Breeding Of L-Histidine Mutant And Its Optimizing Conditions In Fermentation

L-histidine is an alkalescency amino acid with an imidazole group. It's a semi-essentialamino acid in human and animal, but it is an indispensable to infants. It has extensiveapplications in medicament. In our country, L-histidine is mainly produced by hydrolyze pigblood powder, and applications in forage. But in medicament, L-histidine has to depend onimporting.In developed countries, L-histidine is produced with fermentation. In 1970s', L-histidinefermentation was researched in Japan. Now there are several histidine mutant used to fermenthistidine, such as Corynebacterium glutamicum, Brevibacterium flavum, Serratia marcescens.They can accumulate histidine 15.4g/L, 12.5g/L, 17.0g/L and the invert rate is 15.4%, 12.5%,10.0% in respective. After 1980s', gene technology was used on breeding mutant in Japan, soamino acid fermentation has made rapid progress. But in our country, L-histidine is not able tobe produced by fermentation. So it is very important to study this subject.Mutants resistant to 3-amino-1,2,4-triazole, histidinehydroxamate and analogs of purinewere derived from Corynebacterium glutamicum ATCC13761 by mutagenic treatment withdiethyl sulfate (DES), nitrous acid and so on. Then a selected mutant DH-8 was isolated fromhistidase-less and transketolase-less mutants. The level of histidine production by DH-8 reaches2.1g/L.The effect of the medium compositions and culture conditions on L-histidine productionwas investigated. According to experimental results, the better medium compositions weredetermined as(g/L): glucose 80, (NH4)2SO4 10, corn steep liquor 5, KH2PO4 1.5, MgSO4·7H2O1, CaCO₃ 10 g/L, pH 7.0, and the better fermentation was performed at 30℃ on a rotary shaker(100 r/min )with 15mL culture broth inoculated by 1mL. DH-8(TRZR, HisdxR, hisase-, 2,6-CPR,Trkase-) produced 2.5g/L of L-histidine under above conditions.NaF and citric acid were added into fermentation medium to change metabolize of DH-8.Because NaF can weaken EMP pathway, and then glucose is digested through HMP pathway toincrease the quantities of the matter which can synthesize L-histidine. But cells seldom surviveunless citric acid added into the fermentation medium, since citric acid is one of the TCAintermediate, which can offer nutriments and ATP to support cells growing. Finally the level ofhistidine production by DH-8 reaches 2.55g/L.