Metabolic Engineering Of Corynebacterium Glutamicum SYPS-062for L-serine Production

L-serine is a nonessential amino acid and plays an important role in life activities, and itis widely used in the pharmaceutical and cosmetic industries. The current production ofL-serine relies mainly on chemical synthesis，hydrolysis of protein and enzymatic or cellularconversion from the precursor plus one carbon substance. However, there is rarely report onthe production of L-serine by direct fermentation form sugar substances. This paper focuseson the L-serine producer Corynebacterium glutamicum SYPS-06233a selected from soil andtreated with random mutagenesis, which can accumulate L-serine from sucrose. The mainpurpose of this work is to improve the cellular performance and enhance the L-serineproductivity of33a by rational and systematic metabolic engineering with several rouds ofgenetic modifications and evolutions in chromosome level.Biosynthetic pathway of L-serine in C.glutamicum, like in other bacteria, is originedfrom3-phosphoglycerate--a glycolytic intermediate, which is also the source of pyruvate,followed by enzymatic transamination and finally dephosphorylated to form L-serine. Cellseldom accumulate L-serine under normal condition probably due to its central intermediaterole in numerous cellular reactions and the rigidly regulated biosynthetic pathway. Forexample, high conectration of L-serine inhibits the activity of3-phosphoglyceratedehydrogenase which catalyzes the fist step of L-serine formation, besides that, the supplyingof one carbon unit and transformation to other cellular metabolies such as tryptophane. Theconcentration of L-alanine and L-valine reaches a high level in the culture mixture. Accordingto the fact that the unbalanced distribution of metabolic flux at the3-phosphoglycerate nodecontributes to the low productivity of L-serine and the constant carbon skeleton leakage toby-products formation, the metabolic engineering strategy became more clarified and detailedthat redistribute the carbon flow at the critical node by combining enhancing the performanceof L-serine biosynthetic pathway and depressing the formation of by-products. All the resultsare as following respects:1.3-PGDH which catalyzes the first step in the biosynthesis of L-serine. It is Vmax-typeallosterically inhibited by L-serine a heterotropic allosteric negative effector of the enzymeactivity. It contains C-terminal regulatory domain which corresponds to the ACT domain andincludes the L-serine bingding site. Successfully designed a stem-loop structural sequencewhich make an excellent function both as an artificial linker for crossover PCR and theterminational sequence for protein expression, and constructed the recombinant plasmid usedfor the deletion of serA-act which encoding the ACT domain of3-PGDH. Finally, obtainedthe recombinant strain33a serA△act （33a△S1） that the serA-act fragment has been knockedout precisely in chromosome of33a and the mutant enzyme3-PGDH-M preserves theoriginal function. In shake flask, the resulting strain33a serA△act produced25.419± 2.681g/L L-serine with a YP/Sof0.778mol L-Serine per mol Sucrose. The growth condition ofthe recombinant strain was improved substantially and the biomass improved78.52%campared with the wild type strain33a. Further more, the productivity of33a△S1growed ina wild extent up to21.559±2.370mM·（g CDW）^-1and improved87.86%.2. L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate andammonia. L-serine encounter intracellular degradation catalyzed by one type of L-serinedeaminases encoding by sdaA identified in the chromosome of C.glutamicum. The centralfragment encoding605bp nucleotides of sdaA was deleted based on33a and33△S1. thesdaA gene deleted mutant strain33a△S2accumulated14.762±0.497g/L with the same YP/Sas the wild type strain. However, the productivity of by-products transformed from pyruvateincreasing in great deal, and in more detail the productivity of L-alanine and L-valineenhanced37.8%and50.1%. after culture96h, the biomass of33a△S2reached the highestlevel at63.798±3.372g/L among the L-serine producer derivatized from33a.Subsequently, successfully constructed the novel strain33a serA-△act&△sdaA（33a△SS） that combining the inactivity of L-serine deaminase and introduction of the L-serineunsensisitive3-PGDH mutant. The recombinant strain performs excellent L-serineproductivity of25.274±1.866mM/（g CDW） with the YP/Sof0.823±0.061mol L-serine per molSucrose. Compered with33a and△S2，L-serine productivity improved1.12and1.24timesrespectively, at the same time, the yield of L-serine from sucrose also enhanced2.674timesand71.1%. On the contrary, the formation of by-products was depressed.3. L-alanine and L-valine was the main by-products during the production of L-serine byCorynbacterium glutamicum33a△SSA. For the purpose of decreasing the accumulation ofthese two by-products, the work focused on the transamination course of the biosyntheticpathway. Firstly, the alaT gene deletion mutant was constructed and the target strainaccumulated less L-serine and L-alanine.In order to study the effect of ilvE deletion on the accumulation of L-valine, ilvE genedeficient mutant strain Corynbacterium glutamicum33a△SSA△ilvE was constructed byhomologous recombination. The central fragment encoding987bp nucleotides of ilvE wasdeleted. The growth of the△ilvE phenotype on the chemical defined medium wassignificantly retarded due to the reduced biosynthesis capability of branch-chain amino acids（Val, Ile, Leu）. However, growth condition of the ilvE deleted mutant can be significantlyimproved when supplemented with250mg/L branched-chain amino acids. L-Ile was thelimiting factor for the growth of△SSAI. The results suggested that the productivity ofL-valine decreased90%compared with the wild type strain, the concentration of L-valine wasdecreased to0.5g/L in the96h culture.