#### 5.1.3.2. Sucrose 6-phosphate phosphatase.
To date, the sucrose 6-phosphate phosphatase (S6PP) from cyanobacterial *Synechocystis* sp. is the only reported enzyme of this class.<sup>352,474</sup> This enzyme has been extensively studied including experimental structural data of its unliganded form and complexes with sucrose, sucrose 6-phosphate, sucrose and phosphate, glucose, trehalose, cellobiose, and maltose.<sup>352,474</sup> S6PPs share the structural fold observed for T6PPs, showing a high degree of similarity with the corresponding HAD-like and CAP domains. Nevertheless, subtle changes can be observed including a short α-helix in the transition between the HAD-like and CAP domains that replaces the last β-sheet in T6PPs. S6PPs present a shorter loop emerging from the HAD-domain first β-strand that account for a more open cleft, compared to T6PPs. The S6PP complexes with sucrose and sucrose 6-phosphate indicate that this loop is not required to position the disaccharide into the substrate binding site since the sucrose arranges in a different orientation when compared with trehalose 6-phosphate in T6PP.<sup>352,474</sup> The glucose moiety of sucrose binds buried into the cleft plays a critical role in substrate recognition and specificity. Supporting this notion, the affinity for the glucose moiety accounts for inhibition by sucrose and glucose.<sup>352,474</sup> The structural and biochemical data support a common catalytic mechanism for S6PPs and T6PPs mediated by the nucleophilic attack of the substrate to form a covalent phospho-enzyme intermediate.

#### 5.3.4. Maltokinase Mak/Pep2.
Currently, several crystal structures of the mycobacterial enzyme have been reported, including Mak/Pep2 from *M. tuberculosis* in its apo form and in complex with maltose,<sup>375</sup> Mak/Pep2 from *Mycobacterium vanbaalenii* in its apo form, and in complex with ATP, and the non-hydrolizable ATP analog AppCp,<sup>228</sup> and Mak/Pep2 from *M. smegmatis*, as a component of the Mak/Pep2-TreS complex.<sup>232</sup> The structure of Mak/Pep2 is curious in the context of other enzyme folds participating in the α-glucan metabolism. Mak/Pep2 resembles the bacterial S-methylthioribose kinase, despite low global amino acid sequence identity,<sup>228</sup> displaying many conserved structural motifs associated with nucleotide binding and enzymatic activity.<sup>228,475</sup>

The mycobacterial Mak/Pep2 structures reveal a two-domain fold architecture comprising the N-terminal CAP subdomain, containing three antiparallel β-strands that form a curved β-sheet and enclosing the N-terminal α-helix and a β-hairpin. A second intermediate subdomain contains a central seven-stranded antiparallel β-sheet having two lateral α-helical segments. Finally, the C-terminal domain is composed of two central four α-helical bundles, a β-hairpin, and a two-stranded β-sheets<sup>375,228</sup> (Figure 15). Both N- and C-terminal domains remain strongly bound by salt bridges and hydrogen bonds. The active site of Mak/Pep2 enzymes is located within a deep pocket between the intermediate subdomain and the C-terminal domain. Reported structural complexes in the presence of ATP and non-hydrolizable analogs clearly define the site where the adenine group locates. Importantly, a strictly conserved Lys149 interacts directly with the α-phosphate, and the γ-phosphate is held by the so-called 'P-loop' residue Ser136. Mg<sup>2+</sup> sites comprise side chains of Gln310 and Asp322, making bridges with ATP phosphates. On the other hand, maltose accommodates mainly into a pocket located in the C-terminal domain.<sup>375</sup>

The N-terminal CAP subdomain presents large B-factors, suggesting conformational flexibility.<sup>228</sup> The possible struc-

tural/biological function of this subdomain is regulation as well as representing an anchoring point to TreS.<sup>231,232</sup> It is worth noting that Mak/Pep2 and TreS form a hetero-octameric complex.<sup>231,232</sup> Although the crystal structure of the Mak/Pep2:TreS complex appears to rule out substrate channeling between the corresponding active sites,<sup>231</sup> the structural arrangement possibly enhances the enzymes recruitment facilitating the succession of isomerization (TreS) and phosphorylation (Mak/Pep2) reactions to transform trehalose into maltose 1-phosphate. In addition, formation of the complex may provide a mean for allosteric effects, as observed for similar complexes.<sup>232,476</sup> Specifically, the Mak/Pep2:TreS complex has an overall diamond-shape, where the TreS tetramer forms the core. Each TreS protomer pairs with a Pep2/Mak protomers at the apices of the tetramer resulting in a 4 + 4 configuration,<sup>232</sup> mainly mediated by interactions with the C-terminal domain of TreS and Mak/Pep2 N-terminal CAP subdomain. Interestingly, both enzymes are part of a single polypeptide in some members of the phylum Chlamydiae,<sup>222</sup> although presenting a monomeric form might present a similar relative arrangement.

## 5.2. TIM Barrel Fold Enzymes (TBF) and α-Glucan Metabolism

The TIM-barrel is a versatile scaffold present in many enzymes involved in pathways of the central metabolism.<sup>477</sup> TIM is an acronym for the glycolytic enzyme triose-phosphate isomerase, the first reported crystal structure for the scaffold.<sup>478</sup> TIMs are α/β proteins displaying an (α/β)<sub>8</sub> topology. Specifically, the central barrel core comprises eight parallel β-strands. Each β-strand interacts with a corresponding α-helix in antiparallel orientation surrounding the barrel core. Overall, α-helices and β-strands display an angle with the β-barrel central axis,<sup>479</sup> whereas the overall fold shows a degree of elliptical shape.<sup>480</sup>

TIM barrel fold (TBF) structures revealed a toroidal shape with pseudo-C8-fold symmetry that defines two faces, where the catalytic face is oriented at the C-terminal end of the β-strands.<sup>479</sup> As a result of the overall directionality of the β-barrel and α-helices, TIM barrel domains have a global dipole across the toroid, although it does not correlate with the location of the active site.<sup>481,482</sup> In addition to the important amino acid sequence differences between TBF enzymes, the overall shape dictates the conservation of intrinsic domain dynamics.<sup>483</sup> Moreover, TBF enzyme families, such as GHs, exhibit preserved long-range electrostatic interactions among charged residues directed toward the barrel center and the interaction energy in the structural residue network.<sup>483</sup> The clustering of TBF enzymes according to geometrical characteristics such as the length of helices and sheets and the location of the elliptical axes indicates that GH α-amylases (GH13) have commonalities with distant enzymes including glycolytic enzymes such as enolase and pyruvate kinase.<sup>480</sup>

As a result of the relevance of α-glucans in biology and the evolutionary versatility of these enzymes, TBF α-glucan processing enzymes present a large number of functions in terms of substrate specificity and α-glycosidic bond reactions.<sup>484,485</sup> Several of these functions are markedly improved by accessory domains such as Carbohydrate Binding Modules (CBM), which themselves comprise a large diversity.<sup>486</sup> These modules involved in the polymer binding contribute to enzyme-polymer heterogeneous catalysis, as stated by the Sabatier principle, meaning enzymes interact with substrates with intermediary strength, permitting binding association-
<figure><img src="image_68.png" /><figcaption>**Figure 16.** Structure and catalysis of TIM barrel fold enzymes. (A) The secondary structure of TBF enzyme families is represented as a diagram showing bars representing α-helices and arrows as strands. From left to right, the scheme offers the four GH families present in the α-glucan</figcaption></figure>
<caption>**Figure 16.** continued</caption>

metabolism, GH13, GH70, GH77, and GH57. Strands in the $(\alpha/\beta)_8$-barrel are highlighted in colors according to N- and C-terminal color key. For the GH13, accessory domains are shown as colored circles, including the N-terminal domain (blue), inserted β3-strand α3-helix domain B (cyan), and the C-terminal β-sandwich domain C (maroon). The GH70 architecture presents three associated domains formed by N-terminal and C-terminal not consecutive segments shown as semicircles called domain V (orange and red), domain IV (dark and bright yellow) and domain B (dark and light green), and domain C inserted between α6 and β6 (maroon). Note the alternative arrangement of $(\alpha/\beta)_8$ barrel starting with an α-helix instead of a β-strand as GH13. GH77 scheme shows an N-terminal domain (blue circle) before the catalytic domain, whereas the $(\beta/\alpha)_8$ barrel presents subdomains inserted. Subdomain B2 is indicated by a yellow circle, whereas the subdomains B1 (cyan) and B3 (pink) are formed by two semicircles. Note for the GH77 the lack of the C-terminal domain C compared to the GH13 family. The GH57 scheme shows the incomplete TBF $(\beta/\alpha)_7$ displaying several secondary elements extra associated with the catalytic fold. Note the β-strands colored with the same color scheme, presenting seven in the barrel and an antiparallel β-strand not belonging to the barrel (yellow). (B) From left to right, two views of the structure of a GH13 branching enzyme from *Cyanothecie* sp. in complex with maltohexaose (PDB 5GQV),⁶⁷³ and in the left panel, a close-up of the TBF active site. The view shows domains colored according to the scheme presented in A. Note an extra N-terminal domain colored in black. The close-up of the GH13 catalytic site indicates the position of strand β1 as a reference, with the rest of the barrel strand numbering increasing counter-clockwise. Catalytic triad residues are indicated in emerging loops extensions in strand β4, aspartate nucleophile (magenta arrow), in β5 glutamate acid/base (pink arrow), and in β7 aspartic stabilizer (blue arrow). (C) Same as in A, but the structure of GH70 *L. citreum* dextransucrase DSR-M inactive mutant N-terminal truncated mutant E715Q in complex with isomaltotetraose (PDB 6HTV).⁵¹² The catalytic domain permutation places the catalytic triad in different C-terminal loop strands in the barrel. Note a glutamine indicated by the pink arrow appears in the position of the acid/base due to the corresponding mutation E715Q. (D) Similar for the structure of the GH77 *E. coli* amyromaltase MalQ in complex with the pseudo-heptasaccharide (PDB 4S3R).³⁶⁶ (E) Structure of the GH57 *Thermococcus litoralis* 4-α-glucanotransferase complexed with acarbose (PDB 1K1Y).⁵²² From B to E, note the close-up of the left panel; the catalytic domain is oriented to place the bound polysaccharide in the same overall orientation.

