Vigilancia Tecnológica

Graphical Abstract and Lay Summary??Caprolactone is an organic chemical compound, which is mostly used as a monomer to synthesize valuable biodegradable polymer in composite materials and drug delivery. A bi?enzyme cascade reaction was constructed in Escherichia coli to synthesize ??caprolactone from cyclohexanol. An RBS combinatorial library was constructed to regulate the ratio of ADH and CHMO simultaneously. Meanwhile, a high?throughput DNPH screening method was established to screen high?performance strains efficiently and conveniently. This work provides a good reference for other in vivo artificial cascade reactions.Abstract??Caprolactone is an important non?toxic compound for polymer synthesis like polycaprolactone which has been widely used in drug delivery and degradable plastics. To meet the demand for a green economy, a bi?enzymatic cascade, consisting of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO), was designed and introduced into Escherichia coli to synthesize ??caprolactone from cyclohexanol with a self?sufficient NADPH?cofactor regeneration system. To further improve the catalytic efficiency, a carbonyl group?dependent colorimetric method using inexpensive 2,4?dinitrophenylhydrazine (DNPH) was developed for assay of cyclohexanone, an intermediate production of cascade reaction. It can be used to screen mutant strains with high catalytic efficiency from high?throughput library by detecting the absorbance value in microtiter plates (MTP) instead of gas chromatography (GC) analysis. Moreover, an RBS combinatorial library was constructed for balancing the expression of ADH and CHMO from two independent transcriptional units. After the high?throughput screening based on intermediate product control, an optimal variant with higher substrate tolerance and long?term stability was obtained from RBS combinatorial library. Through a fed?batch process, ??caprolactone production reached 148.2 mM after 70 h of reaction under the optimized conditions, which was the highest yield achieved to date.