Systematic Metabolic Engineering For Efficient Synthesis Of Trans-4-hydroxy-L-proline In Escherichia Coli

Fermentation of amino acid is the pillar industry of fermentation industry in China.Hydroxylated amino acids,a sort of the high value-added amino acids,are widely used in food,medicine,and material industries.Hydroxyprolines are derivatives of precursor proline(L-Pro)after hydroxylation in five-membered ring,in which trans-4-hydroxy-L-proline (trans-4-Hyp)is an important chiral amino acid used as intermediate of carbapenem antibiotics,and plays an important role in the fields of medicine,health care,food and so on.Producing trans-4-Hyp by microorganisms has great economic and environmental benefits.The production of trans-4-Hyp through microorganism can be divided into de novo fermentation production with cheap carbon sources such as glucose,and pre-feeding L-Pro according to the different substrates of feeding.Nowadays,the research of microbial production of trans-4-Hyp has been focusing on the feeding of L-Pro.However,a large amount of L-Pro needs to be added during the fermentation process,and the separation and purification of L-Pro and trans-4-Hyp is somehow difficult,which greatly increases the cost of industrialized production.De novo fermentation production of trans-4-Hyp with cheap carbon sources can achieve low-cost raw materials and high-yield production,and is gradually becoming the mainstream trend of industrialized production for trans-4-Hyp.Construction of engineered strains with high production hydroxylated amino acid is suffering three problems:The construction of chassis cells producing targeted amino acid precursor,the supply of?-KG,and the catalytic property of amino acid hydroxylase.In this study,we tried to propose a general strategy for the efficient synthesis of hydroxylated amino acids by microorganisms.Multiple strategies involving synthetic biology,metabolic engineering and protein engineering technology were used to systematically modify Escherichia coli for trans-4-Hyp biosynthesis as well as further exploration of novel biosynthetic pathway of trans-4-Hyp,the main research findings are as follows:(1)Based on the usage of L-Pro codons in E.coli and S.marcescens,different numbers of corresponding rare codons were replaced in kan^R and apm^R,respectively.p ET28a-kan^R-A and p ET28a-apm^R-A were constructed in E.coli BL21 and S.marcescens JNB5-1 for selection of mutation libraries,respectively.Mutagenic plasmid MP6 was also constructed in E.coli for MP6 assisted rare codon selection.The L-Pro-producing mutant E.coli PM-14reached 0.816 g/L at the shake flask level.After fed-batch fermentation in a 5 L fermentor,the L-Pro yield of the E.coli PM-14 strain reached 18.2 g/L,with L-Pro yield 0.455 g/L/h.The library of S.marcescens JNB5-1 mutants was processed by ARTP,and the mutant LK-18 was obtained,in which the production of Prodigiosin was 3.3 times higher than that of the wild-type.After removing pig I in both wild-type and mutant strain LK-18,the production of L-Pro in LK-18?pig I was 2.6 times that of S.marcescens JNB5-1?pigI.(2)The novel pathway of biosynthesis of trans-4-Hyp by multi-enzyme cascade catalysis was constructed,which was processed by the modified GetI mutant Get I(QDPYF),ArgI and OCD.A screening strategy combining rare codon selection and pEvolvR was used for directed evolution of OCD.The three most improved mutants,K205G,M86K and T162A,was increased their enzyme activities by 70.3%,64.8%and 55.7%,respectively.The activity of the triple mutant OCD_{M86K/T162A/K205G} was increased by 2.41 times higher than wild-type with the k_cat value of OCD_{M86K/T162A/K205G} 2.85 times that of wild-type.The triple mutant was constructed into recombinant E.coli GAO-4,and the trans-4-Hyp product concentration generated by whole cell catalysis reached 105.5 mg/L,which was 2.35 times of recombinant strain GAO-1.A smart RBS combinatorial library based on Red Libs was further used to optimize the expression of multi-enzyme pathway of trans-4-Hyp biosynthesis,and the recombinant strain EH#1 reached 189.3 mg/L trans-4-Hyp,which was 79.4%higher than GAO-4.Based on the whole cell catalytic system EH#1,LGOX and CAT were co-expressed to eliminate the by-product H₂O₂,NOX was also co-expressed for the NAD⁺cofactor cycle.GAO-5 reached 269.2 mg/L trans-4-Hyp after optimization of the whole cell catalysis optimization.