Additional file 1 of Chemoautotrophic production of gaseous hydrocarbons, bioplastics and osmolytes by a novel Halomonas species

Additional file 1: Figure S1. Plasmid map for propane production in Halomonas. Figure S2. Tolerance of Halomonas isolates for salinity, pH and butyric acid. Figure S3. Growth of H. rowanensis in mineral-based media using polluted water with and without exogenous carbon sources. Figure S4. Superimposition of AlphaFold predicted structures of reverse TCA cycle proteins and the closest DALI homology match. Figure S5. Sulfur oxidation systems for energy generation. Figure S6. Ectoine production by Halomonas rowanensis in the thiosulfate minimal medium with a range of salinity. Table S1. Partial 16S rDNA sequence analysis of ‘Old Biot’ brine spring isolates. Table S2. Extended PHB assay data for Figs. 2C and . Table S3. Putative carbon fixation cycle genes identified within the genome of H. rowanensis by protein sequence homology and AlphaFold structural homology. Table S4. Putative sulfur metabolism genes identified within the genome of H. rowanensis by protein sequence homology and AlphaFold structural homology

Chemoenzymatic Synthesis of the Intermediates in the Peppermint Monoterpenoid Biosynthetic Pathway

A chemoenzymatic approach providing access to all four intermediates in the peppermint biosynthetic pathway between limonene and menthone/isomenthone, including noncommercially available intermediates (−)-<i>trans</i>-isopiperitenol (<b>2</b>), (−)-isopiperitenone (<b>3</b>), and (+)-<i>cis</i>-isopulegone (<b>4</b>), is described. Oxidation of (+)-isopulegol (<b>13</b>) followed by enolate selenation and oxidative elimination steps provides (−)-isopiperitenone (<b>3</b>). A chemical reduction and separation route from (<b>3</b>) provides both native (−)-<i>trans</i>-isopiperitenol (<b>2</b>) and isomer (−)-<i>cis</i>-isopiperitenol (<b>18</b>), while enzymatic conjugate reduction of (−)-isopiperitenone (<b>3</b>) with IPR [(−)-isopiperitenone reductase)] provides (+)-<i>cis</i>-isopulegone (<b>4</b>). This undergoes facile base-mediated chemical epimerization to (+)-pulegone (<b>5</b>), which is subsequently shown to be a substrate for <i>Nt</i>DBR (<i>Nicotiana tabacum</i> double-bond reductase) to afford (−)-menthone (<b>7</b>) and (+)-isomenthone (<b>8</b>)

Enzymatic Menthol Production: One-Pot Approach Using Engineered <i>Escherichia coli</i>

Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants, <i>Mentha</i> spp. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell. We have developed a one-pot (bio)­synthesis of (1<i>R</i>,2<i>S</i>,5<i>R</i>)-(−)-menthol and (1<i>S</i>,2<i>S</i>,5<i>R</i>)-(+)-neomenthol from pulegone, using recombinant Escherichia coli extracts containing the biosynthetic genes for an “ene”-reductase (NtDBR from Nicotiana tabacum) and two menthone dehydrogenases (MMR and MNMR from Mentha piperita). Our modular engineering strategy allowed each step to be optimized to improve the final production level. Moderate to highly pure menthol (79.1%) and neomenthol (89.9%) were obtained when E. coli strains coexpressed NtDBR with only MMR or MNMR, respectively. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds

Biocatalytic Routes to Lactone Monomers for Polymer Production

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer–Villiger monooxygenases (BVMOs) from <i>Pseudomonas</i> sp. HI-70 (CPDMO) and <i>Rhodococcus</i> sp. Phi1 (CHMO_Phi1) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMO_Phi1. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMO_Phi1 for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal–organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers

Biocatalytic Routes to Lactone Monomers for Polymer Production

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer–Villiger monooxygenases (BVMOs) from <i>Pseudomonas</i> sp. HI-70 (CPDMO) and <i>Rhodococcus</i> sp. Phi1 (CHMO_Phi1) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMO_Phi1. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMO_Phi1 for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal–organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers