Systematic bioprospection for cellulolytic actinomycetes in the Chihuahuan Desert: isolation and enzymatic profiling

Introduction

The quest for alternative energy sources to mitigate climate change has driven the search for microbial enzymes that can sustainably use plant cellulose, the most abundant type of biomass, but also a recalcitrant resource of biological energy (van Vuuren et al., 2017). Achieving inexpensive and environmentally sustainable complete deconstruction of lignocellulose is still a pending task. To address this, researchers are tackling the search for new sources of cellulases by (i) developing cellulolytic consortia (Zhang & Dong, 2022), (ii) screening microorganisms that can survive and function in extreme conditions (Barzkar & Sohail, 2020; Champreda et al., 2019; Elleuche et al., 2014; Sysoev et al., 2021; Verma, 2021; Wu et al., 2018) and (iii) synthetically enhancing cellulases by developing chimeric enzymes (Banerjee et al., 2016; Dadwal, Sharma & Satyanarayana, 2020; Guazzaroni, Silva-Rocha & Ward, 2015). The discovery of new genetic resources and diverse functionality in the enzymatic activity will result in cost-effective, low-energy, and environmentally friendly biofuel producing processes.

Extreme environments have shaped biodiversity driven by harsh physicochemical conditions displayed in a myriad of adaptations in microorganisms of high biotechnological value, including microbial enzymes for a plethora of applications (Manni & Filali-Maltouf, 2022; Pascoal, Magalhaes & Costa, 2020; Sysoev et al., 2021; Tatta et al., 2022; Uma et al., 2020; Wani et al., 2022a). Bacterial enzymes possess key features more advantageous than fungal enzymes such as higher growth rates, genetic versatility (Menendez, Garcia-Fraile & Rivas, 2015) and more specific, efficient catalytic activity in harsh conditions; moreover, bacteria produce multienzyme complexes with greater functionality and specificity in broader conditions (Balla et al., 2022; Castiglia et al., 2016; Maki, Leung & Qin, 2009). Cellulases from actinomycetes associated to insects and other extreme and marine prokaryotes have exhibited high specific activity, thermostability, and other important biochemical properties and hence can contend well with the enzymes from terrestrial sources (Behera et al., 2017; Elframawy et al., 2022; Fathallh Eida et al., 2012; Gong et al., 2017; Gong et al., 2020; Goodfellow et al., 2018; Lewin et al., 2016; Saini et al., 2015; Walia et al., 2017; Xie & Pathom-Aree, 2021).

The Cuatro Ciénegas Basin (CCB) in Northern Mexico is an extreme, oligotrophic environment, and a hotspot of biodiversity thriving in endangered oasis in the Chihuahuan desert (Souza et al., 2012b). Here, the proportions of nitrogen and phosphorus (N:P) are extremely skewed, given the great limitation of phosphorus (P) (157:1) (Elser et al., 2005) or very low nitrogen (N) (1.8:1) (Souza et al., 2006) and sulfur and magnesium (Mg) that replicate marine osmolarity, while being low in NaCl (De Anda et al., 2017; Rebollar et al., 2012; Souza et al., 2018; Wolaver et al., 2012). The Churince System is the most unusual hydrological system within the CCB due to its higher altitude within the valley (around 30 m above most of the basin); the system depends on mostly deep ancient water with a magmatic influence (Wolaver et al., 2012) and a calcium soil matrix (Elser et al., 2005). This peculiar environment had favored a great diversity of microorganisms that have thrived in conditions of scarcity and isolation, creating a so-called “lost world” (Souza et al., 2018), including diverse actinobacteria, proposed to be endemic to CCB ponds (Arocha-Garza et al., 2017) and metabolically unique (Gonzalez-Salazar et al., 2023) have been previously described.

Despite accumulating evidence of catalytic novelty in non-culturable independent studies (Georgiadou et al., 2021; Manni & Filali-Maltouf, 2022; Schaller et al., 2022; Sysoev et al., 2021; Wani et al., 2022a; Wani et al., 2022b), and reports from isolated highly valuable microbial collections from unexplored sites (Gonzalez-Salazar et al., 2023; Swiecimska, Golinska & Goodfellow, 2022), there is still a need for efficient and detailed systematic bioprospecting studies for cellulolytic actinomycetes. Work describing scrutinization criteria and the rationale behind the selective methodology of microbial collections could enhance bioprospection efforts in underexplored biomes that will effectively filter “gifted microbes” harboring more valuable enzymes and other natural products. This work aimed to develop a systematic detailed bioprospecting exploration of actinomycetes isolated from the CCB oligotrophic ponds, with high potential of enzymatic deconstruction of cellulose.

