The Construction And Analysis Of Hybrid Promoters With Different Characteristics In Saccharomyces Cerevisiae

Promoter plays an irreplaceable role in regulation of gene expression,and is one of the most important regulatory expression elements.At present,the common natural promoters in Saccharomyces cerevisiae could be divided into two groups:constitutive promoters and induced promoters.However,these promoters are limited in expression strength,even the strongest promoters' expression strength can't reach the demand in research.Moreover,induced promoters were be inducted with additional inducer;besides,the research on promoters which could response to changes in growth situation and induce the gene expression is few and can't satisfied the demand in metabolic engineering ang synthetic biology.Thus,the research and reconstruction of promoters have great significance in regulating gene expression better.To solve these problems,our works were carried out from two aspects:(1)The construction and analysis of constitutive hybrid promotersUsually,the natural constitutive promoters(even the strongest promoter,like PTEF1)can't meet the demand of gene expression,and the range of expression strength of commonly used promoters are narrow.Thus,we constructed a series of hybrid promoters with different strength by tandem of UAS(Upstream Activation Site)elements and the regulation of core promoters.Firstly,10 UASs from constitutive promoters were chosen and were assembled with one core element from CYC1 promoter.Then the transcriptional strength of these hybrid promoters were measured by using yellow fluorescence protein as reporter gene.The UAS elements with higher expression strength(UASTEF1,UASENO2,UASTPI1 and UASTDH3)were selected and then were combined with different UAS elements or assembled by tandem of same UAS element.Fluorescence measurement showed that the transcriptional capacity of most hybrid promoters with two different or same UAS elements were higher than the hybrid promoters with one UAS element;but,except the tandem of three UASENO2 elements,the tandem and combination of three different or same UAS elements didn't improve the transcriptional capacity furthermore.Although this result may be relative to the distance from the UAS to the TSS(transcriptional starting site),the concrete reason of this phenomenon need to be analyzed furthermore.Based on the above construction,four core elements including PTEF1,PTDH3,PGAL1 and PCYC1 were chosen to assemble with UASENO2,and the fluorescence measurement showed that PTEF1 equipped with the highest transcriptional capacity among these core elements.Then,the core element PCYC1 in relatively stronger hybrid promoters was replaced with PTEF1.The fluorescence value exhibited that the transcription capacity of these new hybrid promoters had increased 1 fold further.Among these hybrid promoters,UASENO2-PTEF1 had the highest transcription capacity.The transcriptional capacity of hybrid promoter UASENO2-PTEF1 which was constructed by combining UASENO2(3X)with P TEF1 was 1.4fold to the UASCIT(3X)-TEF1-PTEF1,and was one of the strongest hybrid promoters at present.(2)The constriction and analysis of automatically induced hybrid promoters in post-diauxic shiftWhen the toxic proteins are expressed,or the strong expression of some pathways would bring burden of cell growth,the constitutive expression won't be adopted.Usually,the cell growth need to be maintained firstly for some time,until the density of cell reaches a degree,the expression of relevant genes will be initiated.Such pathways are completed via induced promoters usually.However,the addition of inducer for induced promoters usually increases the production cost to the disadvantages of industrial application.The automatic induced promoters regulated by growth phase could initiate expression without any inducers to realize the separation of cell growth and accumulation of product.But at present,the transcriptional capacity of natural automatic induced promoters are much lower than the weak constitutive promoters,even in the condition of induction.Such characteristic limit the wild application of automatic induced promoters greatly.In our work,6 UAS elements from the promoters repressed glucose:PHXK1 and PMAL32,promoters responding to low pH:PCWP2 and promoters responding to heat shock:PSSA1,PSSA3 and PHSP30 were chose and combined with core element from PCYC1 promoters to constructed hybrid promoters.The transcriptional capacity of UASHSP30-PCYC1?UASMAL32-PCYC1 ? UASSA3-PCYC1 were repressed and very low,even lower than the core element of PCYC1 when the glucose existed(that is,the exponential phase,during which the cell grows vigorously);while the transcriptional capacity of these promoters increased notably and improved 2?6 fold when cell phase shifted to secondary growth period(that is,when glucose exhausted).The transcriptional capacity of UASSSA1-PCYC1? in post-diauxic shift was also higher than the transcriptional capacity in exponential phase,while the basic transcriptional capacity in exponential phase was higher than PCYC1.In order to improve the strength of these promoters,the core promoter from PCYC1 was replaced with core promoter from PTEF1,and the experiment results showed the transcriptional capacity of these new promoters based-PTEF1 was increased 2?3 fold.Among these new promoters based-PTEF1,UASSSA1-PCYC1 had the highest transcriptional capacity,and was comparable to the transcriptional capacity of UASTPI1-PCYC1.Moreover,the transcriptional capacity in post-diauxic shift of UASHSP30-PCYC1 and UASMAL32-PCYC1 were higher 3?6 fold to the exponential phase.These results demonstrated that the hybrid promoters based-PTEF1 realized to respond to the cell growth stage and high expression by automatic induction.To sum up,through the tandem of UAS elements and assemble of core elements,we constructed two types hybrid promoters:constitutive hybrid promoters and automatically induced hybrid promoters responding to growth stage.The construction of these hybrid promoters not only enrich the starting strength of present promoters,and the construction of automatically induced hybrid promoters provided a possibility of the expression regulation during the growth stage.