Thermotoga Maritima Research Articles

Biohydrogen Production and Quantitative Determination of Monosaccharide Production Using Hyperthermophilic Anaerobic Fermentation of Corn Stover

Second-generation biofuels from lignocellulosic biomass remain critical and require several challenges due to lignin compounds’ inefficient degradation and recalcitrate characteristics. In this regard, this study focuses on enzymatic technology as a promising treatment that is beneficial in breaking down the biomass’s hemicellulose and cellulosic parts. Thermostable bacterial species owe thermostable enzymes that are able to degrade complex carbohydrate compounds and produce efficient hydrogen production. The present study investigates the direct utilization of ligninolytic enzymes such as cellulase and xylanase derived from the hyperthermophilic bacteria Thermotoga maritima (ATCC 43589 strain). The results show that xylanase and cellulase enzymes extracted from Thermotoga maritima could depolymerize the lignin bonds of corn stover substrate and release monomers such as Galactose in the media. In conclusion, this study can open a new advanced research window on directly applying a hyperthermophilic consortium of enzymes capable of hydrolyzing lignocellulose material toward hydrogen production.

Structure, function and substrate preferences of archaeal S-adenosyl-l-homocysteine hydrolases

S-Adenosyl-l-homocysteine hydrolase (SAHH) reversibly cleaves S-adenosyl-l-homocysteine, the product of S-adenosyl-l-methionine-dependent methylation reactions. The conversion of S-adenosyl-l-homocysteine into adenosine and l-homocysteine plays an important role in the regulation of the methyl cycle. An alternative metabolic route for S-adenosyl-l-methionine regeneration in the extremophiles Methanocaldococcus jannaschii and Thermotoga maritima has been identified, featuring the deamination of S-adenosyl-l-homocysteine to S-inosyl-l-homocysteine. Herein, we report the structural characterisation of different archaeal SAHHs together with a biochemical analysis of various SAHHs from all three domains of life. Homologues deriving from the Euryarchaeota phylum show a higher conversion rate with S-inosyl-l-homocysteine compared to S-adenosyl-l-homocysteine. Crystal structures of SAHH originating from Pyrococcus furiosus in complex with SlH and inosine as ligands, show architectural flexibility in the active site and offer deeper insights into the binding mode of hypoxanthine-containing substrates. Altogether, the findings of our study support the understanding of an alternative metabolic route for S-adenosyl-l-methionine and offer insights into the evolutionary progression and diversification of SAHHs involved in methyl and purine salvage pathways.

Multifunctional enzymes related to amino acid metabolism in bacteria.

In bacteria, D-amino acids are primarily synthesized from L-amino acids by amino acid racemases, but some bacteria use D-amino acid aminotransferases to synthesize D-amino acids. D-Amino acids are peptidoglycan components in the cell wall involved in several physiological processes, such as bacterial growth, biofilm dispersal, and peptidoglycan metabolism. Therefore, their metabolism and physiological roles have attracted increasing attention. Recently, we identified novel bacterial D-amino acid metabolic pathways, which involve amino acid racemases, with broad substrate specificity, as well as multifunctional enzymes with D-amino acid-metabolizing activity. Here, I review these multifunctional enzymes and their related D- and L-amino acid metabolic pathways in Escherichia coli and the hyperthermophile Thermotoga maritima.

Biocatalytic Performance of β-Glucosidase Immobilized on 3D-Printed Single- and Multi-Channel Polylactic Acid Microreactors.

Microfluidic devices have attracted much attention in the current day owing to the unique advantages they provide. However, their application for industrial use is limited due to manufacturing limitations and high cost. Moreover, the scaling-up process of the microreactor has proven to be difficult. Three-dimensional (3D) printing technology is a promising solution for the above obstacles due to its ability to fabricate complex structures quickly and at a relatively low cost. Hence, combining the advantages of the microscale with 3D printing technology could enhance the applicability of microfluidic devices in the industrial sector. In the present work, a 3D-printed single-channel immobilized enzyme microreactor with a volume capacity of 30 μL was designed and created in one step via the fused deposition modeling (FDM) printing technique, using polylactic acid (PLA) as the printing material. The microreactor underwent surface modification with chitosan, and β-glucosidase from Thermotoga maritima was covalently immobilized. The immobilized biocatalyst retained almost 100% of its initial activity after incubation at different temperatures, while it could be effectively reused for up to 10 successful reaction cycles. Moreover, a multi-channel parallel microreactor incorporating 36 channels was developed, resulting in a significant increase in enzymatic productivity.

