Metabolic Engineering Modifications Of The L-isoleucine Producing Corynebacterium Glutamicum

C. glutamicum JHI3-156is an L-isoleucine producing strain obtained by multiple rounds of random mutagenesis, therefore, it is very difficult to further improve the yield of L-isoleucine in this strain by random mutagenesis. In this work, transcriptomic and proteomic analysis were employed to reveal the L-isoleucine production mechanism in C. glutamicum JHI3-156, and metabolic engineering strategies were employed to further increase its L-isoleucine production level. The main results are listed below:(1) The transcriptome of C. glutamicum JHI3-156was investigated by high-throughput sequencing technology. C. glutamicum JHI3-156and wild type ATCC13868were harvested at the late exponential phage. Total RNA were extracted and reverse transcribed into cDNA using random hexamer-primer, which were sequenced and annotated. Compared with the wild type ATCC13869,406genes were upregulated and237genes were downregulated in C. glutamicum JHI3-156. KEGG pathway analysis showed that the upregulated genes include genes hom and thrB in amino acids biosynthetic pathway, and genes prpB2,prpC2, prpD2, cg2796, sucC, sucD and dtsRl in propionic acid pathway; the downregulated genes include genes soxA, glyA, Cg2455and serC in amino acids biosynthetic pathway, and genes sdhCD, icd, sdhB, acn, mdh, sdhA,fumC,gltD,gltB and glnA in TCA cycle.(2) The proteome of C. glutamicum JHI3-156was investigated by two-dimensional electrophoresis coupled with MALDI-TOF mass spectrometry. C. glutamicum JHI3-156and wild type ATCC13868were harvested at the late exponential phage. Total protein was extracted and separated by two-dimensional electrophoresis. About750protein spots were visualized on two-dimensional electrophoresis gels from both samples. Compared with the wild type ATCC13869,82protein spots were up-regulated and123protein spots were down-regulated in JHI3-156. In addition, there were181protein spots only observed in ATCC13869and197protein spots only observed in JHI3-156.13protein spots from ATCC13869gel and18protein spots from JHI3-156gel were chosen and further analyzed by MALDI-TOF mass spectrometry. Identified several differentially expressed proteins, such as MetX, PrpC2, Sod, Gnd, CysK, FusA and SerS.(3) Heterologous expression and purification of the threonine dehydratase (TD1) and acetohydroxy acid synthase (AHAS1) from C. glutamicum JHI3-156showed they are feedback resistant. Sequence analysis showed that there was only a single amino acid substitution (F383V) in TD1, and there were three mutated amino acids (P176S, D426E and L575W) in AHAS1compared with threonine dehydratase (TD) and acetohydroxy acid synthase (AHAS) from C. glutamicum ATCC13869. The TD, TD1, AHAS and AHAS1were over-expressed in Escherichia coli BL21(DE3), purified and measured enzyme activity. The TD1not only showed completely resistance to L-isoleucine inhibition, but also showed enhanced activity. The AHAS1showed more resistance to L-isoleucine inhibition than the wild type. Over-expression of the TD1or AHAS1in C. glutamicum JHI3-156led to increase of L-isoleucine production。In3L fermentator, JHI3-156/pDXW-8-ilvBN1-ilvA1produced L-isoleucine from24.3g-L1to30.7g·L-1, which was increased by26.3%than the control strain after72-h fed-batch fermentation.(4) Improving L-isoleucine secretion system could increase L-isoleucine productivity in C. glutamicum JHI3-156. The brnFE operon and the lrp gene were cloned into the shuttle expression vector pDXW-8individually or in combination, then the resutant recombinants JHI3-156/pDXW-8-brnFE, JHI3-156/pDXW-8-lrp and JHI3-156/pDXW-8-lrp-brnFE were constructed, and the L-isoleucine production in these different strains was compared and analysed. More L-isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in L-isoleucine production was observed when Lrp and BrnFE were expressed in combination. In3L fermentator, JHI3-156/pDXW-8-lrp-brnFE produced L-isoleucine from24.3g·L-1to30.7g·L-1, which was increased by11.0%than the control strain, and the specific yield of L-isoleucine increased72%in72-h fed-batch fermentation. RT-PCR analysis showed that the Lrp expression not only increase brnFE transcriptional level, but also increase the L-isoleucine biosynthesis key genes, lysC, hom, thrB, ilvA and ilvBN, transcriptional levels.(5) Improving biosynthesis pathway, secretory system and coenzyme supply could further increase L-isoleucine productivity in C. glutamicum JHI3-156. The recombinant strains JHI3-156/pDX-W-8-ppnkl-lrp-brnFE, JHI3-156/pDXW-8-ilvBN1-ilvA1-lrp-brnFE, JHI3-156/pDXW-8-ilvBN1-ilvA1-ppnk1and JHI3-156/pDXW-8-ppnkl-ilvBN1-ilvAl-lrp-brnFE were constructed, and the L-isoleucine level and activities of several key enzymes were determined. Compared with the control strain JHI3-156/pDXW-8, L-isoleucine production was increased in all the four strains. The highest L-isoleucine production was obtained in the strain JHI3-156/pDXW-8-ilvBN1-ilvA1-ppnk1, it can produce L-isoleucine from24.3g·L-1to32.3g·L-1, which was increased by32.9%than the control strain after72-h fed-batch fermentation.