Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000× more sensitive to an exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 μg/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ∼6 μg/L. The high sensitivity and range of OplR is a powerful tool toward the development of novel routes to the biological synthesis of caprolactam.

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Identification, Characterization, and Application of a Highly Sensitive Lactam Biosensor from Pseudomonas putida.

Microorganisms can utilize biomass to produce valuable chemicals, showing sustainable, renewable and economic advantages compared with traditional chemical synthesis. As a potential five-carbon platform polymer monomer, 5-aminovalerate has been widely used in industrial fields such as clothes and disposable goods. Here we establish an efficient whole-cell catalysis for 5-aminovalerate production with ethanol pretreatment. In this study, the metabolic pathway from L-lysine to 5-aminovalerate was constructed at the cellular level by introducing L-lysine α-oxidase. The newly produced H2O2 and added ethanol both are toxic to the cells, obviously inhibiting their growth. Here, a promising strategy of whole-cell catalysis with ethanol pretreatment is proposed, which greatly improves the yield of 5-aminovalerate. Subsequently, the effects of ethanol pretreatment, substrate concentration, reaction temperature, pH value, metal ion additions and hydrogen peroxide addition on the whole-cell biocatalytic efficiency were investigated. Using 100 g/L of L-lysine hydrochloride as raw material, 50.62 g/L of 5-aminovalerate could be excellently produced via fed-batch bioconversion with the yield of 0.84 mol/mol. The results show that a fast, environmentally friendly and efficient production of 5-aminovalerate was established after introducing the engineered whole-cell biocatalysts. This strategy, combined with ethanol pretreatment, can not only greatly enhance the yield of 5-aminovalerate but also be applied to the biosynthesis of other valuable chemicals.

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Efficient whole-cell catalysis for 5-aminovalerate production from L-lysine by using engineered Escherichia coli with ethanol pretreatment.

Microbial engineering for the production of C2-C6 organic acids.

Progress in the metabolic engineering of bio-based lactams and their ω-amino acids precursors.

Bioproduction of 5-aminovalerate (5AVA) from renewable feedstock can support a sustainable biorefinery process to produce bioplastics, such as nylon 5 and nylon 56 In order to achieve the biobased production of 5AVA, a 2-keto-6-aminocaproate-mediated synthetic pathway was established Combination of L-lysine α-oxidase from Scomber japonicas, α-ketoacid decarboxylase from Lactococcus lactis and aldehyde dehydrogenase from Escherichia coli could achieve the biosynthesis of 5AVA from biobased L-lysine in Escherichia coli The H2O2 produced by L-lysine α-oxidase was decomposed by the expression of catalase KatE Finally, 5224 g/L of 5AVA were obtained through fed-batch fermentation Moreover, homology modelling, molecular docking and molecular dynamic simulation analyses were used to identify mutation sites and propose a possible trait-improvement strategy: the expanded catalytic channel of mutant and more hydrogen bonds formed were beneficial for the substrates stretch In summary, we have developed a promising artificial pathway for efficient 5AVA synthesis

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Ge Hu

Papers

Production of nonnatural straight-chain amino acid 6-aminocaproate via an artificial iterative carbon-chain-extension cycle.

Coenzyme self-sufficiency system-recent advances in microbial production of high-value chemical phenyllactic acid

Electrochemical sensor for determination of methylprednisolone as an anabolic steroid used in doping