Construction Of Metabolic Engineering For Fermentative Production Of 5-Hydroxyleucine In Corynebacterium Glutamicum

5-Hydroxyleucine can be used as a chiral precursor,intermediate and final products for the biosynthesis of a number of natural products,and is due to its structural and functional versatility.Currently,chemical synthesis and enzymatic synthesis are the main methods used for the preparation of 5-hydroxyleucine,all of which have limitations.In this study,an engineered strain with a clear genetic background for continuous improvement was constructed by introducing the 5-hydroxyleucine biosynthetic pathway in Corynebacterium glutamicum,and its yield was improved by systematic metabolic modification.The main findings are as follows.（1）To heterologous introduce L-leucine hydroxylase in Corynebacterium glutamicum,the genes（ldo1,ldo2）,which encoding L-leucine 5-hydroxylase from N.piscinale and N.punctiforme NIES-2108,was expressed in wild-type Corynebacterium glutamicum by p XMJ19.The results showed that the gene encoding L-leucine 5-hydroxylase from N.punctiforme NIES-2108 was codon-optimized and functionally expressed in Corynebacterium glutamicum with a 5-hydroxyleucine yield of 0.52 g/L.（2）To enhance the supply of L-leucine,the gene ilv BN,which is regulated by the high-intensity promoter P_tuf,was integrated and expressed to enhance the branched-chain amino acid synthesis pathway,and the accumulation of L-valine in strain H-3 reached 5.57 g/L;the accumulation of L-leucine in strains H-4 and H-5 respectively reached 4.69 g/L and 5.17 g/L by overexpression of the anti-feedback variant leu A^M;the accumulation of L-valine in strain H-6 decreased by 3.6%and the concentration of L-leucine increased by 10.6%by deleting the region encoding of the transcriptional regulator Ltb R.（3）To construct a 5-hydroxyleucine heterologous pathway in Corynebacterium glutamicum,L-leucine 5-hydroxylase from N.punctiforme NIES-2108 was optimized for induction-type expression and constitutive expression in L-leucine producing bacteria H-6.All three recombinant bacteria were able to synthesize and accumulate 5-hydroxyleucine.The accumulation of 5-hydroxyleucine was increased by 74.2%,79.7%and 81.2%,respectively,compared to strain H-2.（4）To enhance the supply ofα-ketoglutarate,the overexpression of icd,the gene encoding isocitrate dehydrogenase,and glt A,the gene encoding citrate synthase,enhanced the metabolic flow of TCA cycle,and the accumulation of 5-hydroxyleucine in strain H-11reached 3.94 g/L;to further enhance the supply ofα-ketoglutarate,the gene encoding isocitrate lyase ace A was knocked out to block the glyoxylate cycle,and the accumulation of5-hydroxyleucine in strain H-12 reached 4.11 g/L;replacing theα-ketoglutarate dehydrogenase encoding gene odh A promoter to weaken theα-ketoglutarate metabolic flow,the accumulation of 5-hydroxyleucine in strain H-13 was increased by 25.8%compared with strain H-12.