Strain Improvement Of Sporolactobacillus Inulinus By Evolutionary Engineering And Genome Shuffling

In this paper, evolutionary engineering was integrated with genome shuffling technology to generate the evolved Sporolactobacillus inulinus (SP-BME126) with acid tolerance and high lactic acid production in laboratory-scale fermentation.Eight mutants with high genetic stabilities were obtained by using ultraviolet radiation, DES mutations and pH gradient screenings were used as the start population for genome shuffling. The conditions for protoplast preparation and regeneration were studied by response surface methodology (RSM), and the obtained optimal condition was: seed age 12 h, lysozyme concentration 7.75 mg/l, treating time 1.59 h and 38℃, which resulted that the product of protoplast preparation and regeneration was above 59.6 %. Then, eitht protoplasts were obtained based on the optimized condition from RSM. The effects of PEG concentration, Ca2+ concentration, fusion time and temperature on protoplast fusion were studied. A D-lactic acid producing mutant F3-3 was obtained by three rounds of genome shuffling. Under the fermentation condition (pH5.0) in MRS culture broth, the strain F3-3 could produce 66.5 g/L D-lactic acid.At a 5 L fermentor, F3-3 acquired high acid tolerance by evolution engineering. The generation time and selection pressure of F3-3 were determined and it was found that the efficiency of pH-gradient selection for obtaining strains with higher acid tolerance was much better than that of immobilized pH selection. Under the fermentation condition, the evolved strain EV-1 could produce D-lactic acid 85.2 g/L at pH5.0.The 96-well plate was integrated with biosensor to determine the content of lactic acid in the colonies. The culture time of strains in 96-well plate was 3 days. Sterile water was employed as solvent for the abstract of lactic acid from the colonies. The solution of 0.015 % bromcresol purple was applied to strain screening in 96-well plate. So a platform was established for high-throughput screening of strains producing extracellular metabolite.A scheme of metabolomics analysis by GC/MS was developed for detecting the metabolites in the broth. Fourteen intracellular metabolites were confirmed according to the GC/MS data. A quantitative metabolic flux model was developed and tested for Sporolactobacillus inulinus. Accordingly, the metabolic flux distribution was determined between the evolved strain EV-1 and the parental strain SP-BME126 at different fermentation phase.The optimal fermentation condition for D-lactic acid production was investigated by single factor experiment, Plackett-Burman design, central composite design and response surface methodology (RSM). The obtained optimal condition was: glucose 95 g/L, peptone 22 g/L, seed age 24 h, inoculum size 5 %, calcium carbonate 3 g/100mL, medium volume 112 mL/500mL and temperature 35℃. Under this condition, the evolved strain EV-1 showed 96.5 % yield efficiency, led to 7.63 % increase in D-lactic acid production from 85.2 g/L to 91.7 g/L.