Materials & Methods

Sampling site and actinomycetes collection

The complete actinomycetes collection consisted of 196 isolates from seven ponds in the Cuatro Ciénegas Basin (Fig. 1). Isolates belong to the Microbiological Collection of Instituto de Biotecnología-UANL and the data is described in (Table S1). Nearly 90% of isolates had been previously reported (Arocha-Garza et al., 2017; Arocha-Garza, 2018). Access and permission to Natural Protected Area was given by SEMARNAT in document id.no. SGPA/DGVS/01644/19. Only 20 actinomycetes from the collection are newly reported in this work. Briefly, 5 g of sediment from the pond were retrieved with approximately 35 mL of the water column from the pond. Samples were not refrigerated, instead, samples were immediately (2–6 h after sampling) transferred by plating 200 µL of the sediment-water suspension to isolation media (Table S1). Plates were incubated at 28°–30 °C for 7–10 days or until single colonies were able to be picked and transferred to a clean plate for secondary isolation.

Screening and selection of highly cellulolytic actinomycetes

We first screened our CCB actinomycetes collection for cellulolytic isolates. We designed and applied a systematic process that we summarize in Fig. 2.

Primary screening of cellulolytic activity

A primary screening of the cellulolytic potential of axenic cultures of actinomycetes was performed by the petri plates assay method. Actinomycete collection strains were inoculated on carboxymethylcellulose medium-CCM1-(Carboxymethylcellulose sodium salt (CMC) 10 g/L, K₂HPO₄ 2 g/L, (NH₄)₂ SO₄ 0.35 g/L, urea 0.0748 g/L, agar 15 g/L, KENT^® Marine Reef Salt 25 g/L, a pH 7.0 ± 0.1) as follows: (i) an agar plug of 0.5 cm diameter from actively growing actinomycete–sporulated ISP2 culture was transferred to the center of a CCM1 agar plate. (ii) plates were incubated at 30 °C for 10 days; (iii) growth was considered positive when active vegetative growth was >2 mm of length around the agar-plug inoculation area; (iv) Congo-red test was used to measure the diameter of the hydrolysis zone after ten days of culture as an indicator for cellulose degradation (Gupta, Samant & Sahu, 2012). The experiment was performed using three replicates for each strain. The average of three experiments was calculated with the corresponding standard error of the mean. Isolates displaying growth on CMC1 (>2 mm) and a hydrolysis zone revealed by Congo red -(>30 mm diameter), were selected for secondary screening evaluation. Strains displaying growth <2 mm on CCM1, or a cellulose degradation zone <30 mm diameter, were discarded. In addition to the previously described biological collection, two positive controls were included: a strain of Streptomyces coelicolor, previously described as cellulose degrading and a Trichoderma harzianum strain from Instituto de Biotecnología Microbial Collection. Negative controls were also included: an Escherichia coli strain, and non-inoculated plate was used as negative control.

Secondary screening of cellulolytic actinomycetes by determination of Enzymatic Index

Twelve isolated actinomycete strains with larger hydrolysis zones (>30 mm diameter) were selected for secondary screening. These microorganisms were incubated for seven days at 27 °C on CCM1 medium previously described. The measurement of the hydrolysis halo and of the colony was performed with the ImageJ software (version 1.2) analysis program by calibrating the program with the measurement scale present in each acquired image. The enzyme index (EI) (da Cruz Ramos et al., 2016; Florencio, Couri & Farinas, 2012; de Paula et al., 2016; Soy, Nigam & Sharma, 2019), of each strain was calculated as: $EI=\frac{Hydrolysiszonediameter}{Colonydiameter}$

Statistical analysis of Enzymatic Index determination experiment

The presented experiment was performed using three replicates for each strain. The average of three replicates was calculated and the standard error of the mean (SEM). The statistical adequacy of the model was determined through analysis of variance (ANOVA) and Tukey test, with 95% confidence performed Minitab Statistical software (Version 17). The visualization of results was graphed using GraphPad Prism software (version 9.2.0).

