Research On Constructing A Promoter Library Of Lactobacillus Paracasei Based On RNA Probes And High Quality Lactic Acid Producing Strain

Lactic acid is an important organic acid,which is widely used in food,biological,chemical and other industries.In recent years,with the continuous enhancement of environmental protection awareness,the rapid development of the polylactic acid industry has also further increased the demand for high-quality lactic acid monomers.How to efficiently produce high-quality lactic acid is a problem that needs to be solved urgently.Adaptive evolution and metabolic engineering can directly engineer the strain performance.In terms of metabolic engineering,both the construction of expression systems and the optimal expression of products are inseparable from the promoter regulation.The lack of promoters with different regulatory activities in lactic acid bacteria is a major obstacle to precise regulation of cell metabolism.In this study,high-throughput screening of promoters was conducted by constructing and applying RNA probes,and a library of promoters with different regulatory activities suitable for Lactobacillus paracasei was established.In addition,the strain was modified through adaptive evolution and CRISPR-Cas9 gene editing strategies to meet the requirements of industrial lactic acid producer.Firstly,the RNA probe Pepper was used to construct the RNA level promoter probe in lactic acid bacteria.By comparing the strength and analyzing the correlation of the promoters characterized by eGFP and Pepper,the feasibility and reliability of Pepper as a reporter gene applied in lactic acid bacteria was proved.On this basis,an RNA-level promoter probe was constructed.Otherwise,the results showed that the promoter strength could be initially tested in Escherichia coli,and then transformed into Lb.paracasei to verify the regulatory activity.In addition,by optimizing the inoculation amount and culture time of lactic acid bacteria,a suitable fluorescence measurement method for Pepper was established.It was found that when the initial inoculation amount was 5%.the best fluorescence detection value could be obtained after 4 hours of cell cultivation.Secondly,a promoter library of Lb.paracasei.based on Pepper probe was constructed.The P32 promoter mutant fragment was obtained by error-prone PCR.and the constructed RNA probe Pepper was used to characterize the promoter strength.At first,high-throughput screening was performed in E.coli and a total of 950 mutants were screened.10 transformants whose fluorescence intensity was within the range of 0.5-8 times that of the P32 and with gradient changes in regulatory intensity were selected and subsequently transformed into Lb.paracasei.The correlation coefficient between their promoter strengthes in lactic acid bacteria and E.coli reached 0.94,which finally constituted a library of functional promoters of Lb.paracasei.Finally,a high-temperature resistant and high-quality L-lactic acid producer was obtained by using adaptive evolution strategy.After 2160 hours of continuous subculture,a strain designated as Lb.paracasei(HT)that can withstand high temperatures of 45? was obtained.After 24 hours of cultivation,the HT exhibited similar fermentation performance at 45? as the starting strain at 37?.Under the condition of open fermentation at a high initial sugar concentration(250 g/L),the HT could produce L-lactic acid concentration 221 g/L with a productivity of 7.51 g/L/h as well as a yield 0.96 g/g,moreover,the optical purity and chemical purity were both higher than 99.0%.The fermentation performance is better than other lactic acid producer reported in the literature,and it is expected to become a potential industrial high-quality L-lactic acid producer.In addition,in order to further improve the optical purity of L-lactic acid and meet the demand of polylactic acid for high-quality lactic acid monomers,a plasmid targeting D-lactic acid deletion was constructed based on CRISPR-Cas9 gene editing technology.However,it may be limited by the electrotransformation condition or the design of the CRISPR-Cas9 expression element,the corresponding knockout strains have not been obtained yet.Hopefully,this study laid the foundation for the further engineering of lactic acid producer with high optical purity.