Chain Length Of Substrate Research Articles

GH13 Glycogen branching enzymes can adapt the substrate chain length towards their preferences via α-1,4-transglycosylation

Glycogen branching enzymes (GBEs; 1,4-α-glucan branching enzyme; E.C. 2.4.1.18) have so far been described to be capable of both α-1,6-transglycosylation (branching) and α-1,4-hydrolytic activity. The aim of the present study was to elucidate the mode of action of three distantly related GBEs from the glycoside hydrolase family 13 by in depth analysis of the activity on a well-defined substrate. For this purpose, the GBEs from R. marinus (RmGBE), P. mobilis (PmGBE1), and B. fibrisolvens (BfGBE) were incubated with a highly pure fraction of a linear substrate of 18 anhydroglucose units. A well-known and characterized branching enzyme from E. coli (EcGBE) was also taken along. Analysis of the chain length distribution over time revealed that, next to hydrolytic and branching activity, all three GBEs were capable of generating chains longer than the substrate, clearly showing α-1,4-transglycosylation activity. Furthermore, the GBEs used those elongated chains for further branching. The sequential activity of elongation and branching enabled the GBEs to modify the substrate to a far larger extent than would have been possible with branching activity alone. Overall, the three GBEs acted ambiguous on the defined substrate. RmGBE appeared to have a strong preference towards transferring chains of nine anhydroglucose units, even during elongation, with a comparably low activity. BfGBE generated an array of elongated chains before using the chains for introducing branches while PmGBE1 exhibited a behaviour intermediate of the other two enzymes. On the basis of the mode of action revealed in this research, an updated model of the mechanism of GBEs was proposed now including the α-1,4-transglycosylation activity.

Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes.

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. The condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1-7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in β-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

PenA, a penicillin-binding protein-type thioesterase specialized for small peptide cyclization.

Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified group of peptide cyclases that catalyze head-to-tail macrolactamization of nonribosomal peptides. PenA, a new member of this group, is involved in the biosyntheses of cyclic pentapeptides. In this study, we demonstrated the enzymatic activity of PenA in vitro, and analyzed its substrate scope with a series of synthetic substrates. A comparison of the reaction profiles between PenA and SurE, a representative PBP-type TE, showed that PenA is more specialized for small peptide cyclization. A computational model provided a possible structural rationale for the altered specificity for substrate chain lengths.

Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFASs) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes that are homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KSc) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, provides a foundation for understanding the mechanism of substrate recognition and chain length control by the KS-CLF didomain as well as its interaction with a cognate acyl carrier protein partner.

Structure and Mechanism of the Ketosynthase-Chain Length Factor Didomain from a Prototypical Polyunsaturated Fatty Acid Synthase

Long-chain polyunsaturated fatty acids (LC-PUFA) are essential ingredients of the human diet. They are synthesized by LC-PUFA synthases (PFAS) expressed in marine bacteria and other organisms. PFASs are large enzyme complexes homologous to mammalian fatty acid synthases and microbial polyketide synthases. One subunit of each PFAS harbors consecutive ketosynthase (KS) and chain length factor (CLF) domains that collectively catalyze the elongation of a nascent fatty acyl chain via iterative carbon-carbon bond formation. We report the X-ray crystal structure of the KS-CLF didomain from a well-studied PFAS in Moritella marina. Our structure, in combination with biochemical analysis, has provided new insights into substrate recognition and chain length control by the KS-CLF as well as its interaction with a cognate acyl carrier protein partner.

Electrochemical investigation on adsorption kinetics of long chain alkylsilanes and influence of solvents on their self-assembly and electron transfer behavior on indium tin oxide

In situ adsorption kinetics of hexadecyltrimethoxy silane (HDTMS) and octadecyltrimethoxy silane (ODTMS) on indium tin oxide (ITO) surface at short time intervals were measured by electrochemical impedance (EIS) spectroscopy technique at constant frequency. The studies suggest that, self-assembly of silanes on ITO proceeds through two distinct adsorption steps, a fast first step followed by a slower second step. The inhibition of electron transfer by self-assembled silanes prepared in ethanol, toluene and hexane solvents were evaluated by cyclic voltammetry (CV) and EIS using [Fe(CN)6]3−/4− and [Ru(NH3)6]3+/2+ redox species. It is shown that, the quality of silane films are influenced by the polarity of solvent, chain length of silane and ITO substrate–solvent interactions. The effect of annealing on electron transfer barrier property of silane monolayers after their formation were studied. The pinhole characteristics of silane films on ITO prepared from various solvents under study were measured using EIS. This study demonstrates a strategy of producing an ultra-smooth and close-packed silane films on ITO surfaces which can have potential applications towards high performance organic electronics.