dissociation kinetics are optimal to permit effective catalysis.⁴⁸⁷,⁴⁸⁸ The double inversion mechanism is central to all activities, and the reaction can be either under thermodynamic or kinetic control.²⁴⁶,³¹³,³⁴⁰ For example, the comparison of debranching enzymes architecture and accessory domains belonging to very different TBF GH-fold families reveals considerable differences,⁴⁸⁹ whereas debranching/branching enzymes can be clustered inside the same GH subfamily with the seemingly same structure.²³⁵,⁴⁹⁰,⁴⁹¹ Furthermore, it has been proven that subtle differences as a single point mutation can account for changes in function.⁴⁹² Since function may hold for structural extreme differences, but nuances can also alter the outcome function, it is challenging to extract a rationale using a reductionist approach. With this in mind, in the following sections, we present the overall makeup of GH families' structural organization for selected functions. Later, we present a parallel comparison of TBF-substrate complexes to highlight commonalities in enzyme-metabolite coevolution for these enzymes.³⁷⁸

**5.2.1. TIM-Barrel α-Glucan GH13 Family.** TBF enzymes classified into the GH13 family comprise the most significant number of specificities related to α-glucan metabolism.²⁴⁶,³⁴⁰,⁴⁹³ The GH13 family is one of the most studied from a structural point of view, and it is further subdivided into several subfamilies. Structures corresponding to selected functions associated to glycogen and disaccharide metabolism include (i) 4-α-glucanotransferase (EC 2.4.1.25);²⁸⁹ (ii) GlgE α(1→4)-glucan phosphate α-maltosyltransferase (EC 2.4.99.16);²¹⁶⁻²¹⁹,²²¹,⁴⁹⁴ (iii) GlgB glycogen branching enzyme (EC 2.4.1.18); (iv) amylosucrase (EC 2.4.1.4),¹⁵⁶,³³²,³³³,⁴⁹⁵⁻⁴⁹⁷ sucrose phosphorylase (EC 2.4.1.7);³⁶²,⁴⁹⁸⁻⁵⁰⁰ (v) sucrose 6-phosphate phosphorylase (EC 2.4.1.329);⁵⁰¹ (vi) AmyA α-amylase (EC 3.2.1.1);⁵⁰²⁻⁵⁰⁵ (vii) GlgX glycogen debranching enzyme (EC:3.2.1.196);²³⁵ and (viii) malto-oligosyltrehalose trehalohydrolase TreZ (EC 3.2.1.141),³⁴⁸ trehalose synthase TreS (EC 5.4.99.16),²³¹,³⁶⁹ and malto-oligosyltrehalose synthase TreY (EC 5.4.99.15).⁵⁰⁶

The canonical GH13 enzyme comprises a characteristic TBF $(\beta/\alpha)_8$ barrel domain (A) including the catalytic center of the

enzyme and an accessory domain (B) inserted into the loop between the β3-strand and α3-helix of the barrel, whereas a third C-terminal domain (C) completes the enzyme core architecture.⁵⁰⁷,⁵⁰⁸ The GH13 presents a TBF with a linear order of secondary structures, with a topology N-β1-α1-β2-α2-β3-α3-β4-α4-β5-α5-β6-α6-β7-α7-β8-α8-C. The B-domain presents a variable structure and length, whereas the C domain is formed by a β-sandwich domain. In addition to the core fold, often one or two additional C-terminal domains, D and E, are present.⁴⁹³ The E domain is characterized as a starch-binding domain. These domains correlate with GH13 function, specificity, and taxonomy⁴⁹³ (Figure 16).

**5.2.2. The Alternative TBF GH70 Family.** The GH70 family comprises a number of enzymes that use sucrose as a sugar donor to synthesize α-glucans, so-called glucansucrase (GSuc), oftentimes catalyzing more than one type of glucosyl transfer. Selected activities include (i) α(1→6)-dextransucrase (EC 2.4.1.5),⁵⁰⁹⁻⁵¹² α(1→3)/α(1→6)-alternansucrase (EC 2.4.1.140),³³⁸,⁵¹³ and the α(1→4)/α(1→6)-reuteransucrase (EC 2.4.1.-).⁵¹⁴

GSuc enzymes are usually large extracellular proteins that consist of four different regions, including (i) a signal peptide, (ii) an N-terminal variable region (VR) containing other repeat units, (iii) the TBF catalytic domain, and (iv) a C-terminal glucan-binding domain (GBD) also comprising repeating units. GH70s have a characteristic circularly permuted TBF compared to the GH13 topology.¹²⁶ The GH70 $(\alpha/\beta)_8$ barrel starts with an α-helix that corresponds to α3 in the GH13 (instead of starting in β1), in a relative order N-α3-β4-α4-β5-α5-β6-α6-β7-α7-β8-α8-β1-α1-β2-α2-β3-C. Nevertheless, these enzymes still present the same catalytic amino acid motifs of the GH13 family enzymes, with TBF sequence position accounting for the circular permutation. In addition, crystal structures of truncated GH70 GSuc enzymes reveal five domains in a U-shaped form, with equivalent A, B, and C domains as observed in the GH13 members making the catalytic core, and domains IV and V are unique to GH70 enzymes.¹²⁶ Interestingly, domain B is composed of two discontinuous inserts and is next to domain A, while domain C is a single domain formed by the N- and C-termini and is
involved in substrate binding. Domain IV is involved in oligomerization, whereas domain V is located at the C-terminus and is believed to be involved in glucan binding<sup>126</sup> (Figure 16).

**5.2.3. The TBF GH13-like GH77 Family.** Reported bacterial crystal structures of the GH77 family members are limited to the 4-α-glucanotransferase (EC 2.4.1.25).<sup>341,366,515-517</sup> The GH77 TBF has a similar topology to the GH13 (N-β1-α1-β2-α2-β3-α3-β4-α4-β5-α5-β6-α6-β7-α7-β8-α8-C), reflecting a high evolutionary connection.<sup>518</sup> Nevertheless, the separated classification family is supported by the divergence in several structural factors.<sup>485,519</sup> The core of the GH77 enzymes comprises the TBF with three insertions, resulting in the formation of three subdomains so-called B1, B2, and B3. The B1 and B3 subdomains correspond to domains B and C in the GH13 family. The B2 domain is unique to the GH77 family, possibly playing a role in substrate recognition. In addition, the B3 subdomain does not present the classic β-sandwich as in domain C of GH13 enzymes. This structural arrangement of accessory domains generates a narrower and deeper catalytic cleft compared to GH13 enzymes, where domains clamp the α-glucan substrates. Moreover, although the catalytic triad is conserved, some GH77 enzymes present differences in the residue environment at the catalytic site.<sup>520</sup> Finally, some enzymes in this family present an additional N-terminal immunoglobulin-like fold domain presumably implicated in α-glucan binding<sup>519</sup> (Figure 16).

**5.2.4. The Incomplete TBF GH57 Family.** The GH57 family comprises an expanding number of activities since the discovery of α-amylases with unrelated sequences to the GH13 family.<sup>274,521</sup> There are a limited number of bacterial and archeal GH57 enzymes with known three dimensional structures associated to the α-glucan metabolism: the 4-α-glucanotransferase (EC 2.4.1.25)<sup>522</sup> and the glycogen branching enzyme (EC 2.4.1.18).<sup>281,364</sup> The reported enzymes comprise an N-terminal incomplete TBF, containing a short helix (often called subdomain B) emerging from β2, an intermediate five-helix domain, and a C-terminal β-super sandwich domain.<sup>523</sup> The incomplete TBF has a (β/α)<sub>7</sub> topology including a β-barrel with a distorted toroidal shape. The β6 does not participate in the barrel, being located in an antiparallel orientation with respect to β7. Therefore, the lack of a strand in the β-barrel generates a more open conformation because there are two gaps between β3 and β4 and between β7 and β8.<sup>523</sup> As in the other GHs, the TBF C-terminal β-strands define the active site, displaying a more significant loop variability, including the insertion of additional secondary elements (Figure 16).

# 6. POLYSACCHARIDES AT THE DAWN OF LIFE

How polymers metabolism has emerged is a central question in biology. In the case α-glucans, their ubiquitous presence in present biology suggests that they have deep evolutionary roots, possibly dating back to the earliest life forms or before. Miller and Urey, whose famous experiment supported the prebiotic chemical origin of life, proposed that polymerization may be catalyzed by adsorption on clay and mineral surfaces.<sup>524</sup> Orgel and colleagues proposed abiotic polymerization of amino acids and nucleotides on mineral surfaces, suggesting that these rocky interphases could provide a "library" for molecular evolution.<sup>525,526</sup> Polysaccharides may arise first among other polymers in prebiotic times, suggesting

that they were present in a glycoworld from which the RNA world emerged.<sup>527,528</sup> Furthermore, it has been speculated that high-energy poly-phosphates may be served as a scaffold for the assembly of major polymers,<sup>527,529</sup> thus facilitating the condensation of the first polysaccharides.

The putative occurrence of neutral polysaccharides, such as glucans, might help in original phase separation in the primordial soup, allowing the concentration of other charged proto-biopolymers and coalescence of dispersed particles.<sup>528</sup> Indeed, carbohydrate polymers may appear on prebiotic earth as a prerequisite for life's subsequent development.<sup>527</sup> Long and colleagues showed that dextrans can contribute in creating compartments with properties of synthetic cells.<sup>530</sup> Although lessons and the latest advances from synthetic chemistry pinpoint first principles for synthesizing α-glucans,<sup>531,532</sup> it appears that further exploration of α-glucan prebiotic origin is needed, which potentially could offer fascinating views. The presumed existence of prebiotic glucose, as well as the demonstrated abiotic phosphorylation of glucose forming α-D-glucose 1-phosphate,<sup>533,534</sup> opens the door to think about the fate of these sugars phosphates in abiotic times.