Results

Screening and selection of highly cellulolytic bacteria

This study performs a systematic bioprospection of the Cuatro Ciénegas Basin (CCB) for the isolation and evaluation of actinomycetes retrieved from an extreme environment with demonstrable cellulolytic capacity (Fig. 2). The study explored an extensive and diverse actinomycete collection from the CCB for the characterization of β-1,4-glucanase–producing actinomycete strains. Seven ponds in CCB were sampled: Pozas Rojas (PR), Pozas Azules (PA), Mojarral (MO), Becerra (BE), Archaean Domes (AD), Anteojo (AN), and Intermediate Lagoon in the Churince System (CH) (Fig. 1) and 196 morphologically distinct actinomycete isolates were subjected to primary screening using CCM1 media (Fig. 2A).

In this primary screening, 156 of the 196 evaluated strains were considered cellulolytic (78% of total actinomycetes strains) (Table S1), because they had the ability to grow on CCM1 media. Two large groups of cellulolytic microorganisms, those whose hydrolysis halo size was <30 mm in diameter, were considered to have less cellulose degradation. Actinomycetes whose halo was >30 mm in diameter were identified with the greatest potential to degrade cellulose under the conditions of this study (Fig. 2B). According to our study, the positive control (S. coelicolor) strain obtained a hydrolysis halo <30 mm in diameter.

Of the 156 cellulolytic isolates, 12 showed a significantly large diameter of hydrolysis zone values (>30 mm) and were selected for further evaluation in plate assay method in a secondary screening (Fig. 2B). After calculating enzymatic index (EI), (da Cruz Ramos et al., 2016; Florencio, Couri & Farinas, 2012; de Paula et al., 2016; Soy, Nigam & Sharma, 2019), our results showed two major groups in our collection, one with an enzyme index above two and one with values below two as shown in Fig. 3. Statistical analysis showed significant difference of isolates with EI values below two (p-value = 0.043), i.e., group b (PR105, PR108-2). However, the isolated STCH565-A stands out among other isolates not only for its highest enzymatic index (EI = 3.8) but also for the larger halo diameter zone (Fig. 3).

Measurement of enzyme activity

The strain Streptomyces sp. STCH565-A was finally selected to continue with the measurement of endoglucanase activity (Fig. 2C). In a submerged culture, biomass presented several development events. At the beginning, dry weight increases probably due to the immediate consumption of glucose, as it is a less complex carbon source and implies less energy expenditure for the microorganism, therefore, a preference for it is assumed. After a brief fall, biomass stabilizes, followed by a second fall before increasing and finally a fall before a last increase, which coincides with the sporulation of the microorganism. Endo- β-1,4-glucanase activity (in terms of volumetric activity) values are reported from day 4 of culture, coinciding with the consumption of glucose available in the medium; these values ranged from 0.15 U/mL to 0.27 U/mL (Fig. 4A). Regarding the enzymatic activity, three important moments to consider were reported, with values ranging from 56 U/mg to 149 U/mg (Fig. 4B).

Morphological characterization

Actinomycete strain STCH565-A formed a well-developed aerial mycelium with good sporulation on ISP solid medium. The morphological characteristics of STCH565-A growth, substrate mycelium color, aerial mycelium color, and pigment are presented (Table 1). Noticeably, STCH565-A isolate displayed melanin pigment in ISP6 and ISP7 agar medium (Table 1). Sucrose was the best carbon source exhibited maximum growth, as well as STCH565-A exhibited growth on arabinose, xylose, mannitol, fructose, Rhamnose and effectively on cellulose. While on inositol STCH565-A did not show growth (Table 1).

Below there is a general description of the observations of strain STCH565-A in liquid culture medium in Fig. 5A. Three stages of development can be identified in the microorganism at the macroscopic level: the first corresponds to vegetative growth on the substrate with yellow biomass, followed by the beginning of sporulation which is observed in white and finally a stage of aerial growth (sporulation) lilac white, lilac, or light violet depending on the color scale used (Table 1).

Table 1:

Characteristics of Streptomyces sp. STCH565.A grown in different media.

Characters studied	Features
Morphological characteristics	Substrate mycelium	Aerial mycelium
Tryptone-yeast extract broth (ISP1)	Yellow	White
Yeast malt extract agar (ISP2)	Light yellow	Lilac
Oatmeal agar (ISP3)	Transparent Ivory	Ivory
Inorganic salt agar (ISP4)	Light yellow	Yellow orange
Glycerol asparagine agar (ISP5)	Yellow	Cream-white
Physiological characteristics	Diffusible pigments
Peptone yeast extract agar (ISP6)	+
Tyrosine agar (ISP7)	+
Carbon utilization test	*Colony Growth
Arabinose	+
Sucrose	++
Xylose	+
Inositol	-
Mannitol	+/-
Fructose	+
Rhamnose	+
Cellulose	+

DOI: 10.7717/peerj.16119/table-1

Notes:

Assay were carried out as mentioned in Shirling and Gottlieb (1966).

