title: Functional Exchangeability of Oxidase and Dehydrogenase Reactions in the Biosynthesis of Hydroxyphenylglycine, a Nonribosomal Peptide Building Block.
abstract: A key problem in the engineering of pathways for the production of pharmaceutical compounds is the limited diversity of biosynthetic enzymes, which restricts the attainability of suitable traits such as less harmful byproducts, enhanced expression features, or different cofactor requirements. A promising synthetic biology approach is to redesign the biosynthetic pathway by replacing the native enzymes by heterologous proteins from unrelated pathways. In this study, we applied this method to effectively re-engineer the biosynthesis of hydroxyphenylglycine (HPG), a building block for the calcium-dependent antibiotic of Streptomyces coelicolor, a nonribosomal peptide. A key step in HPG biosynthesis is the conversion of 4-hydroxymandelate to 4-hydroxyphenylglyoxylate, catalyzed by hydroxymandelate oxidase (HmO), with concomitant generation of H2O2. The same reaction can also be catalyzed by O2-independent mandelate dehydrogenase (MdlB), which is a catabolic enzyme involved in bacterial mandelate utilization. In this work, we engineered alternative HPG biosynthetic pathways by replacing the native HmO in S. coelicolor by both heterologous oxidases and MdlB dehydrogenases from various sources and confirmed the restoration of calcium-dependent antibiotic biosynthesis by biological and UHPLC-MS analysis. The alternative enzymes were isolated and kinetically characterized, confirming their divergent substrate specificities and catalytic mechanisms. These results demonstrate that heterologous enzymes with different physiological contexts can be used in a Streptomyces host to provide an expanded library of enzymatic reactions for a synthetic biology approach. This study thus broadens the options for the engineering of antibiotic production by using enzymes with different catalytic and structural features.
authors.forename: Veronica
authors.initials: V
authors.lastname: Diez
authors.forename: Mark
authors.initials: M
authors.lastname: Loznik
authors.forename: Sandra
authors.initials: S
authors.lastname: Taylor
authors.forename: Michael
authors.initials: M
authors.lastname: Winn
authors.forename: Nicholas J W
authors.initials: NJ
authors.lastname: Rattray
authors.affiliation: #ThermoFisher Scientific, 1 Boundary Way, Hemel Hempstead, Herts, HP2 7GE, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Podmore
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
authors.forename: Marnix H
authors.initials: MH
authors.lastname: Medema
authors.forename: Ulrike
authors.initials: U
authors.lastname: Müller
authors.forename: Roel
authors.initials: R
authors.lastname: Bovenberg
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.forename: Eriko
authors.initials: E
authors.lastname: Takano
mesh_terms: D000429:Alcohol Oxidoreductases / Q000145:classification / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D006038:Glyoxylates / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000145:classification / Q000378:metabolism*; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000737:chemistry / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D006038:Glyoxylates; C030921:4-hydroxyphenylglyoxylic acid; C023010:4-hydroxyphenylglycine; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C021538:L-mandelate dehydrogenase; D005998:Glycine
keywords: HmO; MdlB; UHPLC-MS; calcium-dependent antibiotics; l-HPG biosynthesis; nonribosomal peptide; synthetic biology