Kinetic and inhibition investigations of TM1785: An acetylornithine aminotransferase from Thermotoga maritima

Kinetic and inhibition investigations of TM1785: An acetylornithine aminotransferase from Thermotoga maritima

Uridine Bisphosphonates Differentiate Phosphoglycosyl Transferase Superfamilies.

Complex bacterial glycoconjugates drive interactions between pathogens, symbionts, and their human hosts. Glycoconjugate biosynthesis is initiated at the membrane interface by phosphoglycosyl transferases (PGTs), which catalyze the transfer of a phosphosugar from a soluble uridine diphosphosugar (UDP-sugar) substrate to a membrane-bound polyprenol-phosphate (Pren-P). The two distinct superfamilies of PGT enzymes (polytopic and monotopic) show striking differences in their structure and mechanism. We designed and synthesized a series of uridine bisphosphonates (UBPs), wherein the diphosphate of the UDP and UDP-sugar is replaced by a substituted methylene bisphosphonate (CXY-BPs; X/Y = F/F, Cl/Cl, (S)-H/F, (R)-H/F, H/H, CH3/CH3). UBPs and UBPs incorporating an N-acetylglucosamine (GlcNAc) substituent at the β-phosphonate were evaluated as inhibitors of a polytopic PGT (WecA from Thermotoga maritima) and a monotopic PGT (PglC from Campylobacter jejuni). Although CHF-BP most closely mimics diphosphate with respect to its acid/base properties, the less basic CF2-BP conjugate more strongly inhibited PglC, whereas the more basic CH2-BP analogue was the strongest inhibitor of WecA. These surprising differences indicate different modes of ligand binding for the different PGT superfamilies, implicating a modified P-O- interaction with the structural Mg2+. For the monoPGT enzyme, the two diastereomeric CHF-BP conjugates, which feature a chiral center at the Pα-CHF-Pβ carbon, also exhibited strikingly different binding affinities and the inclusion of GlcNAc with the native α-anomer configuration significantly improved binding affinity. UBP-sugars are thus revealed as informative new mechanistic probes of PGTs that may aid development of novel antibiotic agents for the exclusively prokaryotic monoPGT superfamily.

Structure-based insights into the mechanism of [4Fe-4S]-dependent sulfur insertase LarE.

Several essential cellular metabolites, such as enzyme cofactors, contain sulfur atoms and their biosynthesis requires specific thiolation enzymes. LarE is an ATP-dependent sulfur insertase, which catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two types of LarE enzymes are known, one that uses a catalytic cysteine as a sacrificial sulfur donor, and the other one that uses a [4Fe-4S] cluster as a cofactor. Only the crystal structure of LarE from Lactobacillus plantarum (LpLarE) from the first class has been solved. We report here the crystal structure of LarE from Methanococcus maripaludis (MmLarE), belonging to the second class, in the cluster-free (apo-) and cluster-bound (holo-) forms. The structure of holo-MmLarE shows that the [4Fe-4S] cluster is chelated by three cysteines only, leaving an open coordination site on one Fe atom. Moreover, the fourth nonprotein-bonded iron atom was able to bind an anionic ligand such as a phosphate group or a chloride ion. Together with the spectroscopic analysis of holo-MmLarE and the previously reported biochemical investigations of holo-LarE from Thermotoga maritima, these crystal structures support the hypothesis of a reaction mechanism, in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand in place of the chloride anion, thus generating a [4Fe-5S] intermediate, and transfers it to the substrate, as in the case of [4Fe-4S]-dependent tRNA thiolation enzymes.

Correlating Conformational Equilibria with Catalysis in the Electron Bifurcating EtfABCX of Thermotoga maritima.

