Multilayer Engineering of Enzyme Cascade Catalysis for One-Pot Preparation of Nylon Monomers from Renewable Fatty Acids

Abstract

Enzyme cascade catalysis has critical problems in obtaining the high concentrations of products, such as the low stabilities and activities of biocatalysts and the inhibition by hydrophobic reactants at high concentrations to biocatalysts. Here, we performed multilayer engineering of enzyme cascade catalysis to produce C11 nylon monomers at commercially viable concentrations from ricinoleic acid. The catalysis was driven by engineered Escherichia coli-based whole-cell biocatalysts and cell-free enzymes (i.e., lipases). Stabilities and activities of the biocatalysts were improved by engineering the bottleneck enzyme Baeyer− Villiger monooxygenase and introducing a cofactor regeneration system. The inhibitory effects of the byproducts n-heptanoic acid and pyruvate on the cascade enzymes were overcome, and the products were simply recovered in situ by engineering of reactions with the addition of an adsorbent resin. We obtained 248 mM undecanedioic acid or 232 mM 11-aminoundecanoic acid as a nylon monomer from 300 mM ricinoleic acid by multilayer engineering. The concentration was 500-and 640-fold higher than those produced by nonengineering, respectively. We have also simply isolated the C11 nylon monomers via solvent extraction to a rather high purity. This study will contribute to the industrial enzyme cascade synthesis of nylon monomers in an environmentallyfriendly route from renewable biomass.

Rational Engineering of a Multi‐Step Biocatalytic Cascade for the Conversion of Cyclohexane to Polycaprolactone Monomers in Pseudomonas taiwanensis

“…Increasing the expression and stabilizing the activity of Baeyer–Villiger monooxygenases also led to higher 9‐hydroxynonanoic acid and C11 nylon monomer concentrations in 4‐ and 5‐step catalytic cascades, respectively. [ 7,16 ]…”

“…[ 11–13,14 ] A holistic approach comprising catalyst and reaction engineering allows controlling the product formation patterns. [ 15 , 16 ]…”

Fatty Acids and their Derivatives as Renewable Platform Molecules for the Chemical Industry

“…Such ac ascade was extended to make 11aminoundecanoic acid from 7a,leading to 77 %product from 300 mm 7a. [206] Thesynthesis of w-aminododecanoic acid from lauric acid on apilot scale was also reported using awhole-cell system for enzymatic oxidation and transamination. [207] Nowadays,c omplex products are produced in suitable microbial hosts that have been designed by metabolic engineering,for example,byintroducing artificial or improving existing pathways.T hus,b iofuels,f atty acid derivatives, and surfactants such as sophorolipids can be made directly in microorganisms.A sa ne xample,a ne ngineered pathway to produce the polyunsaturated fatty acid EPA( 15 a)-commonly obtained from fish oil-in the oleagineous yeast Yarrowia lipolytica was reported.…”

Novel oleate hydratases and potential biotechnological applications

“…By optimising the protein expression and by using an engineered BVMO, a yield of nnonanoic acid and 9-hydroxynonnoic acid of 6 mmol/g dry cells was obtained. Another effective strategy based on the same cascade was to combine different whole cell biocatalysts and cell free enzymes in one pot for the production of C11 n y l o n m o n o m e r s ( u n d e c a n e d i o i c a c i d a n d 1 1aminoundecanoic acid) from ricinoleic acid (12-hydroxy-9cis-octadecenoic acid) (Kim et al 2020). The key for the effective biotransformation was to use adsorbent polymeric beads (Sepabeads S825) to bind the hydroxyl fatty acid and products, which ameliorated their enzyme inhibitory effects.…”