Modification Of Lysine Branch Pathway And Evolutionary Engineering Of 4-hydroxyisoleucine Biosynthesis In Corynebacterium Glutamicum

4-Hydroxyisoleucine(4-HIL)is a promising drug for treating diabetes.In our previous study,4-HIL was synthesized from self-produced L-isoleucine(Ile)in Corynebacterium glutamicum by expressing an Ile dioxygenase gene.L-lysine(Lys)is a by-product of the 4-HIL biosynthesis in C.glutamicum.In this study,the Lys branched pathway of 4-HIL biosynthesis was modified and evolved.On the one hand,the lysE gene which encodes the Lys exporter protein was knocked out to attenuate the Lys biosynthesis and increase 4-HIL production.On the other hand,the Lys inductive evolutionary control system was constructed and used to drive the programming evolution of 4-HIL-producing strain,thereby the positive mutant strains with increased 4-HIL production or decreased Lys production could be rapidly screened out.The main studies and results are as follows.1.To reduce the accumulation of Lys,lysE was knocked out in SN02,resulting in the strain SY01,and then ddh was knocked out in SY01,generating the strain SY02.The Lys production in SY01 significantly decreased by 80.0%compared with SN02,but the 4-HIL production also decreased by 37.1%.The 4-HIL production of SY02 was 60.87±4.25 m M,38.9%lower than that of SZ06,and its Lys production was comparable to that of SY01.To enhance the supply of Ile,lys C was expressed in SY01,resulting in the strain SY03.Ile was accumulated,but the conversion of Ile to 4-HIL was reduced,thereby the 4-HIL production decreased by 57.2%,and the Lys production also decreased by 40.3%.Thus knockdown of lysE and ddh can reduced Lys production,but the total amount of the aspartic acid family amino acids was also reduced.2.The Lys biosensor LysG-P_lysE was used to drive the expression of the fluorescent reporter gene egfp and the mutagenic gene cdd,and the resulting plasmid p MK for expressing this Lys inductive evolutionary control system was constructed and then introduced into a recombinant 4-HIL-producing strain SZ06 to obtain the programming evolutionary control strain SZ06/p MK.The response concentration toward Lys and the modulation range of the LysG-P_lysE biosensor in this strain were characterized.When the Lys concentration was 5-30m M,the biosensor activated the expression of egfp by 1.09-to 1.37-fold.Thus,the LysG-P_lysEbiosensor can sense Lys and activate the expression of its downstream gene according to the Lys concentration.It has a high sensitivity to Lys,but the activation fold for downstream genes is low.3.The evolutionary control strain SZ06/p MK was subjected to several rounds of successive evolution,and the fluorescence changes of the bacterium were monitored.After fluorescence decreased,dozens of mutant strains were randomly selected.According to the 4-HIL production,the positive mutation rate reached 95.5%after eight rounds of evolution(16days),and the highest titer of 4-HIL was 63.98 m M.To shorten the evolution time,three rounds of evolution(6 days)were conducted.The positive mutation rate reached 54.5%,and the highest titer of 4-HIL was 70.67 m M.Thus,the Lys inductive evolutionary control system can sense the Lys concentration to programmedly control the evolution process of SZ06/p MK,and thus promote the synthesis of 4-HIL.It can also generate positive mutant strains in a shorter time.4.Nine strains MK1-MK9 with the highest 4-HIL production were selected from the dozens of positive mutant strains produced by evolution,and were then fermented in shake flasks.The growth and glucose consumption of these nine strains were similar to that of the control strain SZ06,and their 4-HIL production increased to 92.72-146.29 m M,with a maximum increase of 89.5%.However,the content of by-product Lys also increased slightly to21.14-33.70 m M.The evolutionary control plasmid p MK was removed from MK1-MK9,and the genetically stable mutant strains K1-K9 were obtained and fermented in shake flasks.The growth and glucose consumption of K1-K9 were similar to that of the control strain SZ06,and the 4-HIL production increased by 10.6-28.4%,with K3 exhibiting the highest 4-HIL production of 152.19±14.60 m M.However,the Lys concentration of K1-K9 also increased slightly.Thus,the utilization of this Lys biosensor-driven programmed evolution can rapidly screen out mutant strains with significantly increased 4-HIL production.5.The genomes of K1-K9 were resequenced and the common mutation sites in these nine strains were identified.By combining with RT-PCR analysis,the-111T insertion mutation in the upstream of dap C was found to be able to upregulate the transcriptional level of dap C,which may lead to the accumulation of Lys.The T-476C point mutation in the upstream of CGB98?RS08395 leads to the upregulation of its transcriptional level,which may affect the synthesis of peptidoglycan and cell wall.