Electron bifurcation (BF) is an evolutionarily ancient energy coupling mechanism in anaerobes, whose associated enzymatic machinery remains enigmatic. In BF-flavoenzymes, a chemically high-potential electron forms in a thermodynamically favorable fashion by simultaneously dropping the potential of a second electron before its donation to physiological acceptors. The cryo-EM and spectroscopic analyses of the BF-enzyme Fix/EtfABCX from Thermotoga maritima suggest that the BF-site contains a special flavin-adenine dinucleotide and, upon its reduction with NADH, a low-potential electron transfers to ferredoxin and a high-potential electron reduces menaquinone. The transfer of energy from high-energy intermediates must be carefully orchestrated conformationally to avoid equilibration. Herein, anaerobic size exclusion-coupled small-angle X-ray scattering (SEC-SAXS) shows that the Fix/EtfAB heterodimer subcomplex, which houses BF- and electron transfer (ET)-flavins, exists in a conformational equilibrium of compacted and extended states between flavin-binding domains, the abundance of which is impacted by reduction and NAD(H) binding. The conformations identify dynamics associated with the T. maritima enzyme and also recapitulate states identified in static structures of homologous BF-flavoenzymes. Reduction of Fix/EtfABCX's flavins alone is insufficient to elicit domain movements conducive to ET but requires a structural "trigger" induced by NAD(H) binding. Models show that Fix/EtfABCX's superdimer exists in a combination of states with respect to its BF-subcomplexes, suggesting a cooperative mechanism between supermonomers for optimizing catalysis. The correlation of conformational states with pathway steps suggests a structural means with which Fix/EtfABCX may progress through its catalytic cycle. Collectively, these observations provide a structural framework for tracing Fix/EtfABCX's catalysis.

In Vivo Behavior of Systemically Administered Encapsulin Protein Nanocages and Implications for their use in Targeted Drug Delivery

AbstractEncapsulins, self‐assembling protein nanocages derived from prokaryotes, are promising nanoparticle‐based drug delivery systems (NDDS). However, the in vivo behavior and fate of encapsulins are poorly understood. In this study, the interactions between the model encapsulin from Thermotoga maritima (TmEnc) and key biological barriers encountered by NDDS are probed. Here, a purified TmEnc formulation that exhibits colloidal stability, storability, and blood compatibility is intravenously injected into BALB/c mice. TmEnc has an excellent nanosafety profile, with no abnormal weight loss or gross pathology observed, and only temporary alterations in toxicity biomarkers are detected. Notably, TmEnc demonstrates immunogenic properties, inducing the generation of nanocage‐specific IgM and IgG antibodies, but without any prolonged pro‐inflammatory effects. An absence of antibody cross‐reactivity also suggests immune‐orthogonality among encapsulin systems. Moreover, TmEnc forms a serum‐derived protein corona on its surface which changes dynamically and appears to play a role in immune recognition. TmEnc's biodistribution profile further reveals its sequestration from the blood circulation by the liver and then biodegrades within Kupffer cells, thus indicating clearance via the mononuclear phagocyte system. Collectively, these findings provide critical insights into how encapsulins behave in vivo, thereby informing their future design, modification, and application in targeted drug delivery.

Connected Component Analysis of Dynamical Perturbation Contact Networks.

Describing protein dynamical networks through amino acid contacts is a powerful way to analyze complex biomolecular systems. However, due to the size of the systems, identifying the relevant features of protein-weighted graphs can be a difficult task. To address this issue, we present the connected component analysis (CCA) approach that allows for fast, robust, and unbiased analysis of dynamical perturbation contact networks (DPCNs). We first illustrate the CCA method as applied to a prototypical allosteric enzyme, the imidazoleglycerol phosphate synthase (IGPS) enzyme from Thermotoga maritima bacteria. This approach was shown to outperform the clustering methods applied to DPCNs, which could not capture the propagation of the allosteric signal within the protein graph. On the other hand, CCA reduced the DPCN size, providing connected components that nicely describe the allosteric propagation of the signal from the effector to the active sites of the protein. By applying the CCA to the IGPS enzyme in different conditions, i.e., at high temperature and from another organism (yeast IGPS), and to a different enzyme, i.e., a protein kinase, we demonstrated how CCA of DPCNs is an effective and transferable tool that facilitates the analysis of protein-weighted networks.

