.6 times respectively compared with the wild-type strain.Then we developed an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences inspired by the experimental results of activation of nitrogen fixation genes transcription by NifA protein.In this strategy,the nascent positive transcriptional regulatory protein can bind to the upstream activating sequence(UAS)of its own promoter and activate transcription of itself,thus forming a positive feedback regulatory transcription cycle,which makes positive regulatory protein be continuously synthesized,to achieve the effect of overexpression of positive transcriptional regulatory gene.According to this,strategy,we used firefly luciferase reporter gene to verify its feasibility.The results showed that the gene transcription activation strategy was feasible and more advantages in overexpressing genes.Subsequently,the nifA gene was overexpressed using efficient gene transcription activation strategy.The results showed that the nitrogenase activity of DSM4166-Pnif-nifA strain which applied transcription activation strategy was significantly higher than that of the DSM4166-P12445-nifA strain.Among these,the DSM4166-PnifH-nifA strain had the highest nitrogenase activity and extracellular ammonium concentration,which were 6.3-fold and 4.6-fold higher than DSM4166-P12445-nifA strain,respectively.The NifA efficient gene transcription activation system developed in this study can not only improve the nitrogenase activity,but also be used for heterologous expression of natural products.The indole-3-acetic acid(IAA)and prodigiosin were selected to explore the application potential of NifA efficient gene transcription activation system.An efficient IAA biosynthetic strain was constructed by overexpressing the IAA biosynthetic pathway using N ifA efficient gene transcription activation system.Then the NifA efficient gene transcription activation system was optimized,and the highest yield of IAA reached 9.0 g/L.The NifA efficient gene transcription activation system was applied to heterologous expression of the biosynthetic gene cluster of prodigiosin.The yield of prodigiosin was significantly higher than that obtained by using strong constitutive promoter.After optimization,the highest yield of prodigiosin was 1.3 g/L,which was the highest yield of heterologous synthesis of prodigiosin.In order to broaden the application of gene transcription activation strategy,the efficient synthesis of salinomycin using this strategy was studied in Streptomyces albus DSM 41398.The salinomycin biosynthetic gene cluster contains a positive transcription regulatory gene slnR.The SlnR efficient gene transcription activation system was constructed in S.albus DSM 41398,and all DSM41398-Psin-slnR transcription-activated strains had significantly higher salinomycin production than the wild-type strain.Compared with the control strain DSM41398kasOp-slnR which overexpressed slnR using strong constitutive promoter kasOp*in Streptomyces,the salinomycin productions of DSM41398-PslnF-slnR and DSM41398-PslnT1slnR strains were significantly higher than that of DSM41398-kasOp-slnR strain.These results indicated that efficient gene transcription activation strategy can be widely used in a variety of bacteria.In the study of prodigiosin biosynthesis using NifA efficient gene transcription activation system,it was found that promoter UAS-PnifE,which had efficient transcription activation effect,did not produce no prodigiosin when it was used to express prodigiosin biosynthetic gene clusters.This study proposed a theory that the sequence of the first gene downstream of the promoter(promoter-proximal sequence)may affect the transcription efficiency to explain this phenomenon.Placing the firefly luciferase reporter gene in the position of a second gene downstream of the promoter,when the change of the promoter-proximal sequence results in the change of the secondary structure of the DNA,the expression level of the reporter gene was also changing.We concluded that the stronger the DNA secondary structure of the promoterproximal sequence was,the lower the transcription level was.This study initially revealed that the transcription level not only depends on the strength of the promoter itself,but also on the DNA sequence of the promoter-proximal sequence,providing a new idea for the better use of synthetic biology element promoters in the future.In conclusion,this study proposed an efficient gene transcription activation strategy based on pathway-specific positive transcriptional regulators for the first time.The efficient gene transcription activation systems in the gram-negative bacteria P.stutzeri DSM 4166 and grampositive bacteria S.albus DSM41398 were constructed to achieve a significant increase in nitrogenase activity and salinomycin production,respectively.At the same time,the NifA efficient gene transcription activation system was developed into a synthetic biological element with strong promoter function.and IAA and prodigiosin were biosynthesized in DSM 4166 using this system.The effect on the transcription efficiency of the promoter-proximal DNA sequence was also investigated preliminarily,which provided new ideas and methods for researchers to use promoters better. |
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