## 6.1. Extracellular Polysaccharides and Biofilms at the Origin of Living Cells

The evolution of organisms selected confirmed metabolic processes taking advantage of available compounds and synthesizing others that benefit survival under environmental pressure in a defined niche. α-Glucan metabolism provides a framework for understanding how physiological functions associated with cellular structure and energy storage exhibited in different bacterial lifestyles. Biofilms seem to be the most frequent lifestyle, with ca. 80% of bacterial and archeal cells living in this form, which may serve as diversity incubators; planktonic lifestyle might be a secondary form.<sup>535,536</sup> Biofilms provide functions such as harvest and access to nutrients, shelter protection against environmental stresses, water retention to maintain extracellular enzyme activities, social cooperation, and horizontal gene transfer.<sup>537,538</sup> The occurrence of biofilm structures appears early in the fossil record (ca. 3.2 giga annum; Ga), a characteristic shared by prokaryote 'living fossils' present in hot springs and sea hydrothermal vents;<sup>539</sup> bacteria and archaea lineages emerged at a similar time frame (ca. 3.4 Ga).<sup>540</sup> Diverse glucans have been shown to form biofilms. β-Glucans, such as cellulose<sup>541,542</sup> and β-(1→3)-glucans,<sup>543</sup> are common biofilm-forming exopolysaccharides. α-Glucans<sup>119,544-548</sup> are components of the extracellular matrix, capsules and biofilms, often linked to glycogen metabolism. Interestingly, hot spring-living Geobacillus tepidamans forms a glucan-containing biofilm,<sup>549</sup> whereas Geobacillus sp. expresses an extracellular thermostable (4→6)-α-glucanotransferase.<sup>550</sup> Moreover, biofilm-forming uncultivated SM1 Euryarchaeon isolated from subsurface biotopes of sulfidic springs can synthesize glycogen.<sup>551</sup> Based on these pieces of evidence, the presence of α-glucans in ancestral biofilms can be conjectured.

## 6.2. Considerations on the Origin of Glycogen Metabolism in Bacteria

The emergence of metabolic systems and their evolution represents a central question in biochemistry and biology. Efforts are dedicated to reconstruct the metabolic make-up of the Last Universal Common Ancestor (LUCA), the hypothetical prokaryotic common organism antecedent to all existing life. Inferring gene repertoires arising billions of
<figure><img src="image_69.png" /><figcaption>A</figcaption></figure>

Linear
α-glucan

Branched
α-glucan

Linear
α-glucan

Branched
α-glucan

Linear
α-glucan

Mal

<figure><img src="image_70.png" /><figcaption>C</figcaption></figure>

Extracellular
branched
α-glucan

Extracellular
branched
α-glucan

Extracellular
branched
α-glucan

Linear α-glucan

Mal

Tre

<figure><img src="image_71.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_72.png" /></figure>

NDP-glu

<figure><img src="image_73.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_74.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_75.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_76.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

**Figure 17.** Evolutionary basis of α-glucan chemistry and metabolic pathway diversity. Schematics depict an exercise based on different aspects and models for the evolution of metabolic pathways onto α-glucan metabolism. (A) Hypothetical scenario for the divergent evolution of one gene into three enzymes with different functions. Considering organisms with a coding gene (grey arrow) for an enzyme responsible for processing an α-glucoside A into B. Due to gene cumulative mutations and selection by environmental pressure (in the same or different organisms) the enzyme gene diverges into three distinct gene/enzymes (red arrows and boxes), with a different active site suited for different cellular functions involving α-glucosyl bond processing. Over time, these mutations became fixed in the population, creating three distinct enzyme lineages. This case may reflect the diversification of the GH13 family into different functions. (B) Hypothetical case of convergent evolution, involving three different species of
**Figure 17. continued**

bacteria that live in similar environments where the processing of an α-glucan provides an adaptive advantage to survive. Each species had evolved independently and contained genes (grey arrows with different red hatching) that do not share a recent common ancestor, encoding enzymes responsible for processing unrelated α-glucosides. The selection of mutations in these three genes converges on enzymes with the same function. This case may reflect the convergence of enzymes from GH13, GH77, and GH57 families into enzymes 4-α-glucanotransferase (EC 2.4.1.25). (C) A speculative scenario of an anterograde evolution pathway⁵⁸¹ in which an organism utilizes a branched α-glucans using a gene/enzyme (red arrow and box) that processes the substrate in linear chains to be able to harvest it. An event of gene duplication and posterior divergence results in a second gene/enzyme (pink arrow and box) that processes the linear α-glucan into maltose, providing an advantage due, i.e., cluster to the same operon for utilization of nutrients. The same process repeats to acquire a gene/enzyme (purple) to transform maltose into trehalose, reaching a more efficient way to produce the functional disaccharide. (D) Speculative scenario of retrograde evolution⁵⁷⁵ where an extracellular α-glucan is critical for survival, which can be produced by an organism gene/enzyme (red arrow and box) from oligosaccharides obtained from the environment. An undergoing depletion of the disaccharide generates the selection, under growing pressure, of a new gene/enzyme (pink arrow and box) originated by gene duplication and divergence, that can synthesize oligosaccharides from disaccharides available in the environment. The same process repeats to acquire a gene/enzyme (purple) to generate disaccharides from monosaccharides. (E) A hypothetical case of an ancestral gene encoding promiscuous enzyme (blue arrow and hexagon),⁵⁸²,⁵⁸³ i.e., an NSPase produces different sugar donors NDP-glucose. The enzyme through gene duplication and subsequent divergence evolves into three differentiated enzymes (light, medium, and dark blue arrow and hexagons) with higher specificity to synthesize ADP-glucose, UDP-glucose, and GDP-glucose. (F) The differentiation of promiscuous enzymes recruited in an ancestral pathway (blue hexagon, and yellow circle) under gene duplication and subsequent divergence can lead to the arrangement of specific enzymes into different pathways "patchwork". (G) Hypothetical enzyme-substrate co-evolution involving an enzyme (yellow circle) that synthesizes a linear α-glucan in a pathway comprising a second enzyme (red box) to generate branched α-glucans. Eventual errors or promiscuity lead the first enzyme to generate the byproduct maltose 1-phosphate. In a second moment, the new metabolite scaffolds the evolution of a specific enzyme (salmon circle). A higher level of maltose 1-phosphate is removed by unspecific enzymes by oligomerization. In a third moment, the enzyme (pink box) is recruited and evolves to catalyze such reaction specifically.

years ago is extraordinarily challenging since orthologous genes/proteins are subject to continuous vertical inheritance, gene loss, and horizontal gene transfer.⁵⁵² Nevertheless, it could be argued that primitive α-glucan pathways appeared early in the gene makeup of organisms due to the importance of survival to bacteria.⁹⁴ Supporting this notion, glycogen metabolism provides an advantage in enduring famine, resisting different abiotic stresses and helping in colonizing niches.⁸²,⁹⁵,¹⁷³,⁵⁴⁴,⁵⁵³ The structure of bacterial glycogen with a small average chain degrades slowly and serves as a resilience mechanism, prolonging survival.¹⁰¹ Finally, glycogen plays a role in awakening bacteria from dormancy, a near-dead state.⁵⁵⁴ Therefore, glycogen metabolism could have played a role in the early stages of life evolution.

From the point of view of synthetic biology, it has been proposed that the construction of small bacterial genomes containing a minimal set of proteins deduced from existing genomes can reveal the essential set of survival genes compatible with cellular life,⁵⁵⁵ thus strongly suggesting a requisite gene repertoire in the LUCA. Nevertheless, the metabolic routes are strongly context-dependent, and it seems difficult to find protein-coding genes that are entirely conserved in all prokaryotic genomes.⁵⁵⁶ The minimal genome synthetic approach appears unsuitable for evaluating early glycogen metabolism since it is not essential in some bacteria.⁵⁵⁷,⁵⁵⁸ Interestingly, the presence of GBEs and GDEs in all kingdoms of life indicates glycogen metabolism is an early process;⁵⁵⁹ while GP has also been predicted to be part of the LUCA.⁵⁶⁰ Furthermore, meta-consensus analysis of metabolic pathways predicted early enzyme functions and properties that may exist in the core metabolism of early life forms, with several enzymes related to starch metabolism.⁵⁶¹

The LUCA has been predicted/proposed to be an anaerobe.⁵⁶²⁻⁵⁶⁴ Several strict anaerobes are known to produce glycogen,⁵⁶⁵,⁵⁶⁶ while glycogen metabolism enzymes were identified in anaerobic hyperthermophile bacteria from the genera *Thermotogales* and *Aquifex*.⁵⁶⁷,⁵⁶⁸ The reconstruction of the tree of life also suggests that the LUCA was a thermophile,⁵⁶⁹ which is consistent with the hypothesis of

the origin of life at hydrothermal vents.⁵⁷⁰ *Thermotogales* and *Aquifex* are at the base of the bacterial clade⁵⁷¹ and therefore are thought to resemble ancient bacteria. In addition, the anaerobic thermophilic archaea *Methanocaldococcus jannaschii* shares a metabolism similar to bacteria, comprising genes of the glycogen metabolism,⁵⁷² supporting the hypothesis that α-glucan metabolism predates separation between archaea and bacteria. Interestingly, symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides including glycogen.²⁶³ The giant tube-worm hydrothermal vents-living *Riftia pachyptila*, utilizes glycogen as the main anaerobic substrate during extended anaerobiosis and a key molecule in chemoautotrophic endosymbionts,⁵⁷³ suggesting glycogen plays relevant roles in extreme niches associated with life origin. Altogether, these views support glycogen metabolism as an ancient process.

## 6.3. The α-Glucan Pathways in the Light of Metabolism Evolution Models

From the putative LUCA metabolism, enzymatic networks have diversified into apparently disparate systems. Several models for the evolution of pathways attempt to explain the emergence of metabolic networks on prebiotic and ancestral cellular metabolism.⁵⁷⁴ In this regard, the α-glucan and glucose disaccharide metabolism present a framework that could be evaluated *a priori* by these models while subjected to a simple chemical scaffold. Hereof, we discuss the emergence of the α-glucan biosynthetic machinery based on these models (Figure 17).