Biosynthesis of Lacto-N-biose I from starch and N-acetylglucosamine via an in vitro synthetic enzymatic biosystem

Human milk oligosaccharides (HMOs) are very distinctive components in human milk and are beneficial for infant health. Lacto-N-biose I (LNB) is the core structural unit of HMOs, which could be used for the synthesis of other HMOs. In this study, an ATP-free in vitro synthetic enzymatic biosystem contained four thermostable enzymes (alpha-glucan phosphorylase from Thermococcus kodakarensis, UDP-glucose-hexose-1-phosphate uridylyltransferase from Thermotoga maritima, UDP-glucose 4-epimerase from T. maritima, lacto-N-biose phosphorylase from Clostridium thermobutyricum) were constructed for the biosynthesis of LNB from starch and N-acetylglucosamine (GlcNAc). Under the optimal conditions, 0.75 g/L and 2.23 g/L LNB was produced from 1.1 g/L and 4.4 g/L GlcNAc and excess starch, with the molar yield of 39% and 29% based on the GlcNAc concentration, respectively, confirming the feasibility of this in vitro synthetic enzymatic biosystem for LNB synthesis and shedding light on the biosynthesis of other HMOs using LNB as the core structural unit from low cost polysaccharides.

The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima.

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

Alpha-1,4-transglycosylation Activity of GH57 Glycogen Branching Enzymes Is Higher in the Absence of a Flexible Loop with a Conserved Tyrosine Residue.

Starch-like polymers can be created through the use of enzymatic modification with glycogen branching enzymes (GBEs). GBEs are categorized in the glycoside hydrolase (GH) family 13 and 57. Both GH13 and GH57 GBEs exhibit branching and hydrolytic activity. While GH13 GBEs are also capable of α-1,4-transglycosylation, it is yet unknown whether GH57 share this capability. Among the four crystal structures of GH57 GBEs that have been solved, a flexible loop with a conserved tyrosine was identified to play a role in the branching activity. However, it remains unclear whether this flexible loop is also involved in α-1,4-transglycosylation activity. We hypothesize that GH57 GBEs with the flexible loop and tyrosine are also capable of α-1,4-transglycosylation, similar to GH13 GBEs. The aim of the present study was to characterize the activity of GH57 GBEs to investigate a possible α-1,4-transglycosylation activity. Three GH57 GBEs were selected, one from Thermococcus kodakarensis with the flexible loop and two beta-strands; one from Thermotoga maritima, missing the flexible loop and beta-strands; and one from Meiothermus sp., missing the flexible loop but with the two beta-strands. The analysis of chain length distribution over time of modified maltooctadecaose, revealed, for the first time, that all three GH57 GBEs can generate chains longer than the substrate itself, showing that α-1,4-transglycosylation activity is generally present in GH57 GBEs.

Structure of reverse gyrase with a minimal latch that supports ATP-dependent positive supercoiling without specific interactions with the topoisomerase domain.

Reverse gyrase is the only topoisomerase that introduces positive supercoils into DNA in an ATP-dependent reaction. Positive DNA supercoiling becomes possible through the functional cooperation of the N-terminal helicase domain of reverse gyrase with its C-terminal type IA topoisomerase domain. This cooperation is mediated by a reverse-gyrase-specific insertion into the helicase domain termed the `latch'. The latch consists of a globular domain inserted at the top of a β-bulge loop that connects this globular part to the helicase domain. While the globular domain shows little conservation in sequence and length andis dispensable for DNA supercoiling, the β-bulge loop is required for supercoiling activity. It has previously been shown that the β-bulge loop constitutes a minimal latch that couples ATP-dependent processes in the helicase domain to DNA processing by the topoisomerase domain. Here, the crystal structure of Thermotoga maritima reverse gyrase with such a β-bulge loop as a minimal latch is reported. It is shown that the β-bulge loop supports ATP-dependent DNA supercoiling of reverse gyrase without engaging in specific interactions with the topoisomerase domain. When only a small latch or no latch is present, a helix in the nearby helicase domain of T. maritima reverse gyrase partially unfolds. Comparison of the sequences and predicted structures of latch regions in other reverse gyrases shows that neither sequence nor structure are decisive factors for latch functionality; instead, the decisive factors are likely to be electrostatics and plain steric bulk.

Single-stranded DNA recruitment mechanism in replication origin unwinding by DnaA initiator protein and HU, an evolutionary ubiquitous nucleoid protein.