*Strongly positive utilization (++) when growth on tested carbon in basal medium is equal to or greater than growth on basal medium plus glucose.

Positive utilization (+) when growth on tested carbon is significantly better than on basal medium without carbon, but somewhat less than on glucose.

Utilization doubtful (+/-) when growth on tested carbon is only slightly better than on the basal medium without carbon and significantly less than with glucose.

Utilization negative (-) when growth is like or less than growth on basal medium without carbon. (Always record utilization as negative if growth is not better than no-carbon control.

Phylogenetic reconstruction of Streptomyces sp. STCH565-A

Taxonomic annotation of contigs was generated using the Genome Taxonomy Database (GTDB-Tk) v1.0.2 with a set of 115 unique genes and markers predicted by Prodigal v2.6.3 and HMMER v3.1b1 respectively, to be finally concatenated and compared by GTDB-Tk as well as an Average Nucleotide Identity (ANI). After a taxonomic classification, the genome was more closely related to Streptomyces olivaceus (GCF_000721235.1) with a 97.91% FastANI comparison followed by Streptomyces sp. MH60 (GCF_002939385.1) with 90.27%, Streptomyces rubrogriseus (GCF_003112595.1) with 90.04% and Streptomyces violaceorubidus (GCF_000717995.1) with 89.99%. The visualization of the phylogenetic tree (Fig. 6) of Streptomyces STCH565-A also showed a close relationship with Streptomyces coelicoflavus (GCF_003112555.1) and Streptomyces sp. BK208 (GCF_004364245.1).

Discussion

The Cuatro Ciénegas Basin (CCB) harbors diverse and unique microbiological diversity in its multiple ponds (Medina-Chávez et al., 2023; Moreno-Letelier et al., 2012; Rodriguez-Torres et al., 2017; Rodriguez-Verdugo et al., 2012; Taboada et al., 2018; Tapia-Torres et al., 2016; Velez et al., 2016). In particular, the Churince hydrological system is an unusual site of a marked oligotrophy but a great endemic microbial biodiversity (Cerritos et al., 2011; Elser et al., 2018; Minckley & Cole, 1968; Perroni et al., 2014; Taboada et al., 2018; Velez et al., 2016). Unique lineages of cultivable Streptomyces had previously been reported to thrive in the oligotrophic intermediate lagoon of Churince (Arocha-Garza et al., 2017), at least, before it completely dried up (Carson, 2022).

Our results show that 78% of evaluated CCB actinomycetes produce extracellular hydrolytic enzymes using carboxymethylcellulose (CMC) as the only carbon source. Few screening studies that find enzymatically “gifted microbes” able to hydrolyze cellulolytic substrates report the process behind their findings, we have compared our collection (Table S1) to other similar reports of bioprospection (Table S2). Compared to others, the CCB collection reveals a high potential for cellulolytic enzymes. CCB waters, particularly in the Churince, had shown unusual extreme stoichiometric nutrient ratios, generating nutritional stresses that dictate population dynamics perhaps more akin to earlier times in Earth’s history rather than more extant environments. Extreme oligotrophy has likely contributed to biological endemism (Souza et al., 2008; Souza et al., 2012a), which might explain the enriched community of highly cellulolytic actinomycetes, since this evaluated collection was selectively isolated for bioprospection without a carbon-usage bias.

A very recent study reveals the relationships between competition and polysaccharide decomposition, in which authors demonstrate that microbial diversity and competition affect the stability and function of cellulose-degrading communities where antagonism is prevalent in highly cellulolytic enrichment lines (Lewin et al., 2022). These results support our hypothesis that CCB is an enriched source of highly cellulolytic microorganisms that, because of oligotrophy and carbon limitation, harbor valuable microbial communities of gifted microbes.

Because nearly 80% of our collection was able to use CMC as only carbon source, we evaluated cellulose degradation in CCM1 agar medium in terms of enzymatic index (EI) to select the best possible cellulolytic candidate. Calculating EI is used to evaluate enzyme-producing prospects within the same genus (Teather & Wood, 1982) and was previously used for this purpose in other studies (Barzkar & Sohail, 2020; Fatokun, Nwodo & Okoh, 2016; Lewin et al., 2022; Sarkar & Suthindhiran, 2022). In this study, 12 out of 196 strains showed high EI values (≥ 3.0) and were considered as highly cellulolytic, so they were selected for further quantitative enzymatic assays (Fig. 4).

