De Novo Biosynthesis Of Nylon 12 Monomer ω-Aminododecanoic Acid

Nylon 12,one of the valuable representatives of aliphatic polyamides,is extensively applied in offshore pipelines and parts of automobiles due to its extraordinary resistance to heat,abrasion,chemical,UV and scratch.However,the Nylon 12 industry in China is hindered by the high technical barrier,long synthetic route and high production cost,and almost completely depends on imports.Although there have been some studies on the biosynthesis of Nylon 12 monomer,the efficiency is low,and all were based on conversion of dodecanoic acid(DDA)or its derivatives.De novo biosynthesis of Nylon 12 monomer from glucose has not yet been achieved.In this study,aiming to explore efficient biosynthesis of the Nylon 12 monomer ω-aminododecanoic acid(ω-AmDDA).ω-AmDDA-producing Escherichia coli strain was developed by combining enzyme mining,expression regulation,protein engineering,cofactor engineering and tolerance engineering.In order to construct an engineered strain of E.coli for effcient conversion of DDA to ω-AmDDA.the enzymes catalyzingω-hydroxylation,oxidation and amination of DDA were first screened.The chimeric P450 enzyme CYP153A-NCP,the alcohol dehydrogenase BsADHC257L,and the ω-aminotransferase(ω-TA)CV2025 obtained by enzyme screening were assembled into two catalytic modules M1 and M2,and placed on four compatible plasmids of different copy numbers.The multi-enzyme cascade pathway for biocatalytic synthesis of co-AmDDA from DDA was successfully constructed in E.coli BL21(DE3)by adjusting the expression intensity of the enzymes.Subsequently,simultaneous regeneration of the redox equivalents.PLP and L-alanine required in the artificial pathway was enabled by its interfacing with the native metabolism of the host using glucose dehydrogenase(GDH1),L-alanine dehydrogenase(AlaDH2)and an exogenous ribose 5-phosphate(R5P)-dependent PLP synthesis pathway yaaDE as bridges.Further engineering of the host by blocking βoxidation and enhancing substrate uptake generated strain P1-1(E-M1-3/C-M2-2).withω-AmDDA yield of 96.5%when 5.0 mM DDA was used as the substrate.Subsequently,by introducing a saturated C12 acyl-ACP-specific thioesterase BTE into the heterologous multi-enzyme cascade catalytic pathway for ω-AmDDA biosynthesis from DDA,the natural central metabolic pathway of E.coli and the heterologous multi-enzyme pathway were successfully connected.The de novo synthesis pathway of ω-AmDDA enabled the fermentative synthesis of ω-AmDDA via DDA in E.coli using the inexpensive renewable carbon source glucose.In order to further improve the de novo synthesis yield of ω-AmDDA,the metabolic network of the chassis was engineered,the alcohol dehydrogenase and the slection marker were replaced and the reaction conditions were optimized.The yield of ω-AmDDA reached 27.5 mg/L with 2%glucose as carbon source,2 g/L NH4HCO3 as ammonium source and 0.1 mM IPTG as inducer at 30℃ and pH 7.5.After strengthening of PLP synthesis,and using AlkJ as alcohol dehydrogenase and aminoglycoside resistance as the selection marker,80.9 mg/L of ω-AmDDA was produced.Finally,by modularizing and optimizing the expression of the acetyl-CoA supply pathway,the acetyl-CoA activation pathway,and the fatty acid synthesis FAS Ⅱ pathway involved in DDA synthesis,as well as the DDA-derivatized heterologous cascade pathway,the synthesis and further conversion of DDA were promoted.The final strain P1D5(EApr-M1-B3/C-M2-A)produced 243 mg/L of ω-AmDDA in shake-flask fermentation.Ultimately,through hierarchal regulation of the rate-limiting DDA hydroxylation reaction(pathway layer),redox and metabolic energy-homeostasis(cofactor layer)and oxidative stress defense network(intracellular environment layer),the de novo synthesis of ω-AmDDA in E.coli was further improved.Rational and semi-rational design methods were used to improve the catalytic activity of the rate-limiting enzyme CYP153A-NCP to strengthen the flux of DDA hydroxylation.Meanwhile.the metabolic energy homeostasis in the cellular factory was optimized by deletion of the ATP-consuming non-essential enzyme FecE,while the redox homeostasis of the cell factory was regulated by enhancing the supply of reducing equivalence and adjusting its rational distribution via overexpression of the non-phosphorylated glucose transporter Glf and the transhydrogenase PntAB.Moreover,the intracellular oxidative stress defense network was tuned by overexpression of the reactive oxygen species(ROS)-responsive transcription factor OxyR.The final recombinant strain PDANR(EApr-Ml-B3mut/C-M2-A)produced 471 mg/L of ω-AmDDA from glucose in shakflask fermentation.This work successfully constructed the de novo biosynthesis pathway of Nylon 12 monomer,which would lay a foundation for the fermentative production of Nylon 12.Meanwhile,the multi-enzyme cascade catalysis,cofactor engineering,tolerance engineering and enzyme engineering strategies developed in this study would provide reference for the synthesis of other fatty acid derivatives in E.coli.