The Escherichia coli replication origin oriC contains the initiator ATP-DnaA-Oligomerization Region (DOR) and its flanking duplex unwinding element (DUE). In the Left-DOR subregion, ATP-DnaA forms a pentamer by binding to R1, R5Mand three other DnaA boxes. The DNA-bending protein IHF binds sequence-specifically to the interspace between R1 and R5M boxes, promoting DUE unwinding, which is sustained predominantly by binding of R1/R5M-bound DnaAs to the single-stranded DUE (ssDUE). The present study describes DUE unwinding mechanisms promoted by DnaA and IHF-structural homolog HU, a ubiquitous protein in eubacterial species that binds DNA sequence-non-specifically, preferring bent DNA. Similar to IHF, HU promoted DUE unwinding dependent on ssDUE binding of R1/R5M-bound DnaAs. Unlike IHF, HU strictly required R1/R5M-bound DnaAs and interactions between the two DnaAs. Notably, HU site-specifically bound the R1-R5M interspace in a manner stimulated by ATP-DnaA and ssDUE. These findings suggest a model that interactions between the two DnaAs trigger DNA bending within the R1/R5M-interspace and initial DUE unwinding, which promotes site-specific HU binding that stabilizes the overall complex and DUE unwinding. Moreover, HU site-specifically bound the replication origin of the ancestral bacterium Thermotoga maritima depending on the cognate ATP-DnaA. The ssDUE recruitment mechanism could be evolutionarily conserved in eubacteria.

The cell envelope of Thermotogae suggests a mechanism for outer membrane biogenesis

The presence of a cell membrane is one of the major structural components defining life. Recent phylogenomic analyses have supported the hypothesis that the last universal common ancestor (LUCA) was likely a diderm. Yet, the mechanisms that guided outer membrane (OM) biogenesis remain unknown. Thermotogae is an early-branching phylum with a unique OM, the toga. Here, we use cryo-electron tomography to characterize the insitu cell envelope architecture of Thermotoga maritima and show that the toga is made of extended sheaths of β-barrel trimers supporting small (~200nm) membrane patches. Lipidomic analyses identified the same major lipid species in the inner membrane (IM) and toga, including the rare to bacteria membrane-spanning ether-bound diabolic acids (DAs). Proteomic analyses revealed that the toga was composed of multiple SLH-domain containing Ompα and novel β-barrel proteins, and homology searches detected variable conservations of these proteins across the phylum. These results highlight that, in contrast to the SlpA/OmpM superfamily of proteins, Thermotoga possess a highly diverse bipartite OM-tethering system. We discuss the implications of our findings with respect to other early-branching phyla and propose that a toga-like intermediate may have facilitated monoderm-to-diderm cell envelope transitions.

Screening the Thermotoga maritima genome for new wide-spectrum nucleoside and nucleotide kinases

Enzymes from thermophilic organisms are interesting biocatalysts for a wide variety of applications in organic synthesis, biotechnology, and molecular biology. Next to an increased stability at elevated temperatures, they were described to show a wider substrate spectrum than their mesophilic counterparts. To identify thermostable biocatalysts for the synthesis of nucleotide analogs, we performed a database search on the carbohydrate and nucleotide metabolism of Thermotoga maritima. After expression and purification of 13 enzyme candidates involved in nucleotide synthesis, these enzymes were screened for their substrate scope. We found that the synthesis of 2'-deoxynucleoside 5'-monophosphates (dNMPs) and uridine 5'-monophosphate from nucleosides was catalyzed by the already known wide-spectrum thymidine kinase and the ribokinase. In contrast, no NMP-forming activity was detected for adenosine-specific kinase, uridine kinase, or nucleotidase. The NMP kinases (NMPKs) and the pyruvate-phosphate-dikinase of T.maritima exhibited a rather specific substrate spectrum for the phosphorylation of NMPs, while pyruvate kinase, acetate kinase, and three of the NMPKs showed a broad substrate scope with (2'-deoxy)nucleoside 5'-diphosphates as substrates. Based on these promising results, TmNMPKs were applied in enzymatic cascade reactions for nucleoside 5'-triphosphate synthesis using four modified pyrimidine nucleosides and four purine NMPs as substrates, and we determined that base- and sugar-modified substrates were accepted. In summary, besides the already reported TmTK, NMPKs of T.maritima were identified to be interesting enzyme candidates for the enzymatic production of modified nucleotides.

