Analysis Of Physiological Characteirstics Of Vitamin C Producing Strains And Optimization

This thesis studied the strains for the industrial production of2-keto-L-gulonic acid(2-KLG, the precursor of vitamin C), Ketogulonicigenium vulgare WSH001and Bacillusmegaterium WSH002. The genome-scale metabolic models (GSMMs) of K. vulgare and B.megaterium, iWZ663and iMZ1055, were studied. And the biosynthetic capabilities of K.vulgare and B. megaterium were predicted. Wet experiments were conducted to validate thepredictions, establish the minimal chemically defined medium (MCDM), optimizefermentation by supplying glyoxylic acid, and find the new respect of the relationshipbetween K. vulgare and B. megaterium. The main results were described as follows:1. K. vulgare WSH001is predicted to be deficient in the biosynthetic pathways ofL-asparagine, L-glycine, L-cysteine, L-methionine, L-tryptophan, adenine, guanine, thymine,thiamine, pantothenate, pyridoxine and folic acid. In wet experiments, individually omittingL-glycine, L-cysteine, L-methionine, L-tryptophan, adenine, thymine, thiamine andpantothenate from complete chemically defined medium (CDM), caused biomass formationof K. vulgare to decrease to1%,21%,16%,1%,26%,57%,73%and24%, respectively. Afterresupplying single-omission-media with the corresponding nutrients，the cell growth of K.vulgarere restored to41.9%、107.1%、82.3%、99.4%、81.3%、94.5%、102.9%and112.9%of the CDM, respectively. Based on these results, a MCDM was developed that supportedmonoculture of K. vulgare (cell growth of0.28OD600and2-KLG production of3.59g·L-1),and co-culture (cell growth of2.15OD600and2-KLG production of51.5g·L-1).2. Based on the nutrient requirements of K. vulgare WSH001, new stratagies forfermentation optimization were proposed. When glyoxylic acid was supplied into theglycine-omission-medium, the growth and2-KLG production of K. vulgare WSH001wererestored to126.1%and111.2%of CDM, respectively. It shows that glyoxylic acid circumventthe need for L-glycine. Therefore,0.5g·L-1glyoxylic acid was supplied into corn steep liquorpowder medium, resulting in increase in K. vulgare cell growth,2-KLG production, and2-KLG productivity of46.8%,25.0%, and24.7%respectively; co-culture cell growth,2-KLGproduction, and2-KLG productivity of33.3%,5.1%, and5.7%respectively.3. Model iMZ1055shows that B. megaterium WSH002metabolizes glucose other thansorbose. It was found that carbon source outside cells postpones sporulation and that with theconcentration of sorbose increase, B. megaterium sporulation rate decrease. With the help ofgene BLAST, PI staining, and Flow Cytometry, B. megaterium was found to cannibalize thespoOA-inactive cells when sorbose existed, and the cannibalization degree increased when theconcentration of sorbose decrease. It was found that the sporulation rate and cannibalizationdegree of B. megaterium in co-culture are both higher than B. megaterium pure culture. That’sbecause the concentration of sorbose become suitable for sporulation and cannibalization afterK. vulgare consuming sorbose, enhancing the lysis of B. megaterium. Thus, a new aspect ofthe relationship between K. vulgare and B. megaterium was found: the K. vulgare inco-culture consumes sorbose, enhancing the lysis of B. megaterium; B. megaterium lyses,providing nutrients to K. vulgare, and enhancing its growth; the growth of K. vulgare enhances the lysis of B. megaterium more strongly, resulting in a benifitial cycle for K.vulgare growth and2-KLG production.