Production of multiple terpenes of different chain lengths by subcellular targeting of multi-substrate terpene synthase in plants

Multi-substrate terpene synthases (TPSs) are distinct from typical TPSs that react with a single substrate. Although in vitro activity of few multi-substrate TPSs have been reported, in vivo characterization has not been well investigated for most of them. Here, a new TPS from Cananga odorata, CoTPS5, belonging to TPS-f subfamily was functionally characterized in vitro as well as in vivo. CoTPS5 reacted with multiple prenyl-pyrophosphate substrates of various chain lengths as a multi-substrate TPS. It catalyzed the formation of (E)-β-ocimene, (E,E)-α-farnesene and α-springene from geranyl pyrophosphate, (E,E)-farnesyl pyrophosphate and geranylgeranyl pyrophosphate, respectively. Upon transient expression in Nicotiana benthamiana, CoTPS5 localized to cytosol and produced only (E,E)-α-farnesene. However, expression of plastid-targeted CoTPS5 in N. benthamiana resulted in biosynthesis of all three compounds, (E)-β-ocimene, (E,E)-α-farnesene and α-springene. Similarly, transgenic Arabidopsis plants overexpressing plastid-targeted CoTPS5 showed stable and sustainable production of (E)-β-ocimene, (E,E)-α-farnesene and α-springene. Moreover, their production did not affect the growth and development of transgenic Arabidopsis plants. Our results demonstrate that redirecting multi-substrate TPS to a different intracellular compartment could be an effective way to prove in vivo activity of multi-substrate TPSs and thereby allowing for the production of multiple terpenoids simultaneously in plants.

Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

Unexplored lipolytic activity of Escherichia coli: Implications for lipase cloning

Recent investigations on cloned bacterial lipases performed in our laboratory revealed the presence of lipolytic activity that was not due to the cloned lipase-coding gene but was probably the result of an intrinsic activity of Escherichia coli itself. To confirm such a hypothesis, we assayed the activity of frequently used E. coli strains by fast paper tests, zymograms and spectrofluorometry. A band of Ca. 18−20 kDa showing activity on MUF-butyrate was detected in zymogram analysis of crude cell extracts in all E. coli strains assayed. Moreover, the spectrofluorometric results obtained confirmed the presence of low but significant lipolytic activity in E. coli, with strain BL21 showing the highest activity. Detailed characterization of such a lipolytic activity was performed using E. coli BL21 cell extracts, where preference for C7 substrates was found, although shorter substrates were also hydrolysed to a minor extent. Interestingly, E. coli lipolytic activity displays traits of a thermophilic enzyme, showing maximum activity at 50 °C and pH 8, an unexpected feature never described before. Kinetic and inhibition analysis were also performed showing that activity can be inhibited by several metal ions or by Triton X-100® and SDS, used in zymogram analysis. Such properties ‒ low activity, preference for medium chain-length substrates, and high operational temperature ‒ might justify why this activity had gone unexplored until now, even when many lipases and esterases have been cloned and expressed in E. coli strains in the past. From now on, lipase researchers should take into consideration the presence of such a basal lipolytic activity before starting their lipase cloning or expression experiments in E.coli.