Orientation of Cel5A and Xyn10B in a fusion construct is important in facilitating synergistic degradation of plant biomass polysaccharides

One approach to achieve efficient and economical saccharification of plant biomass would be using thermostable and multifunctional enzymes from hyperthermophiles such as Thermotoga maritima. Thus, the bifunctional constructs, Cel5A-Xyn10B and Xyn10B-Cel5A, were produced by fusing cellulase Cel5A at the N- and C-terminals of xylanase Xyn10B, respectively. The Cel5A-Xyn10B fusion construct showed cellulase activity of 1483 Uμmol-1 against carboxymethyl cellulose, which was nearly the same as that of Cel5A in the free form. However, xylanase activity of this construct increased by 2-fold against beechwood xylan as compared to that of Xyn10B in free form. The synergistic effect between Cel5A and Xyn10B in the form of Cel5A-Xyn10B fusion resulted an overall increase in the release of reducing sugars. However, Xyn10B-Cel5A showed about 60% decrease in activities of both the component enzymes as compared to their activities in the free form. Both the fusion constructs were active in a wide range of pH from 4.0 to 9.0 and temperatures from 50 to 90°C. Nearly 80% of cellulase and xylanase activities were retained in Cel5A-Xyn10B fusion after incubation at 60°C for 1h. Secondary structures of the component enzymes were retained in the Cel5A-Xyn10B fusion as observed by circular dichroism spectroscopy. Docking and simulation studies suggested that the enhanced xylanase activity in Cel5A-Xyn10B was due to the high binding energy, favorable orientation of the active sites, as well as relative positioning of the active site residues of Cel5A and Xyn10B in closer vicinity, which facilitated the substrate channeling.

Engineering TM1459 for Stabilisation against Inactivation by Amino Acid Oxidation

AbstractOxidative alkene cleavage is a highly interesting reaction to obtain aldehydes and ketones. The Mn‐dependent protein TM1459 from Thermotoga maritima can catalyse alkene cleavage of styrene derivatives in the presence of tert‐butyl hydroperoxide. Despite the high thermal stability of the enzyme, it gets inactivated during the reaction. The data reported here indicate that auto‐oxidation is responsible for the low stability of TM1459 in the oxidative environment required for the alkene cleavage reaction. By targeting the exchange of residues prone to oxidation, this phenomenon was successfully prevented. Importantly, the stability to oxidation conveyed by the amino acid exchanges led to increased enzyme activity. However, the exchanges resulted in slightly modified positions of two of the four metal‐binding amino acids, thereby strongly impacting metal binding.

Co-expression of thermophilic pectinases in a single host for cost-effective pectin bioconversion into D-galacturonic acid

Co-expression of enzymes allow to produce multiple enzymes in a single host, representing a cost-effective alternative in biocatalytic processes which can be used for pectin bioconversion. Pectin-rich biomass is an abundant by-product from the fruit and sugar industries that is usually disposed in landfill or sold as a low value feedstock. The aim of this work was to co-express a thermophilic pectin methyl esterase (PME) and exo-polygalacturonases (exo-PGs) in a single host for pectin bioconversion into D-galacturonic acid (GalA) using different pectic substrates such as apple, citrus and sugar beet pectin. To achieve this, a PME from Bacillus licheniformis (BLI09) with either an exo-PG from Thermotoga maritima (TMA01) or from Bacillus licheniformis (BLI04) were cloned in pETDuet-1 and co-expressed in E. coli BL21 (DE3). Four co-expression plasmids containing both pectinases were constructed and factors such as the effect of the genes’ cloning order and their expression were evaluated. Co-expression constructs 3 and 4 (pETDuet-TMA01-BLI09 and pETDuet-BLI04-BLI09, respectively) showed better expression of both pectinases compared to co-expression constructs 1 and 2 (pETDuet-BLI09-TMA01 and pETDuet-BLI09-BLI04, respectively). Co-expression constructs 3 and 4 were the most efficient for pectin bioconversion into GalA reaching 3 and 2.5 mM GalA, respectively from apple and citrus pectin after 4 h reaction. In conclusion, this work demonstrates that the co-expression of pectinases can potentially contribute to reduce the cost associated to their production and purification as well as to increase their applicability for exploiting pectin-rich biomass to obtain bio-based chemicals.