The volumetric enzymatic activity (Fig. 4) report shows us that the selected strain Streptomyces sp. STCH565-A presents higher values (0.27 U/mL) than the Streptomyces CC48 strain (0.11 U/mL) (Celaya-Herrera et al., 2020), previously isolated also from Cuatro Ciénegas (both values obtained when their optimization has not been carried out).

Streptomyces sp. STCH565-A volumetric activity values seem barely satisfactory when compared to other reports of non-optimized processes (Budihal, Agsar & Patil, 2016; Grigorevski de Lima et al., 2005). However, in this work, Streptomyces sp. STCH565-A- specific enzymatic activity measured for the crude extract (CE) of a non-optimized process was 149 U/mg after 15 days of non-fed submerged culture. Furthermore, specific enzymatic endoglucanase activity measured in crude extract, is also elevated after several days in the growth curve. After four days of culture, a specific enzymatic activity of 66.7 U/mg was found and after seven days, a specific enzymatic activity of 56.8 U/mg. Detailed studies of the characterization of the enzyme could give an approximation to an apparent affinity for the substrate. We can say that Streptomyces sp. STCH565-A has a high specific enzymatic activity when compared to another cellulolytic Streptomyces. For instance, a previous study reports 0.205 U/mg of cellulolytic activity (Fatokun, Nwodo & Okoh, 2016) while another measurement eaches 4.38 U/mg (Sujatha & Hemalatha, 2020).

The specific enzymatic activity is relevant in biotechnological terms (Budihal, Agsar & Patil, 2016) Because. Although there are different commercial cellulases obtained from fungal strains, their enzymatic activity is limited to volumetric activity, but not to its specificity to the substrate. This volumetric activity implies an increase in production costs because large amounts of these cellulases are required to achieve complete hydrolysis of polymeric matrices susceptible to enzymatic attack (Quinlan, Teter & Xu, 2010). Although high values of cellulolytic activity were reached under the evaluated conditions, the fermentation time necessary for Streptomyces sp. STCH565-A could be considered an obstacle for scaling processes because of the low bioconversion speed that leads to conversion: co-production of inhibitory compounds added to the size of the infrastructure (Manzanares, 2010). An alternative solution to consider as a research perspective would be the isolation and heterologous expression of the genes that were identified as responsible for the enzymatic activity (Arsov, Petrov & Petrova, 2021).

Regarding identification of selected strain, a previous work had already alerted the scientific community of the risks of under-appreciating diversity between strains with identical or nearly rRNA genes (Antony-Babu et al., 2017). Although this work phylogeny was based on the analysis of 115 marker genes, Streptomyces sp. STCH565-A was found to be closely related to Streptomyces olivaceus.

By and large, the degradation of polysaccharides into sugar monomers requires the synergistic action of several classes of carbohydrate-active enzymes (CAZyme). Among the CAZymes, glycoside hydrolases (GH) are the main family of enzymes involved in the degradation of polysaccharides such as starch and cellulose (Sidar et al., 2020).

Genomic analysis of the CAZyome showed that this Streptomyces sp. STCH565-A encodes the genes necessary for cellulose hydrolysis. The genes found in the CAZy database coincide with an important family of enzymes to degrade cellulose (Endoglucanase, GH12) as well as families of glycosyl-hydrolases such as GH5-GH6 (celobiohydrolases) and GH1-GH3 (β-glucosidases), forming a robust cellulolytic complex.

In the CAZy database, endoglucanases are classified into 15 different families, GH5, GH6, GH7, GH8, GH9, GH10, GH12, GH26, GH44, GH45, GH48, GH51, GH74, GH124 and GH148. Exoglucanases are divided into 5 families of GH5, GH6, GH7, GH9 and GH48 (Nguyen et al., 2018). Two of the most abundant domains found in the Streptomyces sp. STCH565-A genome belong to the GH1 and GH3 families (β-glucosidases [BG]), which are found in at least five gene copies; this is a considerable abundance compared to the CAZyome of the soil-isolated Streptomyces yeochonensis CN732 recently reported (Malik et al., 2020).

Endoglucanase variations in active site topology result in different substrate specificities. It was found that GH6 PaCel6B showed increased activity on CMC, which is a highly specific substrate for Endo-acting cellulases (Sidar et al., 2020). CMC is decrystallized cellulose and therefore contains more amorphous sites that are ideal for access to the cellulose chain by internally cleaving endoglucanases, whereas exoglucanase GH6 PaCel6A showed greater activity on insoluble microcrystalline Avicel than on CMC. Considering this and finding both GH12 and GH6 in the genome of our Streptomyces sp. STCH565-A, it is plausible that the CMC substrate could be catalytically attacked both in its amorphous structure and in the reducing and non-reducing ends, confirmed by the ability of our studied strain to grow on both substrates (Fig. 5C).