Resolving Free-Energy Contributions of Substrate Delivery to Desaturase by Atomistic Simulations

The plant acyl-acyl carrier protein (ACP) desaturases are a family of soluble enzymes that convert saturated fatty-acyl ACPs into their cis-monounsaturated equivalents in an oxygen-dependent reaction. These enzymes play a key role in biosynthesis of mono-unsaturated fatty acids in plants. The archetype of this group is the ▵9-18:0-ACP desaturase that introduces a cis double bond between carbons 9 and 10 to produce oleoyl-ACP. ACPs are central proteins in the fatty acid biosynthesis that deliver the substrate to the desaturase. They have been reported to show a varying degree of local dynamics and structural variability depending on the substrate length. It has been suggested that the substrate specific change in structure and dynamics is crucial for the decreasing enzymatic activity in the case of the ▵9-desaturase for chains length shorter than 18. We investigated the solution structure and dynamics of ACP with four different substrate lengths (10, 14, 16, and 18) based on published NMR solution structures using extensive all atom molecular dynamics simulations. Umbrella sampling and replica exchange dynamics simulations highlight the chain length depend free energy of substrate binding and chain length depended binding motifs. This suggests that longer substrates have a lower binding energy, but also a much larger ensemble of structures that are accessible. Thereby estimating the effect on changes in the binding affinity of the substrate bound Acyl carrier protein to the ▵9-desaturase will not be able without taking into account the Enzyme itself.

Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering.

Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7-10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.

The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL.

Quorum quenching lactonases are enzymes capable of hydrolyzing lactones, including N-acyl homoserine lactones (AHLs). AHLs are molecules known as signals in bacterial communication dubbed quorum sensing. Bacterial signal disruption by lactonases was previously reported to inhibit behavior regulated by quorum sensing, such as the expression of virulence factors and the formation of biofilms. Herein, we report the enzymatic and structural characterization of a novel lactonase representative from the metallo-β-lactamase superfamily, dubbed GcL. GcL is a broad spectrum and highly proficient lactonase, with kcat /KM values in the range of 104 to 106 m-1 s-1 . Analysis of free GcL structures and in complex with AHL substrates of different acyl chain length, namely, C4-AHL and 3-oxo-C12-AHL, allowed their respective binding modes to be elucidated. Structures reveal three subsites in the binding crevice: 1) the small subsite where chemistry is performed on the lactone ring; 2) a hydrophobic ring that accommodates the amide group of AHLs and small acyl chains; and 3) the outer, hydrophilic subsite that extends to the protein surface. Unexpectedly, the absence of structural accommodation for long substrate acyl chains seems to relate to the broad substrate specificity of the enzyme.

Bioinformatics analysis of diversity in bacterial glycan chain-termination chemistry and organization of carbohydrate-binding modules linked to ABC transporters.

The structures of bacterial cell surface glycans are remarkably diverse. In spite of this diversity, the general strategies used for their assembly are limited. In one of the major processes, found in both Gram-positive and Gram-negative bacteria, the glycan is polymerized in the cytoplasm on a polyprenol lipid carrier and exported from the cytoplasm by an ATP-binding cassette (ABC) transporter. The ABC transporter actively participates in determining the chain length of the glycan substrate, which impacts functional properties of the glycoconjugate products. A subset of these systems employs an additional elaborate glycan capping strategy that dictates the size distribution of the products. The hallmarks of prototypical capped glycan systems are a chain-terminating enzyme possessing a coiled-coil molecular ruler and an ABC transporter possessing a carbohydrate-binding module, which recognizes the glycan cap. To date, detailed investigations are limited to a small number of prototypes, and here, we used our current understanding of these processes for a bioinformatics census of other examples in available genome sequences. This study not only revealed additional instances of existing terminators but also predicted new chemistries as well as systems that diverge from the established prototypes. These analyses enable some new functional hypotheses and offer a roadmap for future research.

Hydrocarbon-contaminated Antarctic soil: changes in bacterial community structure during the progress of enrichment cultures with different n-alkanes as substrate

