Directed evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production

From Springer Nature via Jisc Publications RouterHistory: received 2021-03-02, accepted 2021-07-29, registration 2021-08-20, pub-electronic 2021-09-06, online 2021-09-06, collection 2021-12Publication status: PublishedFunder: EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council); doi: https://doi.org/10.13039/100010663; Grant(s): pre-FAB 695013Abstract: Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group

Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

A finished clone-based assembly of the mouse genome reveals extensive recent sequence duplication during recent evolution and rodent-specific expansion of certain gene families. Newly assembled duplications contain protein-coding genes that are mostly involved in reproductive function

Enzymatic elaboration of oxime-linked glycoconjugates in solution and on liposomes

Oxime formation is a convenient one-step method for ligating reducing sugars to surfaces, producing a mixture of closed ring α- and β-anomers along with open-chain (E)- and (Z)-isomers. Here we show that despite existing as a mixture of isomers, N-acetylglucosamine (GlcNAc) oximes can still be substrates for β(1,4)-galactosyltransferase (β4GalT1). β4GalT1 catalysed the galactosylation of GlcNAc oximes by a galactose donor (UDP-Gal) both in solution and in situ on the surface of liposomes, with conversions up to 60% in solution and ca. 15–20% at the liposome surface. It is proposed that the β-anomer is consumed preferentially but long reaction times allow this isomer to be replenished by equilibration from the remaining isomers. Adding further enzymes gave more complex oligosaccharides, with a combination of α-1,3-fucosyltransferase, β4GalT1 and the corresponding sugar donors providing Lewis X coated liposomes. However, sialylation using T. cruzi trans-sialidase and sialyllactose provided only very small amounts of sialyl Lewis X (sLe(x)) capped lipid. These observations show that combining oxime formation with enzymatic elaboration will be a useful method for the high-throughput surface modification of drug delivery vehicles, such as liposomes, with cell-targeting oligosaccharides

Direct Analysis of Biotransformations with Mass Spectrometry – DiBT-MS

Ambient ionization coupled to mass spectrometry has the advantages of minimal requirements for sample preparation prior to analysis which renders it suitable for high throughput screening. We present a protocol that permits the application of this method in routine biotechnology and chemical biology laboratories which are using engineered enzymes to produce target compounds from substrates. We show how DESI-MS can be used to directly analyse the activity of biotransformations from crude cell lysate which we term DiBT-MS, this method is 10-1000 times faster than LC-MS and uses far less solvent. This protocol demonstrates the impact of solvent spray composition on ionization efficiency of the target analyte, the benefits of a nylon membrane slide and the reusability of sample slides in multiple experiments.