Metabolic Engineering toward Sustainable Production of Nylon-6.

Nylon-6 is a bulk polymer used for many applications. It consists of the non-natural building block 6-aminocaproic acid, the linear form of caprolactam. Via a retro-synthetic approach, two synthetic pathways were identified for the fermentative production of 6-aminocaproic acid. Both pathways require yet unreported novel biocatalytic steps. We demonstrated proof of these bioconversions by in vitro enzyme assays with a set of selected candidate proteins expressed in Escherichia coli. One of the biosynthetic pathways starts with 2-oxoglutarate and contains bioconversions of the ketoacid elongation pathway known from methanogenic archaea. This pathway was selected for implementation in E. coli and yielded 6-aminocaproic acid at levels up to 160 mg/L in lab-scale batch fermentations. The total amount of 6-aminocaproic acid and related intermediates generated by this pathway exceeded 2 g/L in lab-scale fed-batch fermentations, indicating its potential for further optimization toward large-scale sustainable production of nylon-6.

Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity.

In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with alpha-methylleucine amide as substrate within 24h. Conversion was measured on the [M-H]- species of the corresponding alpha-methylleucine (m/z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.

Chiral liquid chromatography-mass spectrometry for high-throughput screening of enzymatic racemase activity.

In finding suitable biocatalysts for processes in chemical industry, expression libraries are constructed containing typically >10,000 clones. Search for a desired activity is done by examination of all the clones in one or more libraries using a high-throughput screening assay. Here we describe a method for the screening of the enzymatic racemase activity of clones from an expression library on alpha-amino-epsilon-caprolactam (ACL) using a fast chiral LC separation and ionspray-MS as the detection technique. After substrate incubation with S-ACL, the 96-well microplates were centrifuged to remove cell material. The conversion of S-ACL to R-ACL was monitored by quantitation of the R-ACL enantiomer. Separation of the two ACL enantiomers was performed on a Crownpak CR+ column within 1 min. A Gilson 215 autosampler with a 889 multiple injection probe was used for injecting the samples into the LC system. The total analysis time for a 96-well microplate was 56 min. The MS was operated in the positive-ion mode using selected ion monitoring at m/z 129 [M+H]+ of ACL. Using this method over 12,000 samples were analyzed without loss in performance of the system. The LC column remained stable without loss of resolution and the MS system did not show loss in sensitivity throughout the screening. Inter-day reproducibility was within 15%.