Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source

Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction

We would like to thank C. Dilßner and M. Neytschev (UFZ) for excellent technical assistance with CoA thioester synthesis, strain cultivation and HPLC analyses. In addition, we thank Birgit Würz (UFZ) for invaluable analytical advice and help with GC mass spectrometry. We are also indebted to L. von Wintzingerode, A. Grunwald, and J. Grabengießer (UFZ) for assistance in the cultivation and enzyme assay experiments. Many thanks to K. Eismann (UFZ) as well, for help with the proteome analysis and fruitful discussions regarding different protein extraction methods.The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2020.00691/full#supplementary-materialThe tertiary branched short-chain 2-hydroxyisobutyric acid (2-HIBA) has been associated with several metabolic diseases and lysine 2-hydroxyisobutyrylation seems to be a common eukaryotic as well as prokaryotic post-translational modification in proteins. In contrast, the underlying 2-HIBA metabolism has thus far only been detected in a few microorganisms, such as the betaproteobacterium Aquincola tertiaricarbonis L108 and the Bacillus group bacterium Kyrpidia tusciae DSM 2912. In these strains, 2-HIBA can be specifically activated to the corresponding CoA thioester by the 2-HIBA-CoA ligase (HCL) and is then isomerized to 3-hydroxybutyryl-CoA in a reversible and B12-dependent mutase reaction. Here, we demonstrate that the actinobacterial strain Actinomycetospora chiangmaiensis DSM 45062 degrades 2-HIBA and also its precursor 2-methylpropane-1,2-diol via acetone and formic acid by employing a thiamine pyrophosphate-dependent lyase. The corresponding gene is located directly upstream of hcl, which has previously been found only in operonic association with the 2-hydroxyisobutyryl-CoA mutase genes in other bacteria. Heterologous expression of the lyase gene from DSM 45062 in E. coli established a 2-hydroxyisobutyryl-CoA lyase activity in the latter. In line with this, analysis of the DSM 45062 proteome reveals a strong induction of the lyase-HCL gene cluster on 2-HIBA. Acetone is likely degraded via hydroxylation to acetol catalyzed by a MimABCD-related binuclear iron monooxygenase and formic acid appears to be oxidized to CO2 by selenium-dependent dehydrogenases. The presence of the lyase-HCL gene cluster in isoprene-degrading Rhodococcus strains and Pseudonocardia associated with tropical leafcutter ant species points to a role in degradation of biogenic short-chain ketones and highly branched organic compounds.Program Topic "Chemicals in the Environment" within the Research Program "Terrestrial Environment" of the Helmholtz Association European Union (EU) 62485

Mechanistic details of the actinobacterial lyase-catalyzed degradation reaction of 2-hydroxyisobutyryl-CoA

Actinobacterial 2-hydroxyacyl-CoA lyase reversibly catalyzes the thiamine diphosphate-dependent cleavage of 2-hydroxyisobutyryl-CoA to formyl-CoA and acetone. This enzyme has great potential for use in synthetic one-carbon assimilation pathways for sustainable production of chemicals, but lacks details of substrate binding and reaction mechanism for biochemical reengineering. We determined crystal structures of the tetrameric enzyme in the closed conformation with bound substrate, covalent postcleavage intermediate, and products, shedding light on active site architecture and substrate interactions. Together with molecular dynamics simulations of the covalent precleavage complex, the complete catalytic cycle is structurally portrayed, revealing a proton transfer from the substrate acyl Cβ hydroxyl to residue E493 that returns it subsequently to the postcleavage Cα-carbanion intermediate. Kinetic parameters obtained for mutants E493A, E493Q, and E493K confirm the catalytic role of E493 in the WT enzyme. However, the 10- and 50-fold reduction in lyase activity in the E493A and E493Q mutants, respectively, compared with WT suggests that water molecules may contribute to proton transfer. The putative catalytic glutamate is located on a short α-helix close to the active site. This structural feature appears to be conserved in related lyases, such as human 2-hydroxyacylCoA lyase 2. Interestingly, a unique feature of the actinobacterial 2-hydroxyacyl-CoA lyase is a large C-terminal lid domain that, together with active site residues L127 and I492, restricts substrate size to ≤C5 2-hydroxyacyl residues. These details about the catalytic mechanism and determinants of substrate specificity pave the ground for designing tailored catalysts for acyloin condensations for one-carbon and shortchain substrates in biotechnological applications.Michael Zahn - Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United KingdomGerhard König - Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth, United KingdomHuy Viet Cuong Pham - Department of Environmental Microbiology, Helmholtz Centre for Environmental Research - UFZ, Leipzig, GermanyBarbara Seroka - Faculty of Chemistry, University of Bialystok, Bialystok, PolandRyszard Łaźny - Faculty of Chemistry, University of Bialystok, Bialystok, PolandGuangli Yang - Organic Synthesis Core Facility, Memorial Sloan Kettering Cancer Center (MSKCC), New York, New York, USAOuathek Ouerfelli - Organic Synthesis Core Facility, Memorial Sloan Kettering Cancer Center (MSKCC), New York, New York, USAZenon Łotowski - Faculty of Chemistry, University of Bialystok, Bialystok, PolandThore Rohwerder - Department of Environmental Microbiology, Helmholtz Centre for Environmental Research - UFZ, Leipzig, GermanyVogel, C., and Pleiss, J. 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Recently evolved combination of unique sulfatase and amidase genes enables bacterial degradation of the wastewater micropollutant acesulfame worldwide

