Screening Of Promoters In Corynebacterium Crenatum SYPA5-5Based On2-DE And Their Functional Research

The stain Corynebacterium crenatum SYPA5-5used by this thesis is an L-argininehyper-producing mutant strain obtained through screening from soil by our studying team.The promoters used in C. crenatum were relatively inefficient. Screening efficient promoterswhich were used for construction of an efficient C. crenatum overexpression system becamean important method. The main results are listed below:(1) In this article, C. crenatum SYPA5-5was fermented in normal condition (stirring speed400r/min, ventilation volume3L/min) and different dissolved oxygen level condition(stirring speed300r/min, ventilation volume2L/min; stirring speed600r/min, ventilationvolume4L/min), respectively. Two-dimensional electrophoresis (2-DE) andmatrix-assisted laser-desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) were used to analyze high-expression proteins and differentialexpression of proteins in different oxygen supply conditions in C. crenatum SYPA5-5,respectively. Based on the genomic information of Corynebacterium, the copy numbers ofthe coding genes of these proteins were analyzed and their expression level of one copywere compared. Five constitutive high-expression proteins, includingN-acetyl-gamma-glutamyl-phosphate reductase, argininosuccinate synthase, ketol-acidreductoisomerase, phosphoglycerate dehydrogenase and ornithine carbamoyltransferase,were identified. Simultaneously, three differential expression of proteins in differentoxygen supply conditions, including Transketolase, Fumarate hydratase, andL-2,3-butanediol dehydrogenase, were also identified.(2) The target promoters were amplified by PCR. Five constitutive promoters, which werenamed P-argC, P-argG, P-argF, P-ilvC and P-serA, and three inducible promters bydissolved oxygen, which named P-tkt, P-fum and P-bdh were obtained. The tac promotersof recombinant pDXW-8-cat and pDXW-8-gfp were replaced by the new promoters, andthe recombinant plasmids were transformed into E.coli JM109and C.crenatum SYPA5-5respectively． The function of promoters were compared by the expression ofchloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP).(3) The recombinant bacteria carrying constitutive promoters were fermented in normalconditions. The results showed that, the activity of the promoters were: P-argG> P-argF> P-ilvC> tac> P-argC> P-serA. The constitutive expression of GFP and CAT underP-argG, P-argF and P-ilvC were more efficient than that under tac promoter. The CATactivity of recombinant C. crenatum were achieved4.84U/mg,4.43U/mg and2.91U/mgin shake flasks, respectively. These were more activity than that under tac promoter whichwas2.22U/mg. A similar result was got by using5L fermentor.(4) On the other hand, the recombinant bacteria carrying inducial promoters were cultureed indifferent dissolved oxygen level. Results showed that, the CAT activity of P-fum was very low. In the high dissolved oxygen level the CAT activity of E. coli JM109/pDXW-Ptkt-catwas2.03U/mg, which was1.80folds than that in the low dissolved oxygen. The CATactivity of C. crenatum SYPA5-5/pDXW-Ptkt-cat in the high dissolved oxygen level was1.08U/mg, which was1.74folds than that in the low dissolved oxygen. The CAT activityof E. coli JM109/pDXW-Pbdh-cat and C. crenatum SYPA5-5/pDXW-Pbdh-cat in the lowdissolved oxygen level were2.0and1.99folds than that in the high dissolved oxygenlevel. A similar result was got by using5L fermentor.