Mining for novel cyclomaltodextrin glucanotransferases unravels the carbohydrate metabolism pathway via cyclodextrins in Thermoanaerobacterales

Abstract

Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (T_opt ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:β:γ-CDs (34:62:4) from soluble starch, as well as G3-G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs.

Figure 1

CGTases with different domain organizations. (A) Schematic representation of conventional five-domain ABCDE_CBM20 CGTases (blue), five-domain ABCDE_arch CGTases (orange), and four-domain ABCE_CBM20 CGTases (red), which are recognized by CAZy. Note that the novel group of 19 CGTases, (CldA/ThmA)-like enzymes from thermophilic C. subterraneus ssp. and Thermoanaerobacter spp., showed a three-domain ABC architecture (magenta). (B) Multiple amino acid sequence alignment of CGTases from GH13_2 with a conventional five-domain ABCDE_CBM20 (blue), five-domain ABCDE_arch (orange), four-domain ABCE_CBM20 (red), and three-domain ABC distribution (magenta), as well as maltogenic starch-acting enzymes (white). Note the CSR I-VII motifs showing functionally critical residues (asterisk) for the GH13 family. The underline indicates the conserved acidic catalytic triad Asp^x, Glu^y, and Asp^z from CSR II, III, and IV, respectively. The conserved aromatic central Tyr/Phe residue (green sphere) and the hydrophobic pair (Phe/Trp/Tyr)/(Phe/Tyr/Met) (H1 and H2 shadow boxes), which are essential for the cyclization activity of CGTases and to distinguish them from α-amylases are also showed^,,. The same color code is used in all other figures.

Figure 2

CldA enzymatic assay. (A) Effect of temperature (filled diamonds) and pH (empty diamonds) on CGTase activity. (B) Production of α-CD (circles), β-CD (squares) and γ-CDs (triangles) from 50 g L⁻¹ soluble starch by the action of CldA at 75 °C and pH 4.0 for 4 h. (C) The relative production of end products from 50 g L⁻¹ soluble starch after 2 h of reaction at 75 °C and pH 4.0. Note that G5-G7 is the sum of the linear oligosaccharides maltopentaose, maltohexaose, and maltoheptaose. The error bars indicate the standard deviation of three replicates.

Figure 3

Phylogenetic analysis of novel three-domain ABC CGTases. Evolutionary relationships were determined by the maximum likelihood method based on the WAG + G model using the full amino acid sequences of 78 CGTases, including the 48 characterized CGTases from GH13_2 recognized in the CAZy database, 19 three-domain ABC (CldA/ThmA)-like CGTases, and 11 putative CGTases. The sequences of 7 α-amylases from GH13 were used as an outgroup. The conventional five-domain ABCDE_CBM20 CGTases (blue), five-domain ABCDE_arch CGTases (orange), four-domain ABCE_CBM20 CGTases (red), and the novel group of 19 three-domain ABC CGTases, (CldA/ThmA)-like enzymes from thermophilic C. subterraneus ssp. and Thermoanaerobacter spp. (magenta) were observed in four different clades. The ABCDE_CBM20 maltogenic starch-acting enzymes (blue dashed line) and α-amylases (black branch) from GH13_2 and GH13, respectively, are also shown in two different clades. Note that while the α-amylases from Aspergillus oryzae and Cordyceps farinosa belong to the GH13_1 subfamily, the α-amylases from bacteria showed an unassigned GH13 subfamily. Bootstrap values (1000 iterations) are indicated for each node. Only bootstrap values above 50% were shown. The tree was drawn using iTOL v4 (http://itol.embl.de).

Figure 4

Comparative view of the gene clusters involved in the CM-CD pathway. Note the genetic organization of the CM-CD gene clusters from K. oxytoca (cym), Thermococcus sp. (cgt), B. subtilis (cyc), C. subterraneus ssp. (cld), Thermoanaerobacter spp. (thm), and Thermoanaerobacterium spp. (thb). Additionally, note the protein-encoding genes involved in the four steps of the CM-CD pathway. (i) Synthesis: CGTases (1, red). (ii) Translocation/Internalization: MdxE (2), MdxF (3), and MdxG (4) in blue. While the MdxX (5) and CDP (6) from G− K. oxytoca (cym) are also blue, the putative msmX-encoding gene is not included. (iii) Degradation: CDase (7), GA (8), and GP (9) in green. (iv) Metabolic assimilation: Pgi (10), PfkA (11), and PykF (12) in orange. AmyB (33) and the AmyEDC transporter system (34–36) from Thermoanaerobacterium spp. (thb), and the putative transcriptional regulator of the ABC transporter system from cym/cyc (37–38) are shown. Note the five groups of protein-encoding genes that are essential for several prokaryotic cell functions: (i) HPr (13), PolIIIα (25), and the CBS domain/Bateman module (24) for carbon catabolite regulation, bacterial genome replication, and sensing cellular energy status, metal ion concentration, and ionic strength. (ii) MurB (14), PHP (15), RapZ (16), RodZ (17), and WhiA (18) for cell wall biogenesis, sporulation, and cell division. (iii) feruloyl esterase (22), 2-phospho-l-lactate transferase (19), the enzyme system (R)-2-hydroxyglutaryl-CoA dehydratase (20, 21), and 4-hydroxy benzoyl-CoA thioesterase (23) for oxidative stress defense, degradation of aromatic compounds, and fatty acid metabolism. (iv) signal-transducing protein PII (26), methylenetetrahydrofolate reductase (29), methionine synthase (30), PepT (27) and the anaerobic transcriptional activator fnr (28) for amino acid metabolism. (v) tRNA(m⁵U₅₄)methyltransferase (31) and MATE (32) for tRNA maturation and detoxification. Genes of unknown function are in gray. Abbreviations are listed in Table S4.

Figure 5

Proposed CM-CD pathway in G− bacteria (I), G+ bacteria (II), and archaea (III). Note the proteins involved in the four steps of the CM-CD pathway. (i) Synthesis: four-domain CGTases in G−, three- and conventional five-domain CGTases in G+ , and five-domain CGTases in archaea with either E_CBM20/E_arch domains at the C-terminal region. (ii) Translocation/Internalization: MdxEFG-(X/MsmX) transporter system. The CDP in G− is also shown. Note that while the cyclo/maltodextrin-binding protein MdxE is an untethered component of the periplasmic space in G−, it is predicted to be anchored to the cytoplasmic membrane outer surface via a lipid moiety in G+ and archaea. Although the MdxX enzyme is a dedicated ATPase in G−, MsmX is a promiscuous ATPase in G+ and archaea. Cyclo/maltodextrin translocation into the cytoplasm by the two permease subunits MdxFG is triggered by the ATPase activity of MdxX/MsmX. (iii) Degradation: CDase, GA, and GP. Hexagons represent individual glucose molecules. (iv) Metabolic assimilation: Pgi, PfkA, and PykF. While Pgm and HK are not included in the CM-CD gene clusters of Fig. 4, the asterisks in HK*, Pgi*, and PykF* represent the modified EMP pathway in archaea. This figure was created with http://BioRender.com.