(3)Comparative transcriptome analysis was further used to analyze the fluctuation in the transcription level of L-Pro related synthetic pathways in the mutant PM-14 including glycolysis,TCA cycle,L-Pro synthetic pathway and L-Pro transport system.Compared with the wild-type,the gene of the glycolysis showed higher expression level.In the TCA pathway,icd and acn encoding isocitrate dehydrogenase and aconitase showed significantly increased expression,thereby enhancing the flux to?-KG.The rate-limiting enzyme pro B gene also showed a higher expression level in the mutant strain.put A,put P,pro P and ace A were knocked out and point mutation of pro B was carry out in further metabolic engineering of PM-14 to promote L-Pro biosynthesis.The L-Pro titer of M10 strain reached 37.5 g/L,which was 2.06 times that of PM-14,and the production efficiency was 0.938 g/L/h.HM10harboring p DXW-10-Datp4h with proline-4-hydroxylase showd 15.7 g/L of trans-4-Hyp,but also resulted 18.1 g/L of L-Pro accumulation.(4)A dynamic regulation strategy based on the quorum sensing gene circuit regulation strategy was designed to moderate ODHC activity.The original promoter of suc A gene was replaced by P_{esa S} promoter,which could be combined with the transcription regulator Esa RI70V to trigger its transcription in the absence of AHL.The accumulation of AHL in the cell destroyed the binding of P_{esa S} promoter and Esa RI70V and dynamically inhibits ODHC activity.The expression of esa I driven by four different P_bs promoters could lead to the dynamic accumulation of AHL,thereby down-regulating the P_{esa S} promoter at different times to achieve better?-KG metabolic flux.The designed quorum sensing gene circuit regulation system can dynamically control GFP expression after verification.Furthermore,the quorum sensing gene circuit regulation system was used to dynamically regulate ODHC activity.HM14 reached 43.2 g/L trans-4-Hyp,which was 2.75 times that of the wide-type.The residual L-Pro decreased by 76.6% compared with the wide-type but still retained the 4.3 g/L L-Pro.(5)Based on the sequence of Dactylosporangium sp.RH1,BLASTP alignment was performed in NCBI,and three putative trans-P4Hs were selected.UB2TP4H out of them with the highest hydroxylation activity reached 83.6 U/mg.According to the structural analysis,total of four single mutation points in the residues 170 and 172 of UB2TP4H were designed.Activities of UB2TP4H_L170A and UB2TP4H_P172N were increased by 16.1%and 27.8%,respectively.Compared with wild-type,the enzyme activity of double mutant UB2TP4H_L170A/P172N was increased by 38.2%,reaching 115.5 U/mg.Structural analysis showed that the substitution of L170A and P172N residues would bring additional hydrogen bonds with the substrate.The molecular dynamics simulations were further processed on the UB2TP4H_WT and double mutant UB2TP4H_L170A/P172N complexes,respectively.UB2TP4H_L170A/P172N displayed larger binding pocket of substrate than that of wild-type,and the conformational fluctuation of the substrate pocket was more stable than that of UB2TP4H_WT.The RMSF of the loop_170-175 of UB2TP4H_L170A/P172N was lower than that of UB2TP4H_WT.Rosetta tool Cartesian?ddg was used to measure the binding free energy??G between the enzyme and L-Pro,and the??G of UB2TP4H_L170A/P172N was-3.27 KJ/mol.HMY carrying recombinant plasmid p DXW-10-ub2tp4h _L170A/P172N reached 54.8 g/L trans-4-Hyp with productivity 0.236 g/g in a 5 L fermentor,which was 26.9%higher than the strain HM14har boring Datp4h,while the concentration of L-Pro remained finally reduced to 0.2 g/L.