The pioneer retrograde model proposed by Horowitz, based on the Oparine-Haldane "primordial soup" hypothesis,⁵⁷⁰ proposed the metabolic evolution of a rudimentary heterotrophic organism under significant selective pressure due to the depletion of the prebiotic compound supply.⁵⁷⁵ Under such circumstances, the primordial cells acquired mechanisms to synthesize functional molecules by assembling the enzymatic machinery in a retrograde manner, originating the metabolism that frees them from exogenous sources of such compounds. In the retrograde view, α-glucans metabolism could arise by harvesting hypothetical proto-glucan compounds as an energy
source or making part of functional compartments/biofilm in
primordial cell communities. Once depleted, selective pressure
enforced the selection of synthetic machinery to obtain in a
retrograde manner the enzymes to synthesize first the α-glucan
polymers or disaccharides, then nucleotide sugars, and so on.
Phylogenetic studies comparing E. coli enzymes and pathways
universally present in all domains of life indicate that the
catabolism of α-glucans is among the most conserved pathways
with enzymes present in Archaea, Bacteria, and Eucarya.⁵⁷⁶
Moreover, the presence of an analogous enzymatic machinery
to the glycogen synthesis for the usage of external α-glucan,
such as the maltose/maltodextrin utilization system present in
distant bacteria,⁴⁶⁴,⁵⁷⁷⁻⁵⁷⁹ could indicate that the evolution of
the α-glucan metabolism first associated to external sources.
The presumed LUCA containing α(1→4)-glucan-phosphor-
ylases is part of the GT35 family (MalP/GP).⁵⁶⁰ The
reversibility of these enzymes³⁶⁰,⁵⁸⁰ insinuates an ancient
pivot function between synthesis and degradation. During
evolution, PLP could be incorporated into a proto-GT-35
NDP-sugar-dependent enzyme, surrogating a nucleoside
monophosphate, and resulting in MalP/GP.³¹⁴ This appears
as a sensible explanation in the context of almost the whole
GT-B family using nucleotide-activated sugars.

Contrary to the retrograde model, Granick proposed that biochemical pathways extended in an anterograde fashion, acquiring successive steps in the metabolic pathway to produce a functional metabolite which provides a survival advantage.⁵⁸¹ Exopolysaccharides may be originated from early communities to form a matrix of biofilms as a survival strategy, allowing colonization of surfaces. From this point of view, the metabolism could have extended from gluconeogenesis toward α-glucans synthesis, possibly supported by activities from the nucleotide and RNA metabolism based on meta-consensus enzyme functions (EC: 2.4.1.- and 2.7.7.-).⁵⁶¹ The putative LUCA presents NSPases and related GT-2 enzymes, including UTP-glucose 1-phosphate thymidylyltransferase.⁵⁶⁰,⁵⁶³ Moreover, phylogenetic analysis highlights GT folds go back in evolution to the LUCA.⁴²⁸ Therefore, this view could support nucleotide-sugars and disaccharides first, instead of a retrograde view of glucans first.

In the evolution of pathways, innovations are the driving
force for enzyme function specialization that permits the
synthesis of new specific metabolites. Ycas proposed earlier
enzymes were less specific (more promiscuous) to catalyze
several related reactions with a smaller number of enzymes.⁵⁸²
Independently, and along the same reasoning, Jensen
postulated a “Recruitment Hypothesis” in which pathway
assembly occurs by recruitment of such primitive enzymes with
a wide range of chemically related substrates, also known as the
“Patchwork Hypothesis”.⁵⁸³ The evolution of an enzyme into
diverse enzymes with specific traits arising from gene
duplication of the ancestral enzyme can be viewed as
functional specialization, or division of labor, an evolutionary
mechanism observed from molecules to organisms.⁵⁸⁴
Promiscuity outcome includes a ‘Flexible Metabolome’, a
concept that suggests genetic and metabolic pathways are
cross-wired in diverse unpredicted forms, therefore inherently
ambiguous and stochastic.⁵⁸⁵ Importantly, enzyme promiscuity
appears widespread in modern α-glucan metabolism. NSPases
and GT-B enzymes often present some degree of usage of
alternative nucleotide triphosphate or nucleotide sugars,
respectively. For example, (i) OtsA *M. thermoresistibile* can
use ADP-glucose and GDP-glucose,⁴⁴⁰ and (ii) GS from *P.*

abyssi can use UDP-glucose and ADP-glucose.⁴⁵⁷ Moreover, NSPase promiscuity has been reported in several bacterial enzymes, including the AGPase from *Rhodococcus jostii* that can use ATP, GTP, and CTP, as well as diverse hexose 1-phosphates.⁵⁸⁶ On the other hand, TIM barrel folds bear a large diversity of functions for α-glucan processing. In the case of GBEs and GDEs (GH13), both are related to gene duplication and paralogous.⁵⁵⁹ Furthermore, functional and phylogenetic studies show the majority of these enzymes classified into the GH13 family comprising 35 subfamilies likely monofunctional, while others appear polyspecific,²⁴⁶ which correlates with the promiscuity to specificity evolution. Moreover, the GH13 family is a member of the clan GH-H, which also contains GH70 (glucansucrase) and GH77 (amylomaltases) families, supporting the evolutionary relatedness of these enzymes.⁵¹⁸

6.4. Structural Relationships between the Enzyme Folds Involved in α-Glucan Biosynthetic Pathways to Other Metabolic Pathways

The catalytic folds involved in α-glucan and glucose
disaccharide synthesis are widespread in the other areas of
the metabolism, which could be thought to represent distant
evolutionary relationships or convergent evolution. Rossmann-
like folds are thought to have evolved prior to LUCA from a
primordial generic nucleotide-binding domain, which partic-
ipates in five out of eight enzymatic reactions of the oldest
metabolic Wood-Ljungdahl pathway.<sup>380,563</sup> TIM barrels have
been hypothesized to allow early evolution of protein-mediated
catalysis since they are flexible to incorporate different
cofactors, providing a scaffold to transition from ribozymes
and peptides catalysts to modern protein enzymes.<sup>587</sup>
Rossmann-like folds and TIM barrels can be considered the
oldest folds based on the hypothesis that protein folds are
more prevalent and more widely shared the more ancestral
they are.<sup>377</sup>

In the case of the NSPase fold, the N-terminal region of the active site, participating in the nucleotide-binding, shows high degree of similarity to enzymes in distant regions of the metabolism, including (i) the molybdopterin guanine dinucleotide synthesis enzyme MobA, a key component in molybdenum cofactor synthesis,⁵⁸⁸,⁵⁸⁹ and (ii) the bifunctional methylenylthritol 2,4-cyclodiphosphate synthase of *Campylobacter jejuni*, which transfers cytidil- groups to 2-C-methyl-D-erythritol 4-phosphate to form CDP-ME2P in the isoprenoid biosynthesis.⁵⁹⁰ Interestingly, the C-terminal pocket portion accounting for the sugar-binding in the NSPase folds presents the largest variability which correlates with differences in the sugar substrate, including (i) the N-acetylmuramic acid α-1-phosphate uridylyltransferase MurU (EC: 2.7.7.99) involved in peptidoglycan metabolism,⁵⁹¹ and (ii) the nucleotide monophosphate CMP-Kdo synthetase (EC: 2.7.7.38), that activates the modified sugar Kdo for the synthesis of bacterial cell wall lipopolysaccharides.⁵⁹² Altogether, the N-terminal region presents variability associated with nucleotide usage, whereas the C-terminal region of the cavity highlights a broad variability associated to several other areas of the metabolism. Finally, the NSPase domain shares similarities to enzymes with other functions. Interestingly, the N-terminal eukaryotic translation initiation factor eIF2B presents an extraordinary degree of architectural similitude.⁵⁹³

The GT-B fold, comprising the two articulated Rossmann
domains, represents an interesting case since several bi-
<figure><img src="image_79.png" /><figcaption>**Figure 18.** Integrated summary of α-glucan metabolic pathways in bacteria. The scheme reflects the overlay of different metabolic routes and enzymes discussed, evoking the idea of an underlying common theme supported by the evolution of an ancestral metabolism. The prokaryotic cell is depicted as enclosed by its membrane (double dotted boundary line) where glucose and other sugars enter and are transformed along the gluconeogenesis pathway eventually leading to the synthesis of glucose 1-phosphate, which is subsequently activated by forming sugar donors NDP-glucose (ADP-glucose or UDP-glucose). Different pathways for intra- and extracellular α-glucan synthesis depart from top to bottom, traversing the synthesis of glucose disaccharides. The area of the metabolism comprises reactions requiring nucleotide-sugar donors (violet background shade) and independent nucleotide-sugar (light blue background shade). The parallel biosynthetic routes follow similar transformations catalyzed by enzymes with typical catalytic domain architecture. Rossmann folds enzymes, preeminently NDP-sugar transferases (blue hexagons), and GT-B fold (yellow circles) drive transformation involving nucleotide derivatives. In contrast, TIM barrel folds (red boxes) take control of all nucleotide-independent transformations. Other intermediate reactions are driven by HAD domain phosphorylases (green parallelograms), which catalyze disaccharide dephosphorylation, and the phosphorylation of maltose carried out by the Maltokinase (violet trapezium).</figcaption></figure>

substrate reactions can profit from the open-to-close mechanism. Therefore, this architecture could have emerged by convergent evolution or very distant divergent evolution in several areas of the metabolism.³⁸⁰ Indeed, a resembling GT-B-like structural organization is observed in non-GT-B enzymes. Many examples can be extracted from glycolytic enzymes such as (i) the phosphofructokinase that catalyzes the ATP-

dependent phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate (EC: 2.7.1.11),⁵⁹⁴ (ii) the phosphoglycerate mutase that catalyzes the transfer of phosphate from 3-phosphoglycerate acid to form 2-phosphoglycerate (5.4.2.12),⁵⁹⁵ and (iii) the phosphoglycerate kinase (EC: 2.7.2.3) that catalyzes the reversible transfer of a phosphate group from 1,3-bisphosphoglycerate to ADP producing 3-
phosphoglycerate and ATP.⁵⁹⁶ Other examples include (i) UDP-glucose 6-dehydrogenase that converts UDP-glucose into UDP-glucuronic acid, the precursor in the biosynthesis of bacterial exopolysaccharides (EC: 1.1.1.22),⁵⁹⁷ and (ii) 3-D-phosphoglycerate dehydrogenase that converts D-3-phosphoglycerate to phosphohydroxypyruvate in the serine synthesis pathway (EC: 1.1.1.95).⁵⁹⁸