Hydrocarbon contamination in soils from extremely cold areas, such as those from Antarctica, requires the development of specific remediation strategies for cleaning up anthropogenic pollution. Previous reports evidenced that after on-site biostimulation process of gasoil-contaminated Antarctic soils, 20% of the initial hydrocarbons remained undegraded (mainly C11–C14 n-alkanes). In the present work, these n-alkanes were added as sole carbon and energy source to enrichment cultures inoculated with the previously treated soil (biostimulation) as microorganism’s source to investigate changes occurring in the bacterial community structure. Three subcultures (8, 16, and 24 days) were performed from each enrichment culture. Changes in bacterial communities among different cultures and its subcultures were evidenced by Denaturing Gradient Gel Electrophoresis (DGGE). Results showed that even differences of one C in the alkane chain-length led to different community structures that evolved divergently from the original one. Clusters analysis showed that while samples grouped mainly by culture time, substrate-dependent differences were also evident. Isolation of biological tools for bioremediation from the cultures showed that Pseudomonadaceae members were omnipresent, whereas Rhodococcus spp. were obtained in cultures with the longest chain-length substrates. Results provided evidence about the presence of certain substrate preference of soil bacteria (even when substrates differed only in one C-atom of their chain-length), leading to different community structures. A collection of psychrotolerant hydrocarbon degrading/tolerant strains was obtained, representing a valuable tool for the design of a bioaugmentation strategy as a second, more specific stage, targeting the remnant hydrocarbons after a first bioremediation process involving biostimulation.

Shift in Bacillus sp. JR3 esterase LipJ activity profile after addition of essential residues from family I.5 thermophilic lipases

We recently described LipJ, a mesophilic esterase clustering in a specific clade of bacterial lipase family I.5, bearing the same structure but several distinct sequence motifs. Two mutagenesis approaches were used here to modify LipJ by addition of common features of family 1.5 lipases, searching for changes on the activity profile. Iterative saturation mutagenesis of a residue possibly involved in Zn2+ coordination produced a LipJ variant –H110N– displaying a remarkable shift in substrate specificity, possibly boosted by changes in number and distance of relevant polar interactions for Zn2+ coordination. Also, variants E27 A, G136 A and M139Q were obtained by site directed mutagenesis as single, double or triple mutants, showing a general shift in specificity towards higher temperature and longer chain-length substrates, all of them displaying higher activity than LipJ on C7 substrates. The results obtained suggest that the N-terminal region of LipJ may indeed contribute to substrate accommodation, whereas mutations at the pentapeptide displayed maximum activity at 50 °C, with the triple mutant showing activity even at 80 °C, greatly enhanced by Mn2+. 3D modelling and molecular docking analysis of selected mutants provided clues for a better accommodation of medium chain-length substrates, and for a modified Zn2+ environment, in agreement with the substrate and temperature profile shifts observed.

Increasing Uniformity of Biosurfactant Production in Starmerella bombicola via the Expression of Chimeric Cytochrome P450s

Sophorolipids are one of the best known microbial biosurfactants and are produced by several yeast species. The best studied producer is Starmerella bombicola, a non-pathogenic yeast associated in nature with bumblebees. Sophorolipids are built up of the rare disaccharide sophorose, which is attached to a fatty acid through a glyosidic bound. Sophorolipids produced by S. bombicola mainly contain oleic acid as the incorporated hydrophobic group. Other chain lengths can, to a certain content, be incorporated by feeding the yeast with substrates of alternative chain lengths. However, the efficiency for such substrates is low as compared to the preferred C18 chain length and defined by the substrate specificity of the first enzymatic step in sophorolipid biosynthesis, i.e., the cytochrome P450 enzyme CYP52M1. To increase product uniformity and diversity at the same time, a new strain of S. bombicola was developed that produces sophorolipids with a palmitic acid acyl chain. This was achieved by heterologous expression of the cytochrome P450 cyp1 gene of Ustilago maydis and feeding with palmitic acid. Optimization of the production was done by protein and process engineering.

