| Lactic acid is the most widely used natural organic acid in nature,and it is also a precursor of the degradable polymer material polylactic acid.In recent years,with the increasing awareness of protecting the ecological environment,the development of the polylactic acid industry has been promoted,thereby expanding the market’s demand for high-quality lactic acid.As a potential industrial lactic acid producer,Lactobacillus paracasei has an efficient L-lactic acid production capacity in previous studies,but the resulting product still contains a small amount of D-lactic acid,which makes L-lactic acid optical purity not reach the level of polylactic acid(>99.0%).In this study,by establishing the L.paracasei CRISPR/Cas9 gene editing platform,gene the ldhD gene in the starting strain was successfully replaced with the high-transcriptional ldhL1,so as to obtain a mutant that efficiently produced high-quality L-lactic acid.And then,the physiological and metabolic characteristics of the mutant under high sugar concentration was preliminarily analyzed.Firstly,the electric conversion conditions of L.pseudocasei were optimized,and the best transformation method suitable for L.paracasei was determined,so that the conversion efficiency could reach more than 104(CFU/μg DNA).Subsequently,RT-PCR was used to analyze the relative transcriptional levels of the three lactate dehydrogenase genes(ldhL1,ldhL2,and ldhD)present in L.paracasei,and it was determined that the ldhL1 gene with high transcriptional level was used as the phenotype gene to construct the CRISPR/Cas9 gene editing system to achieve efficient editing of L.paracasei.Secondly,to investigate the influence of temperature and pH on the optical purity of L-lactic acid produced by L.paracasei in environmental factors,it was found that the optimal optical purity(about 95.0%)was at a temperature of 37℃ and a pH of 6,but this environmental regulation It cannot effectively improve the optical purity of L-lactic acid.Using the CRISPR/Cas9 gene editing method to replace the ldhD gene with the ldhL 1 gene,a high-quality L-lactic acid production strain was obtained.Through shake flask fermentation analysis,the L-lactic acid optical purity of the mutant strain was enhanced from 95.2%to 99.0%,the production rate and was the conversion rate were improved from 1.65 g/L/h and 83.2%to 1.86 g/L/h and 93.0%,respectively.Finally,the ldhL1 overexpression mutant strain and the starting strain were subjected to a 5 L fermentor experiment with an initial glucose concentration of 130 g/L,and the fermentation characteristics of the two strains were analyzed and compared.By fitting the growth kinetics and production kinetics curves,the maximum specific growth rate(μm)was increased from 0.440 h-1 to 0.497 h-1,and the growth correlation coefficient a was increased from 7.46 g/g to 8.20 g/g,indicating that the production of lactic acid is a closely growth-dependent process,moreover,it presented a closer relationship in the mutant.In addition,under high-sugar concentration(200 g/L)condition,the results showed that the mutant strain had a faster cell growth rate to continuously and efficiently produce L-lactic acid,and the production rate and conversion rate were increased by 37.0%and 9.3%respectively,compared with the starting strain.The optical purity of L-lactic acid was also improved from 95.2%to 99.0%,and the content of extracellular by-product acetic acid was significantly reduced,so that the mutant strain not only had high optical purity,but also its chemical purity reached more than 99.0%.By comparing the key performance index parameters of excellent lactic acid producer reported in the literature,the mutant strain constructed in this study not only has good comparability in tolerance to initial sugar concentration,lactic acid yield,and lactic acid conversion rate,but also the lactic acid yield is much higher,making it an industrial lactic acid production strain with great application potential. |
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