Breeding Of L-Isoleucine-Over-Producer From Brevibacterium Flavum On Metabolic Engineering

L-Isoleucine is one of eight essential amino acids for vertebrates. It plays an important role in metabolism. Therefore, it has many potential applications, such as food additives, feed supplements, pharmaceuticals and cosmetics. L-isoleucine production can not meet the rapidly increasing demand of market. In China, the yield of L-isoleucine, conversion rate of sugar and extraction ratio of L-isoleucine are low. Therefore, it is important to develop an L-isoleucine-producing strain, which could secrete high yield of L-isoleucine combined with low yield of by-products. In this study, strain was endowed with genetic marker by traditional method. Several problems were discovered and resolved in L-isoleucine fermentation. Meanwhile, optimizations of fermentation medium and conditions were carried out. On the other hand, bioconversion of threonine to L-isoleucine was also reseacrhed in recombinant C. glutamicum strain. The main findings and contents are summarized as follows:1. The origin strain B. flavum Q-5 was endowed with genetic marker of 3-ATrSucg by ARTP, and tolerances of α-ABr and Ethr were increased from 20 to 50 mg·m L-1 and 3 to 12 mg·m L-1, respectively. Finally, strain ART-I was obtained and L-isoleucine production was 24.1±0.70 g·L-1, about 33.9% higher than strain Q-5.2. Modified threonine dehydrase(TD) to obtain TD variants. The gene of TDART in strain ART-I was cloned and expressed. TDART protein was purified and analyzed. Although TDART reduced feedback inhibition, it was still inhibited by end product. TDV140 M, TDF383 A and TDV140M-F383 A were obtained by site-specific mutagenesis. These TD variants were expressed, purified and analyzed. Specific activity of TDV140 M was 1.5-fold higher than wild-type TD. TDF383 A variant released itseslf from feedback inhibition. TDV140M-F383 A variant had advantages of TDV140 M variant and TDF383 A variant. L-isleucine production was increased to 25.4±0.53 g·L-1 by expressing TD variant in strain ART-I.3. Modified acetohydroxy acid synthase(AHAS) to obtain AHAS variants. The gene of AHASART in strain ART-I was cloned and expressed. AHASART protein was purified and analyzed. Although AHASART reduced feedback inhibition by brainched-chain amino acids(BCAAs), it was still inhibited by end product. AHASA42 V, AHASA89 V and AHASA42V-A89 V were obtained by site-specific mutagenesis. These AHAS variants were expressed, purified and analyzed. AHASA42 V and AHASA89 V reduced feedback inhibition although they were still inhibited by BCAAs. AHASA42V-A89 V almost released itseslf from feedback inhibition. L-isleucine production was increased to 24.9±0.21 g·L-1 by expressing AHASA42V-A89 V variant in strain ART-I. In strain ART-I, co-expressing of TDV140M-F383 A and AHASA42V-A89 V increased L-isoleucine production to 26.0±0.16 g×L-1, 7.9% higher than strain ART-I.4. For recombinant B. flavum strain ART-I-3, optimizations of fermentation medium and conditions were carried out to enhance L-isoleucine production. The best fermentation medium contained 126 g·L-1 glucose, 37 g·L-1(NH4)2SO4, 23 g·L-1 corn steep liquor, 1 g·L-1 KH2PO4, 0.5 g·L-1 Mg SO4·7H2O, 50 μg×L-1 D-biotin and and 30 g·L-1 Ca CO3. The best condition was: 20 m L fermentation medium in a 500 m L triangular flask, 10% inoculum size, p H 7.5, 30℃, 0.5 mmol·L-1 inducer was at 0 h. Finally, recombinant strain ART-I-3 could accumulate 28.2±0.25 g×L-1 L-isoleucine, 8.4% higher than comtrol strain. By fed-batch glucose in 7 L fermentor, L-isoleucine production reached up to 31.3 g×L-1.5. In recombinant C. glutamicum, bioconversion of threonine to L-isoleucine was also investigated by variants of TD and AHAS. In order to reduce consume of pyruvate and accumulation of lactic acid, ldh gene(encoding lactic dehydrogenase) was freely knockouted. Bioconversion of threonine to L-isoleucine was studied by co-expressing of TDV140M-F383 A and AHASA42V-A89 V in C. glutamicum Δldh. The study found that the conversion radio of threonine was 56.5% and L-isoleucine production was 19.3±0.50 g·L-1 under the following conditions: ① Molar ratio of threonine and pyruvate was 2.772; ② Temperatures of first 24 h and last 24 h were 30℃ and 35℃, respectively; ③ Feed-batch was carried out every 6 h; ④ Batches of first 24 h and last 24 h were 1/12 and 1/6, respectively.