The TIM barrel fold is the scaffold of at least 15 different enzymatic functions in all life kingdoms.⁴⁷⁷,⁵⁹⁹ Network interaction-conservation phylogenetic analysis allows classification of different groups of TIM barrel fold enzymes, including remote homologues, highlighting structural and functional evolutionary relationships.⁴⁷⁹ Due to the relationship of the α-glucan to the glycolysis pathways, it is worth mentioning three glycolytic enzymes comprising this fold, (i) the triosephosphate isomerase that interconverts dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate (EC 5.3.1.1),⁶⁰⁰ (ii) the pyruvate kinase that catalyzes the final step of glycolysis producing ATP and pyruvate from ADP and phosphoenolpyruvate (PK, EC: 2.7.1.40),⁶⁰¹ and (iii) the 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase that transforms KDPG into pyruvate and D-glyceraldehyde-3-phosphate (EC 4.1.2.14) in the alternative glycolysis Entner-Doudoroff pathway.⁶⁰² Other remarkable enzymes harboring this fold in other parts of the metabolism are (i) the α-subunit of the tryptophan synthase (EC: 4.2.1.20),⁶⁰³ and (ii) the iron-sulfur-flavoprotein trimethylamine dehydrogenase (EC: 1.5.8.2) part of an electron transfer complex.⁶⁰⁴

## 6.5. The Big Picture of Bacterial α-Glucan and Glucose Disaccharide Metabolisms

The joint analysis of the α-glucan and glucose disaccharide biosynthetic pathways reveals some interesting relationships. At first glance, the synthesis of these molecules follows a similar overall 'vertical' pathway comprising (i) the nucleotide-glucose synthesis mediated by NSPases, (ii) the synthesis of an α-glucosyl-bond mediated GT-B fold enzymes, that may be or not be followed by the removal of phosphate by a phosphatase, and (iii) the editing and interconversion of the α-glucose moieties by TIM barrel enzymes. In that sense, only Pep2/Mak appears foreign to this picture and may represent a novelty in this metabolism acquired after LUCA, as suggested by the lack of detection of this enzyme in *Themotagae* and *Aquifex*.²²⁸ Interestingly, this order suggests that the grouping of the early part of the metabolism is mediated by Rossmann fold enzymes (nucleotide-sugar dependent metabolism), while the latest part of the metabolism is driven by the TIM barrel enzymes (nucleotide-sugar independent metabolism; Figure 18).

A horizontal view of the overall metabolism discloses the direction of evolutionary specialization of the different enzymes participating in the α-glucan and disaccharide pathways, suggesting the common ancestry of these enzymes. Interestingly, as we analyzed, in some species, the level of specialization can virtually separate these pathways; meanwhile, in others, it can be intertwined by the enzyme promiscuity, permitting a level of redundancy that can offer an evolutive advantage. This horizontal view also accounts for the specialization of GT-B disaccharide synthases. It can be speculated an ancestral enzyme to GlgM, a processive GS, and a processive GP that also acquired PLP to achieve the α-glucan depolymerization. In this general context, the synthesis of internal glycogen appears as a specialization that allows virtually enclosing this α-glucan energy storage broadly

distributed in bacteria, implying an important evolutive advantage, whereas the GlgE pathway appears mostly centered on actinobacteria (Figure 18).⁶⁰⁵

Finally, TIM barrels appear associated to the intracellular and extracellular milieu, suggesting a primitive function of the corresponding polysaccharides associated with external energy sources or structural functions. Most of the interconversions mediated by the TIM barrel enzymes in this metabolism are phosphate-independent, and may be originated as part of the recent proposed ancient phosphate-free cryptic core metabolism.⁶⁰⁶ GlgE and S6FP are exceptions to this rule and their functions may have emerged in modern metabolism (Figure 18).

# 7. α-GLUCANS AND MICROBIAL PATHOGENICITY

Genes encoding enzymes involved in biosynthesis and mobilization of α-glucans are widespread among bacterial pathogens employing very diverse pathophysiological strategies. The specific contribution of α-glucan metabolism to virulence, pathogenesis, and potentially immune evasion has been investigated for several important pathogenic bacteria, and our knowledge on these aspects is summarized in the following sub-sections.

## 7.1. Mycobacterium Tuberculosis

The human pathogen *M. tuberculosis* produces α(1→4)-linked and α(1→6)-branched glycogen-like α-glucan exclusively via the GlgE pathway (Figure 3B). Two alternative routes, the TreS-Pep2/Mak and the GlgC-GlgM pathways, provide the substrate maltose 1-phosphate that is converted to the branched polymer by iterative cooperativity between the maltosyltransferase GlgE and the branching enzyme GlgB. The polymer is synthesized intracellularly, and then a portion of it is secreted by an unknown transport mechanism to form the major constituent of the capsular layer.¹¹⁹

Synthesis of glycogen-like α-glucan is relevant for pathogenesis of *M. tuberculosis* in two aspects. First, several genes involved in the α-glucan metabolic network are essential and, thus, represent potential drug target candidates. The maltosyl-transferase GlgE was found to be essential because loss of enzymatic activity leads to intracellular build-up of its substrate maltose 1-phosphate, which is toxic to the bacterial cells for reasons that have not been fully elucidated yet.²¹² Similarly, the branching enzyme GlgB is essential because it is required for iterative production of the glycogen-like α-glucan polymer in cooperation with GlgE. Loss of GlgB enzymatic activity curtails GlgE activity since GlgE alone can only produce linear α(1→4)-linked maltooligosaccharides that become insoluble and provide less non-reducing ends than branched glucans to be extended by GlgE. Due to these reasons, inactivation of GlgB also causes accumulation of toxic levels of maltose 1-phosphate.²¹² In addition, a similar sugar phosphate-related toxicity supports essentiality of the trehalose 6-phosphate phosphatase OtsB2 in *M. tuberculosis*, which produces the trehalose substrate for the TreS-Pep2/Mak pathway, as inactivation causes accumulation of its substrate trehalose 6-phosphate.²²⁵ Furthermore, while the genes encoding trehalose 6-phosphate synthase OtsA and maltose 1-phosphate-producing glucosyltransferase GlgM are individually dispensable, their combined inactivation is synergistically lethal likely because of accumulation of their joined substrate ADP-glucose reaching toxic levels.¹¹⁹
Second, glycogen-like α-glucan was shown to be important for full virulence of *M. tuberculosis* in a mouse infection model. A mutant lacking the gene encoding maltose 1-phosphate-producing glycosyltransferase GlgM (described as glycogen synthase GlgA before knowing the configuration of α-glucan metabolic pathways in *M. tuberculosis*) that has reduced levels of capsular α-glucan showed impaired virulence in mice.⁸² With the recent elucidation of the complete metabolic network and pathways required for α-glucan production in *M. tuberculosis*, it was possible to generate defined mutants completely devoid of α-glucan in a rational way. This was mediated by blocking maltose 1-phosphate synthesis in a ΔglgC ΔtreS double mutant. This double mutant exhibited significant reduction in virulence in mice in contrast to the corresponding single mutants. Since these mutations affected both intracellular and capsular α-glucan likewise, which likely fulfill different biological functions, it is unknown to which degree the loss of each α-glucan type contributed to the observed attenuation phenotype. As the outermost layer of the mycobacterial cell wall and first line of contact with host cells, capsular α-glucans may be important for *M. tuberculosis* pathogenesis by interacting with mammalian host cells and influencing the immune response to *M. tuberculosis*. In fact, in vitro experiments using purified capsular α-glucan from mycobacteria demonstrated that it can interact with complement receptor 3, thus mediating binding of *M. tuberculosis* to phagocytic cells.⁶⁰⁷,⁶⁰⁸ Capsular α-glucan has also been reported to block dendritic cell functions⁶⁰⁹ and to interact with the C-type lectin receptor DC-SIGN on dendritic cells.⁶¹⁰ While it is intriguing to speculate that *M. tuberculosis* uses capsular α-glucans to modulate and evade antimicrobial immune effector functions of phagocytic cells, there is a puzzling complexity and reciprocal promiscuity regarding interaction of mycobacterial capsule components with immune receptors, i.e., one molecule can be a ligand for several receptors and one receptor can be triggered by several ligands.⁶¹¹ Unless the still pending discovery of the mechanism of secretion will allow the generation of *M. tuberculosis* mutants specifically defective only in capsular glycogen-like α-glucans that still produce normal levels of the intracellular polymer, the precise function and importance of capsular glycogen-like α-glucans for pathogenesis of *M. tuberculosis* and other pathogenic mycobacteria remain elusive.