Xenobiotics often challenge the principle of microbial infallibility. One example is acesulfame introduced in the 1980s as zero-calorie sweetener, which was recalcitrant in wastewater treatment plants until the early 2010s. Then, efficient removal has been reported with increasing frequency. By studying acesulfame metabolism in alphaproteobacterial degraders of the genera Bosea and Chelatococcus, we experimentally confirmed the previously postulated route of two subsequent hydrolysis steps via acetoacetamide-N-sulfonate (ANSA) to acetoacetate and sulfamate. Genome comparison of wildtype Bosea sp. 100-5 and an acesulfame degradation-defective mutant revealed the involvement of two plasmid-borne gene clusters. The acesulfame-hydrolyzing sulfatase is strictly manganese-dependent and belongs to the metallo beta-lactamase family. In all degraders analyzed, it is encoded on a highly conserved gene cluster embedded in a composite transposon. The ANSA amidase, on the other hand, is an amidase signature domain enzyme encoded in another gene cluster showing variable length among degrading strains. Transposition of the sulfatase gene cluster between chromosome and plasmid explains how the two catabolic gene clusters recently combined for the degradation of acesulfame. Searching available genomes and metagenomes for the two hydrolases and associated genes indicates that the acesulfame plasmid evolved and spread worldwide in short time. While the sulfatase is unprecedented and unique for acesulfame degraders, the amidase occurs in different genetic environments and likely evolved for the degradation of other substrates. Evolution of the acesulfame degradation pathway might have been supported by the presence of structurally related natural and anthropogenic compounds, such as aminoacyl sulfamate ribonucleotide or sulfonamide antibiotics

Enzyme activity of Aquincola tertiaricarbonis L108 acylating aldehyde dehydrogenase ATN38601 against various aldehyde and acyl-CoA substrates

<b>Method</b><br>Kinetic assays were performed using a photometer (Hitachi U-2000 Spectrophotometer, in SUPRASIL quartz cuvettes (path length 10 mm) at 35°C (or temperature as indicated). As assay buffer a solution of 50 mM HK2PO4/H2KPO4, 50 mM Tris/HCl, 1 mM DTT, 20% glycerol, pH 7.8 (or at pH as indicated) was used. For quantifying the oxidation (Ox) of aldehydes, each 400 µL reaction contained buffer, 2 mM NAD+ (or as indicated), 5 mM CoA (or as indicated), purified enzyme (heterologously expressed in <i>E. coli</i>, N-terminal 6xHis tag; wild type = WT, enzyme variants with A254I, A254P or A254G replacements) at concentrations as indicated and various concentrations of aldehydes. For quantifying the reduction (Red) of acyl-CoAs, each 400 µL reaction contained buffer, 0.625 mM NADH (or as indicated), purified enzyme (WT) at concentrations as indicated and various concentrations of acyl-CoAs. The enzyme activity was monitored by measuring the formation/consumption of NADH at 340 nm using an absorption coefficient of 6.22 mM-1 cm-1. Enzyme activity (U) is expressed as µmol NADH produced/consumed per minute. Steady state kinetic data, obtained with technical repeats, were analyzed by non-linear regression to the Michaelis-Menten (MM) equation using GraphPad Prism software.<br><br><b>Data</b><br>The following data sets are included in this collection:<br><br>Temperature and pH optima<br>Co-factor Km values<br>Kinetic Data for aldehydes and acyl-CoAs<br><br><b>Raw data processing method</b><br><br>The following method was used to process the data:<br><br>-Export raw data from photometer (see file Assay_Rawdata.txt and corresponding meta data file Assay_Metadata.txt)<br>-Analyze slope (Abs340nm/s) by linear regression<br>-Calculate enzyme activity in U/mg enzyme<br>-Calculate mean and SD for technical replicates<br>-Analyze by nonlinear regression (curve fit, MM). <br

Carbon Conversion Efficiency and Limits of Productive Bacterial Degradation of Methyl tert-Butyl Ether and Related Compounds

The utilization of the fuel oxygenate methyl tert-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the Y(ATP) concept. Experiments were conducted to derive realistic maintenance coefficients and K(s) values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g(−1), which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient m(s) and its associated biomass decay term b, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations, S(min), below which growth would not further be supported. S(min) strongly depended on the maximum growth rate μ(ma)(x), and b and was directly correlated with the half maximum rate-associated substrate concentration K(s), meaning that any effect impacting this parameter would also change S(min). The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase K(s) and S(min) for MTBE