Molecular mechanism of substrate preference for ω-3 fatty acid desaturase from Mortierella alpina by mutational analysis and molecular docking

The ω-3 fatty acid desaturase (ω3Des) is a key enzyme in the biosynthesis of polyunsaturated fatty acids (PUFAs). However, the enzyme exhibits a significant preference towards different fatty acid substrates. To examine the molecular mechanism of its substrate specificity, a series of site-directed mutants were constructed based on the membrane topology model and functionally characterised by heterologous expression in Saccharomyces cerevisiae. Our results revealed that the W106F and V137T mutations markedly decreased the enzyme activity which indicated that these two residues were associated with substrate recognition. In contrast, the A44S, M156I and W291M mutations showed significant increments (30 to 40%) of the conversion rate for AA substrate desaturation, which suggests that these residues play a pivotal role in desaturation of longer chain-length substrates. Through homology modelling of 3-dimensional structures and molecular docking of FADS15, we propose that the critical residues that bind to the CoA groups may affect substrate localisation and govern substrate preference and chain-length specificity. Our work increases the understanding of the structure-function relationships of the microbial membrane-bound desaturases. The growing knowledge of the molecular mechanism will also aid in the efficient production of value-added fatty acids.

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

Vibriocholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of β-ketoacyl-acyl carrier protein synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate-binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V.cholerae. Additionally, the distinctive substrate preference of FabH2 in V.cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.

Characterization of Carboxylic Acid Reductases for Biocatalytic Synthesis of Industrial Chemicals.

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids into aldehydes, which can serve as common biosynthetic precursors to many industrial chemicals. This work presents the systematic biochemical characterization of five carboxylic acid reductases from different microorganisms, including two known and three new ones, by using a panel of short-chain dicarboxylic acids and hydroxy acids, which are common cellular metabolites. All enzymes displayed broad substrate specificities. Higher catalytic efficiencies were observed when the carbon chain length, either of the dicarboxylates or of the terminal hydroxy acids, was increased from C2 to C6 . In addition, when substrates of the same carbon chain length are compared, carboxylic acid reductases favor hydroxy acids over dicarboxylates as their substrates. Whole-cell bioconversions of eleven carboxylic acid substrates into the corresponding alcohols were investigated by coupling the CAR activity with that of an aldehyde reductase in Escherichia coli hosts. Alcohol products were obtained in yields ranging from 0.5 % to 71 %. The de novo stereospecific biosynthesis of propane-1,2-diol enantiomer was successfully demonstrated with use of CARs as the key pathway enzymes. E. coli strains accumulated 7.0 mm (R)-1,2-PDO (1.0 % yield) or 9.6 mm (S)-1,2-PDO (1.4 % yield) from glucose. This study consolidates carboxylic acid reductases as promising enzymes for sustainable synthesis of industrial chemicals.

Exploring Lactobacillus reuteri DSM20016 as a biocatalyst for transformation of longer chain 1,2-diols: Limits with microcompartment.

Lactobacillus reuteri metabolises glycerol efficiently to form 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3PDO) by the same mechanism as that for 1,2-propanediol (1,2PDO) conversion to propionic acid and propanol via its propanediol utilization (pdu) pathway. Pdu enzymes are encoded by the pdu-operon, which also contain genes encoding the shell proteins of the microcompartment housing the metabolic pathway. In this work the selectivity and kinetics of the reactions catalysed by L. reuteri DSM20016 Pdu enzymes glycerol dehydratase (GDH), 1,3-propanediol oxidoreductase (PduQ) and coenzyme-A acylating propionaldehyde dehydrogenase (PduP), produced recombinantly, was investigated against corresponding substrates of different chain lengths. Glycerol dehydratase exhibited activity against C2-C4 polyols, with the highest activity against glycerol and 1,2-propanediol (1,2-PDO). A double mutant of the pduC gene of GDH (PduC-S302A/Q337A) was constructed that displayed lowered activity against glycerol and 1,2PDO but extended the substrate range upto C6-diol. The best substrate for both PduQ and PduP was 3-hydroxypropanal (3HPA), although PduP exhibited nearly 10-fold higher specific activity. The enzymes also showed some activity against C3-C10 aliphatic aldehydes, with PduP having higher relative activity. Subsequently, transformation of polyols using whole cells of L. reuteri containing the wild type- and mutated GDH, respectively, confirmed the reduced activity of the mutant against glycerol and 1,2PDO, but its activity against longer substrates was negligible. In contrast, recombinant Escherichia coli BL21(DE3) cells harboring the GDH variant converted diols with up to C6 carbon chain length to their respective aldehydes, suggesting that the protein shell of the microcompartment in L. reuteri posed a barrier to the passage of longer chain substrate.