## 7.2. Chlamydiae

Chlamydiae are Gram-negative bacteria that employ an obligate intracellular lifestyle, which is linked to a massive genome reduction and a biphasic developmental cycle that includes two distinct stages: the elementary body and the reticulate body. While the elementary body is a non-dividing and infectious form adapted to extracellular survival, the reticulate body constitutes a replicating form that is surrounded by a membrane forming an inclusion.⁶¹²⁻⁶¹⁴ The order Chlamydiales includes the *Chlamydiaceae* family and several family-level lineages collectively called *Chlamydia*-related bacteria.⁶¹⁵ The *Chlamydiaceae* family includes human pathogens such as *Chlamydia trachomatis*, which is the most common sexually transmitted infection leading to infertility, extrauterine pregnancy, and miscarriages in females; *Chlamydia pneumoniae*, which causes acute respiratory diseases of the upper and lower respiratory tract, such as pneumonia and bronchitis; and *Chlamydia psittaci*, which is a leading cause of zoonotic avian chlamydiosis and mainly causes respiratory

infections followed by systemic dissemination to the heart, liver, and gastro-intestinal tract. Furthermore, different species of the *Chlamydiaceae* family can also infect different animals such as *Chlamydia muridarum*, which infects rodents.⁶¹⁶

While for other intracellular bacteria the adaptation to a strict intracellular life-style led to the loss of glycogen metabolism pathway,⁵⁵⁷ nearly all sequenced chlamydial genomes with the exception of *Criblamydiaceae* and *Wad-dliaceae* families maintained the GlgC-GlgA-GlgB dependent glycogen pathway.²²² *Chlamydia trachomatis* and the closely related rodent pathogen *Chlamydia muridarum* are unique amongst Chlamydiae phylum as they accumulate α(1→4)-linked and α(1→6)-branched glycogen-like α-glucan not only intracellularly but also extracellularly in the inclusion lumen.⁶¹⁷,⁶¹⁸ Glycogen accumulates first in the inclusion lumen and only later in elementary bodies. While extracellular glycogen deposition was initially thought to result from bacterial cell lysis,⁶¹⁷ it was recently shown to represent a specific process involving two mechanisms.⁶¹⁹ First, the host glycogen synthase Gys1, which is known to tightly bind to glycogen,¹⁰⁹ is translocated into the vacuole, resulting in concomitant bulk import of host glycogen. Translocation of cytoplasmic glycogen likely occurs through invagination of the inclusion membrane, but the underlying mechanisms remain elusive yet. Bulk uptake of host-derived glycogen is a minor pathway for glycogen accumulation in the inclusion lumen and contributes only to ca. 20% of total glycogen accumulation.⁶¹⁹ In contrast, the main source of intraluminal glycogen accumulation was found to result from de novo synthesis of glycogen that is mediated by secretion of the glycogen synthase GlgA⁶²⁰ and the branching enzyme GlgB⁶²¹ by the bacteria inside the inclusion. Furthermore, the host transporter SLC35D2 and possibly further transporters are recruited to the inclusion membrane to allow translocation of UDP-glucose from the host cell cytoplasm into the inclusion lumen.⁶¹⁹ While glycogen synthase GlgA from other bacteria typically prefers ADP-glucose as the substrate, GlgA from *C. trachomatis* can also utilize host-derived UDP-glucose to mediate glycogen formation in the inclusion lumen.⁶¹⁹ Consistently, in contrast to GlgA and GlgB, chlamydial GlgC was not found to be secreted and remains intracellularly in the bacterial cells, highlighting the fact that intraluminal glycogen synthesis relies on host-derived UDP-glucose as the substrate.⁶¹⁹ While secretion of GlgA, GlgB, and other proteins into the inclusion lumen was reported to be mediated by the bacterial type 3 secretion system,⁶¹⁹ another study suggested that *C. trachomatis* employs a plasmid-dependent and type 3 secretion system-independent mechanism to export GlgA.⁶²² Early during infection, the chlamydial cell population largely consists of reticulate bodies, and intraluminal glycogen accumulation via de novo synthesis and bulk import sets in between 16 and 20 h post infection.⁶¹⁹ Later during the infection cycle, reticulate bodies start converting into elementary bodies, which are associated with mobilization of intraluminal glycogen. For this, the chlamydial cells secrete the enzymes required for glycogen degradation, i.e., the debranching enzyme GlgX and the glycogen phosphorylase GlgP into the inclusion lumen, likely in a type 3 secretion system-dependent manner.⁶¹⁹ The resulting depolymerization end product, glucose 1-phosphate, is converted to glucose 6-phosphate by the chlamydial phosphoglucomutase MrsA, which is secreted as well.⁶¹⁹ Glucose 6-phosphate can be imported into *C. trachomatis* cells by the hexose phosphate transporter UhpC
and can be used as source of energy and carbon. Later during conversion of reticulate into elementary bodies, the type 3 secretion system is turned off, allowing for intrabacterial activity of the glycogen metabolism enzymes, and glycogen accumulation in the bacteria.⁶¹⁹ The temporal separation of glycogen synthesis and subsequent mobilization allows *C. trachomatis* to sequester a host metabolite and accumulate it extracellularly in an osmotic inert storage form and to rapidly depolymerize and convert it into a readily usable molecule for the bacterium. While the precise role and importance of glycogen synthesis and degradation for virulence of *C. trachomatis* has not been fully elucidated yet, it was demonstrated that loss of the *glgA* gene in *C. muridarum* resulted in a significant reduction of pathogenicity in the genital tract in a mouse infection model, demonstrating the role of GlgA in *C. muridarum*-mediated induction of hydrosalpinx in mice, which is similar to the pathophysiology of *C. trachomatis* infection in women. The reduced infectivity of the *C. muridarum* *glgA* mutant was associated with a decreased ability to trigger inflammation.⁶²³ Consistent with this finding, treatment of human monocytic cell line THP-1 with GlgA protein from *C. trachomatis* elicited the expression of proinflammatory cytokines interleukin-8 (IL-8), interleukin-1beta (IL-1β), and tumor necrosis factor alpha (TNF-α) in a TLR2- and TLR4-dependent manner.⁶²⁴

While nearly all sequenced chlamydial genomes comprise the genes for a fully functional GlgC-GlgA-GlgB dependent glycogen pathway,²²² the *Criblamydiaceae* and *Waddliaceae* families form an exception since the GlgC-GlgA pathway is defective. This is due to genomic rearrangements causing deletion of both the *glgC* and *glgP* genes and a fusion of the *glgA* and *glgB* genes rendering the branching enzyme domain inactive.²²² Instead, a complete and functional GlgE pathway was identified in five chlamydial species distributed in *Criblamydiaceae*, *Waddliaceae*, and *Parachlamydiaceae* families including the emerging human pathogen *Waddlia chondrophila*,⁶²⁵,⁶²⁶ comprising genes encoding the maltosyltransferase GlgE, the branching enzyme GlgB2, and a bifunctional TreS-Mak fusion protein, mediating glycogen biosynthesis from trehalose.²²² Furthermore, these organisms also encode the enzymatic machinery for degradation of glycogen comprising glycogen phosphorylase GlgP2, debranching enzyme GlgX, and $\alpha(1\to4)$-glucanotransferase MalQ. Thus, glycogen metabolism is remarkably preserved also in environmental Chlamydiae suggesting an important function for their intracellular lifestyle.²²² However, no specific glycogen-defective mutants are available yet to address the role a glycogen metabolism for infectivity and pathogenesis.

Altogether, while not strictly essential for viability, the capability to synthesize and to mobilize glycogen-like $\alpha$-glucan appears to be a conserved metabolic feature of *Chlamydiae* that contributes to the virulence potential of pathogenic members of this phylum. This might represent a storage function particularly important regarding fueling essential metabolic pathways in the extracellular forms, i.e., elementary bodies of *Chlamydiales*.

### 7.3. *Pseudomonas aeruginosa*

*P. aeruginosa* is an important human pathogen and one of the most common causes of nosocomial infections. It is an opportunistic pathogen that primarily affects immunocompromised individuals, notably patients with immunodeficiencies and traumatic burn wounds. It is also a major pneumonia agent

causing both acute and chronic lung infections, the latter particularly associated with cystic fibrosis and the ability of the bacterium to form biofilms.⁶²⁷ Due to its high intrinsic antibiotic resistance, *P. aeruginosa* infections are difficult to eradicate.⁶²⁸ *P. aeruginosa* is also ubiquitously found in the environment in soil and aquatic habitats, and strains of *P. aeruginosa* as well as of related species such as *P. syringae* can be pathogenic to plants.⁶²⁹,⁶³⁰

*P. aeruginosa* is peculiar in producing $\alpha(1\to4)$-linked and $\alpha(1\to6)$-branched glycogen-like $\alpha$-glucan employing both the GlgE pathway and the classical glycogen synthase GlgA as has recently been reported for strain PAO1 (Figure 3C).¹⁷³ Production of glycogen through GlgA is surprising since *P. aeruginosa* PAO1 lacks AGPase GlgC. However, GlgA was shown to be specific for UDP-glucose and thus contrast to typical bacterial glycogen synthases that prefer ADP-glucose. UDP-glucose is produced in this strain from UTP and glucose 1-phosphate by the UGPase GalU to be used as substrate for GlgA and other enzymes.¹⁷³ More surprisingly, it was shown that GlgA can produce linear $\alpha(1\to4)$-linked $\alpha$-glucan with a DP >40, but this polymer is readily degraded and metabolized to trehalose employing the TreY-TreZ pathway in a cellular context before glycogen particles are formed.¹⁷³ In fact, this is the only route to trehalose formation in *P. aeruginosa* PAO1, which lacks the OtsA-OtsB pathway that is dominant in most other bacteria. Part of the trehalose synthesized from GlgA-produced $\alpha(1\to4)$-linked $\alpha$-glucan is then converted to $\alpha(1\to4)$-linked and $\alpha(1\to6)$-branched glycogen-like $\alpha$-glucan by the GlgE pathway comprising a bifunctional TreS-Pep2 fusion protein producing maltose 1-phosphate from trehalose and ATP, the maltosyltransferase GlgE and the branching enzyme GlgB in a process very similar to the one described for *M. tuberculosis*.¹⁷³ There is a second route to maltose 1-phosphate in *P. aeruginosa* PAO1 that involves the $\alpha$-glucanotransferase MalQ, which can produce maltose by transferring a glucose moiety from the non-reducing end of a maltoligosaccharide when glucose is used as an acceptor molecule or when maltotriose is used as the donor substrate. The maltokinase activity of the TreS-Pep2 fusion protein can then yield maltose 1-phosphate in presence of ATP. Thus, MalQ is able to bypass the TreY-TreZ-TreS reactions to provide maltose 1-phosphate for the GlgE pathway.¹⁷³ $\alpha(1\to4)$-linked and $\alpha(1\to6)$-branched glycogen-like $\alpha$-glucan that accumulates in *P. aeruginosa* PAO1 is almost exclusively produced by the GlgE pathway, whereas linear $\alpha(1\to4)$-glucan produced by GlgA is demonstrated to accumulate only when TreY-TreZ is inactivated. Consistent with its role in trehalose production, the *glgA* gene is localized in an operon together with the *treY* and *treZ* genes in addition to *glgX* and *malQ*, separated from the GlgE pathway operon comprising the genes *treS-pep2*, *glgE*, and *glgB*, while the *glgP* gene is an orphan.¹⁷³ It remains elusive why in *P. aeruginosa* PAO1 only the $\alpha(1\to4)$-glucan produced by GlgA is virtually quantitatively degraded and converted to trehalose, while the GlgE pathway produces polymeric glycogen particles. It appears to be a futile cycle to build linear $\alpha$-glucan only for synthesis of trehalose and subsequently to produce $\alpha$-glucan from it again. But in the face of a lack of the OtsA-OtsB route for trehalose, coexpression of the *glgA*, *treY*, and *treZ* genes from the same operon and possibly direct interaction of the corresponding proteins might enable efficient trehalose formation. Trehalose is not quantitatively, but only partially, converted to glycogen-like $\alpha$-glucan and accumulates in the cytoplasm of the bacterial cells itself in response to
certain stress conditions.173 Theoretically, specificity of GlgA for UDP-glucose might allow *P. aeruginosa* PAO1 to sequester this metabolite from host cells during infection similar to what has been described above for *C. trachomatis*. However, this would require the ability of *P. aeruginosa* PAO1 to take up extracellular UDP-glucose, but no such activity has ever been reported for this bacterium.

Both trehalose synthesized by the GlgA-TreY-TreZ route and glycogen-like α-glucan synthesized by the GlgE pathway play important, yet distinct, roles in stress protection in *P. aeruginosa* PAO1. Trehalose is specifically required for tolerance to osmotic stress, whereas the GlgE-derived α-glucan mediates desiccation tolerance. In turn, GlgE-derived α-glucan has a minor effect on osmotic sensitivity, while trehalose does not contribute directly to the desiccation response.173 Both trehalose and glycogen-like α-glucan were shown to be equally important for survival on abiotic surfaces.173 However, it remains to be elucidated to which degree each molecule contributes to virulence and pathogenesis of *P. aeruginosa* PAO1 in various infection models. Previously, deletion of the *treS* and *treY-treZ* genes, respectively, in *P. aeruginosa* strain PA14 was shown to abolish trehalose production and cause attenuation of virulence in a plant infection model, implicating trehalose as a stress protectant and possible virulence factor.631 Similarly, *treS* gene disruption in *P. syringae* pv. tomato resulted in osmotic sensitivity and attenuation during plant infection.632 However, the conclusions back at the time were drawn without knowledge of the full network and close interaction of the metabolism of trehalose and glycogen-like α-glucan in *Pseudomonas*. Given that the mentioned *Pseudomonas* strains very likely exhibit the same metabolic configuration as *P. aeruginosa* strain PAO1, the mutations in both cases would also lead to abolition of α-glucan biosynthesis. Thus, the virulence phenotypes previously attributed to trehalose could in fact rather be mediated at least partially by α-glucan. In contrast to the reported role in phytopathogenesis, deletions of the *treS* and *treY-treZ* genes in *P. aeruginosa* strain PA14 did not attenuate growth in a range of animal infection models.631 However, the used models were unlikely to reflect the significant osmolarity and desiccation stresses present during cystic fibrosis in humans. Therefore, the importance of glycogen-like α-glucan for human pathogenesis of *P. aeruginosa* remains to be fully investigated.

## 7.4. Biofilm-Forming Bacteria (Streptococcus, Neisseria, Aeromonas)

The ability to form biofilms is an important virulence trait of several pathogenic bacteria. While most of them produce an extracellular matrix composed of polysaccharides other than α-glucan, α-glucan is an important component of the EPS of bacterial biofilms related to dental plaque formation and caries development. Within the complex oral microbiome, the facultatively anaerobic gram-positive coccoid bacterium *S. mutans* is a major producer of EPS, the composition of which is complex and changes dynamically depending on food uptake. Within the mixture of EPS, extracellular DNA, and lipoteichoic acid, α-glucan can comprise between 10 and 20% of the dry weight of dental plaque.633–637 Within the complex and dynamically changing polymicrobial oral microbiome, *S. mutans* is not always the most abundant species, and many other bacteria contribute to acidogenesis and cariogenicity.638,639 However, *S. mutans* is a major EPS producer and rapidly modulates the formation of cariogenic biofilms when

appropriate dietary substrates (i.e., sucrose and starch) are available.640–643 The role of extracellular α-glucan production by *S. mutans* in caries development has extensively been reviewed recently.137 Briefly, *S. mutans* secretes three different glucosyltransferases, which have distinct but partially overlapping, redundant functions. GtfB (formerly known as Gtf-I) and GtfC (Gtf-SI) are mutansucrase that synthesize water-insoluble mutan, which is α-D-glucan mainly composed of α(1→3) linkages.644,645 The secreted mutansucrase are incorporated into pellicle (particularly GtfC) but also adsorb on the bacterial cell surface (mainly GtfB), and both utilize dietary sucrose to synthesize water-insoluble mutan in situ. Furthermore, the secreted mutansucrase can also adsorb onto the surface of other oral microorganisms that do not produce glucosyltransferases themselves, turning them into mutan producers. The water-insoluble mutans facilitate adherence of microorganisms to the smooth surface of the tooth enamel. Concomitantly, salivary α-amylase in the pellicle digests dietary starch, releasing maltose and maltooligosaccharides that can serve as acceptors particularly for surface-adsorbed GtfB and are incorporated into the mutan polymer. *S. mutans* produces and secretes a third glucansucrase enzyme (GtfD, formerly known as Gtf-S) catalyzing the synthesis of water-soluble dextran-like α-D-glucan mainly containing α(1→6) linkages,646 which can serve as primers for GtfB.137 The parallel formation of both water-soluble dextran and water-insoluble mutan enhances the adherence of the oral microorganisms to the smooth enamel surface and promotes dental plaque formation. Accordingly, it was shown that the sucrose-dependent adherence of *S. mutans* to the tooth surface was related to all three glucansucrase enzymes at an optimum ratio.647 The insoluble mutan molecules provide ample binding sites for *S. mutans* and other oral bacteria mediating tight bacterial clustering and adherence to the tooth enamel. This is supported by the secretion of several nonglucosyltransferase glucan-binding proteins, inactivation of which has been shown to result in impaired biofilm formation and altered architecture.83 The α-glucan-containing EPS formed in situ enables the assembly of a spatially heterogeneous and cohesive multicellular structure and development of a cariogenic biofilm.648 The importance of α-glucan production for virulence and cariogenicity of *S. mutans* has been demonstrated by studies of mutants lacking glucansucrase activities. Inactivation of any of the glucansucrase GtfB, GtfC, or GtfD resulted in a reduction of smooth-surface carious lesions in a rat model system.649 Similarly, although the nature of mutations is not known, glucan synthesis-defective mutants of *S. mutans* were shown to cause reduced caries development in rats.650,651

In addition to extracellular mutan and dextran-like α-glucan, *S. mutans* also produces intracellular glycogen employing the classical GlcG-GlcA pathway, which may also be important for virulence. A mutant defective in intracellular glycogen accumulation exhibited reduced cariogenic potential in rats.652 Conversely, a transposon mutant overproducing glycogen showed increased cariogenicity.653 However, it remains unclear whether glycogen accumulation contributes to biofilm formation and how it can influence virulence of *S. mutans* during growth in an environment rich in carbohydrates.

Different *Neisseria* species such as *N. polysaccharea*, *N. mucosa*, and *N. perflava* can also be found in human dental plaque. *Neisseria* secrete a GH13 amylosucrase to produce an extracellular glycogen-like polymer mainly composed of α(1→
4) linkages with few $\alpha(1\to6)$ branches.161-164 While this suggests that extracellular glucan produced by these Neisseria contribute to biofilm and dental plaque formation, no glucan deficient mutants have been described to address their relevance for inhabiting the oral environment.

Members of the genus Aeromonas are gram-negative, water-borne bacteria that are ubiquitously found in aquatic environments. As many strains are able to grow and to produce exotoxins at low temperatures, they are mostly infective to poikilothermic animals, but some species are also emerging as human pathogens causing systemic as well as gastrointestinal infections.654 Aeromonas species produce a surface $\alpha$-glucan consisting of $\alpha(1\to4)$-linked glucosyl units with $\alpha(1\to6)$-branches that is intracellularly synthesized by the UGPase GlgC and the glycogen synthase GlgA before being exported in a WecP-dependent fashion.166-168 The surface-exposed glycogen-like $\alpha$-glucan is important for biofilm formation as was revealed by a GlgA deficient mutant of A. hydrophila.168 While this suggests that glycogen-like $\alpha$-glucan may contribute to virulence of this species, no studies in relevant infection models have been performed to address this hypothesis.

### 7.5. Enteric Bacteria (Escherichia coli, Salmonella enterica, Vibrio cholerae)

Despite providing a mucus layer rich in carbohydrates, lipids, and proteins, the mammalian gastrointestinal tract represents an environment characterized by fluctuating availability of nutrients. Thus, both commensal and pathogenic bacteria in the gut are regularly facing prolonged periods of restricted nutrient availability and even starvation.655,656 To cope with a nutrient-limiting environment and to compete with other microorganisms, the ability to accumulate intracellular carbon and energy storages during nutrient-rich periods and to remobilize them during hunger phases is believed to be essential for persistence of intestinal bacteria. Glycogen is thought to be the primary carbon and energy storage molecule for enteric bacteria that employ the classical GlgC-GlgA pathway for glycogen synthesis (Figure 3A).164 Furthermore, after shedding with feces, glycogen may be important for extracellular survival and possibly promotes dissemination of pathogenic enteric bacteria.93

To assess the importance of glycogen biosynthesis and degradation in E. coli, $\Delta glgA$ and $\Delta glgP$ mutants were assessed in a streptomycin-treated mouse infection model and revealed that both are necessary for efficient colonization of the intestine by E. coli.657 In contrast, conflicting data exist regarding the role of glycogen metabolism for virulence of the closely related gram-negative bacterium S. enterica. Supporting the hypothesis of a central role of glycogen for virulence and infectivity of enteric bacteria, one study showed a correlation between the amount of glycogen stored during the preincubation step and the 50% lethal dose in for S. enterica serovar Enteritidis in a chicken infection model.544 In contrast, in another study, no significant differences in intestinal colonization or virulence in chicken were observed for deletion mutants in the glgC gene encoding AGPase generated in three different S. enterica serovars (Gallinarum, Pullorum, Typhimurium).553 However, while the glgC mutant strains were thought to be deficient in glycogen accumulation because they cannot make the glycogen precursor ADP-glucose, more recent findings suggest that glgC mutants of E. coli and S. enterica serovar Typhimurium possess an alternative pathway for ADP-

glucose formation and still can accumulate glycogen under certain conditions.658 While this alternative route has not been identified and characterized enzymatically yet, it is possible that the importance of glycogen accumulation for virulence and infectivity in E. coli and S. enterica is underestimated when the assessment is based on the phenotype of GlgC-deficient mutants. In favor of this, studies with mutants of the pathogenic enteric bacterium Vibrio cholerae defective in glycogen synthesis or degradation support the importance of glycogen metabolism for pathogenesis.95 Like other enteric bacteria, V. cholerae employs the classical GlgC-GlgA pathway for glycogen synthesis but is peculiar in possessing two genes coding for AGPase, glgC1 and glgC2. V. cholerae mutants defective in glycogen biosynthesis ($\Delta glgC1 \ \Delta glgC2$ double mutant) or degradation ($\Delta glgX$) demonstrated that glycogen prolongs survival in nutrient-poor environments that are known ecological niches of V. cholerae, including pond water and rice-water stool. Additionally, defects in glycogen metabolism strongly attenuated pathogenesis of V. cholerae in an infant mouse transmission model of cholera. Lending further credence to the hypothesis of glycogen being an important contributor to virulence, glycogen granules can be found inside bacterial cells present in rice-water stool from cholera patients.95 Together, these findings implicate glycogen metabolism as a relevant survival strategy employed by enteric bacteria to survive the dramatic changes encountered in the host and the environment. This not only applies to pathogenic enteric bacteria, since glycogen has also been shown to support gut retention of probiotic bacteria in a germ-free mouse model, yielding a significant fitness disadvantage of a $\Delta glgA$ mutant strain of the gram-positive lactic acid bacterium Lactobacillus acidophilus.659

## 8. CONCLUDING REMARKS

More than a century of lessons about the biosynthesis of $\alpha$-glucans has disclosed some functional roles and some patterns in the metabolism of these polysaccharides in bacteria. As we highlight in this Review, we show how bacterial metabolism evolved to handle the $\alpha$-d-glucose scaffold to build assorted $\alpha$-glucans with different functions, including energy storage or extracellular structural components as key elements in matrix biofilms formation, which provide protection to colonizing bacteria, and also as virulence factors. Taken together, the chemical landscape from $\alpha$-d-glucose to disaccharides and $\alpha$-glucans, the analysis of their metabolism and the structural/functional diversity of the intervenient enzymes, we provide an overall picture reflecting the underlying evolution of such enzymatic machinery (Figure 18). Our current view unveils a complexity in $\alpha$-glucans diversity, although as we delve deeper into the fundamentals of heterogeneity, we encounter a nuanced landscape formed by few enzymatic folds linked by the evolution of the metabolism. Further examining the synergies of non-catalytic domains, including CBMs, for such $\alpha$-glucans diversity, highlights the need for further research.

There is still an immense unexplored metabolic diversity landscape in the biosphere, likely influenced differently by such polymers in their niche and their metabolic blueprint. For example, the human gut microbiome, arguably the most studied ecosystem, is composed of ca. 3500 species,660 whose population composition is affected by the structure of dietary $\alpha$-glucans.661 This is due to the different arsenal of enzymes of the diverse bacterial species for their processing;662 therefore, environmental $\alpha$-glucans architecture acts as an environmental
selective pressure, a fact that is already used to shift bacterial populations as medical treatment.⁶⁶³,⁶⁶⁴ In that sense, of special relevance is the so-called resistant starch, a type of starch that resists digestion in the small intestine, progressing to the large intestine where it acts as a prebiotic, feeding the beneficial bacteria in the gut microbiota.⁶⁷⁴,⁶⁷⁵ The use of resistant starch by gut bacteria involves several degradation steps, favoring the production of short-chain fatty acids such as butyrate that serves as a primary energy source for colon cells.⁶⁷⁶ Importantly, this process helps to maintain a healthy balance of gut bacteria, contributes to gut health by promoting bowel regularity, and can enhance the immune system. Therefore, foods high in resistant starch are indicated in a balanced diet for maintaining overall health. Extrapolating this to the biosphere, where diverse calculations predict the existence of ca. 9 million eukaryotic species and ca. 1 trillion microbial species,⁶⁶⁵,⁶⁶⁶ the dimension of the biosphere metabolic landscape suggests that we are only observing the tip of the iceberg. Moreover, most of our knowledge on α-glucans metabolism belongs to bacteria species cultured in the laboratory; nevertheless, the existence of the so-called “microbiological dark matter” represents one-quarter of the population of microbes belonging to phyla with no cultured relatives,⁶⁶⁷ indicating that future research will greatly expand on this metabolism architecture, possibly finding different rewirings of their metabolism, also offering an immense repertoire of new biotechnological opportunities.

# AUTHOR INFORMATION

## Corresponding Authors

**Javier O. Cifuente** – Instituto Biofisika (UPV/EHU, CSIC), University of the Basque Country, E-48940 Leioa, Spain;
*orcid.org/0000-0001-5420-121X*;
Email: javier.cifuente@ehu.es

**Marcelo E. Guerin** – Structural Glycobiology Laboratory, Department of Structural and Molecular Biology, Molecular Biology Institute of Barcelona (IBMB), Spanish National Research Council (CSIC), 08028 Barcelona, Catalonia, Spain; *orcid.org/0000-0001-9524-3184*;
Email: mrccri@ibmb.csic.es

## Authors

**Christophe Colleoni** – University of Lille, CNRS, UMR8576-UGSF -Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France

**Rainer Kalscheuer** – Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University, 40225 Düsseldorf, Germany; *orcid.org/0000-0002-3378-2067*

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemrev.3c00811

## Author Contributions

CRediT: **Javier Orlando Cifuente** conceptualization, data curation, investigation, writing-original draft, writing-review & editing; **Christophe Colleoni** conceptualization, data curation, funding acquisition, investigation, writing-original draft, writing-review & editing; **Rainer Kalscheuer** conceptualization, data curation, funding acquisition, investigation, writing-original draft, writing-review & editing; **Marcelo E. Guerin** conceptualization, data curation, funding acquisition, investigation, writing-original draft, writing-review & editing.

# Notes

The authors declare no competing financial interest.

# Biographies

Javier O. Cifuente graduated in Biochemistry at the Universidad Nacional de La Plata, Argentina (2006). He earned a Ph.D. in Biological Sciences under the direction of Dr. Ricardo M. Gomez at the Institute of Biotechnology and Molecular Biology (2009). He conducted postdoctoral research under the direction of Prof. Susan Hafenstein, at Penn State University College of Medicine, USA, (2009–2012). He continued his postdoctoral training under the guidance of Prof. Marcelo E. Guerin at the Structural Glycobiology Laboratory, (Unit of Biophysics, UPV/EHU-CSIC, CIC bioGUNE and IIS-BioCruces Bizkaia), Spain (2014–2023). He is a postdoctoral researcher under the direction of Prof. Iban Ubarretxena-Belandia, in the Structural Biology of Disease Mechanisms Laboratory, at the Biofisika Institute (UPV/EHU-CSIC).

Christophe Colleoni obtained his PhD in microbial genetics under the supervision of Prof. Steven Ball. He completed a post-doctoral fellowship (1999–2003) on the structural and biochemical characterization of storage polysaccharides produced by various sweet-corn cultivars under Prof. Alan Myers in the Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology (BBMB) at Iowa State University, USA. He is a full professor at the University of Lille, France, and leader of the Integrative Biology of Storage Polysaccharides (IBSP) team at the Institute for Structural and Functional Glycobiology (CNRS-UM8576). His research focuses on the evolution of storage polysaccharide metabolisms in prokaryotes and microalgae.

Rainer Kalscheuer graduated in Biology from the University of Münster, Germany (1998). He obtained his Ph.D. in Biology under the supervision of Prof. Alexander Steinbüchel at the Institute of Molecular Microbiology and Biotechnology, University of Münster, Germany (2003). Following a postdoctoral period at the same institute (2003–2005), he conducted postdoctoral studies under the direction of Prof. William R. Jacobs Jr. at the Howard Hughes Medical Institute at the Albert Einstein College of Medicine, Bronx, United States (2005–2009). He was leader of the Jürgen Manchot Research Group for Molecular Host-Pathogen Interaction at the Institute für Medical Microbiology and Hospital Hygiene, Heinrich Heine University Düsseldorf, Germany (2010–2015). Since 2015, he is professor at the Institute of Pharmaceutical Biology and Biotechnology, Heinrich Heine University Düsseldorf, Germany.

Marcelo E. Guerin graduated in Pharmacy from the University of Buenos Aires, Argentina (1997). He earned his Ph.D. in Chemistry under the direction of Prof. Armando J. Parodi, at the Glycobiology Laboratory, the Leloir Institute of Buenos Aires, Argentina (2002). He carried out postdoctoral studies in the Structural Biochemistry Unit, under the direction of Prof. Pedro M. Alzari, at the Institut Pasteur Paris, France (2003–2007). Additionally, he conducted postdoctoral studies in the Mycobacteria Research Laboratories, under the direction of Prof. Mary Jackson, at Colorado State University, United States (2008–2009). He is a full research professor at the Spanish National Research Council (CSIC) and leader of the Structural Glycobiology Laboratory (2009–present), currently at the Institute of Molecular Biology of Barcelona (IBMB), Spain.

# ACKNOWLEDGMENTS

This work was supported by the MICINN grant PID2022-138694OB-I00 (M.E.G.) and National Institutes of Health grant R01AI149297 (M.E.G.); the Jürgen Manchot Founda-
tion (R.K.) and the CNRS, the Université de Lille CNRS, and the ANR grants "Animalga" (ANR-21-CE20-0051; C.C.). J.O.C. acknowledges financial support from the IKUR Strategy (Ikerbasque Foundation and Fundación Biofísica Bizkaia) and the Department of Education of the Basque Government.

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