It was unexpected to find GH10 or GH11 enzymes in the Streptomyces sp. STCH565-A genome (Table 3), because both families had been identified mostly in host-associated Streptomyces and not within environmental strains (Book et al., 2016). What can be considered singular to our strain is to find the simultaneous presence of GH11 and 11 copies of acetyl-xylan-esterase in the genome (Table 3). This particular combination of enzymatic activity was previously found to be synergistic in vitro, improving the hydrolysis of pre-treated lignocellulosic biomass (Marasinghe et al., 2021); this experiment, however, was carried out using a cocktail of a commercially produced GH11 from Streptomyces and acetyl-xylan-esterase obtained from Ochrovirga pacifica. Identifying a bacterial strain harboring such a robust complex of CAZy domains makes Streptomyces sp STCH565-A an obvious candidate for future applications in the industry, either using the whole microorganism in a consortium or its genetic sources for heterologous expression and mixed enhanced cocktails.

Streptomyces sp. STCH565-A can use xylan as its only carbon source (Fig. 5C), which can be explained with the presence of several enzymes codified in the CAZyome: Glycosyl-Hydrolase GH10, GH11, GH26 and GH43 (xylanases), as well as multicopies of CE1 and CE4 (acetyl xylan esterases) (Table 3). These enzymes have been previously reported as auxiliary enzymes in xylan hydrolysis, whose activity eliminates recalcitrant lateral chains and exposes the xylan skeleton, improving the deconstruction process of cellulosic material (Vardakou et al., 2008).

The enzymatic attack is not limited to the catalytic machinery of glycosyl-hydrolases. CBM are ancillary modules attached to GH, and they are improving the catalytic functions of the CAZymes (Lombard et al., 2014) by gaining access to the insoluble substrate fraction of the cellulosic chain (Bernardes et al., 2019; Chalak et al., 2019). In this work, we report that additionally to the GH identified in the genome of Streptomyces sp. STCH565-A, a carbohydrate-binding module (CBM2) is codified in our strain, probably forming the CBM2-GH6 complex. CBMs do not have catalytic activity per se, but function as substrate-binding modules (Boraston Alisdair et al., 2004; Lombard et al., 2014). Studies suggest that the binding characteristic of a CBM enhances the catalytic function of CAZymes by directing the enzyme to the substrate and increasing substrate-enzyme proximity, as well as disrupting the crystallinity of the insoluble substrate fraction (Bernardes et al., 2019; Reyes-Ortiz et al., 2013); therefore, finding this CBM2-GH6 in the analyzed genome arouses great interest for this study. We could suggest that this module is one of the key characteristics to understand the affinity of the Streptomyces sp. STCH565-A strain for the evaluated substrate –CMC—which could be further confirmed by studies showing that removal of the CBM from the enzyme results in decreased enzyme activity and reduced enzyme stability (Cockburn et al., 2018).

Focused analysis on the auxiliary activities that bind to the cellulolytic complex to form a complete model for the enzymatic hydrolysis of cellulose, five copies of AA10 were found in the CAZyome of Streptomyces sp. STCH565-A These copies of AA10 suggest an important role in the results (Table 3). AA10 is a domain assigned by the CAZy database for lytic polysaccharide monooxygenases (LPMOs), which are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes have initially been used in synergy with glycoside hydrolases to enhance the saccharification of plant biomass and have been incorporated into state-of-the-art commercial enzyme cocktails for biofuel production (Johansen Katja, 2016; Moreau et al., 2019). In the STCH565-A genome, these LPMOs are present and naturally assume participation in cellulose degradation, without being added exogenously as occurs in other studies.

Overcoming recalcitrance of lignin hydrolysis is probably one of the urgent challenges in the deconstruction of hardwood and other lignocellulosic residues. Streptomyces sp. STCH565-A genome codifies auxiliary activities of AA1 (laccase) as well as CE1 (acetyl xylan esterase) and AA3 (aryl alcohol oxidase) whose activity has been shown to be essential in lignin hydrolysis (Ozer et al., 2020; Pothiraj, Kanmani & Balaji, 2006). Despite the presence of the lignin-related codified enzymes in its genome, Streptomyces sp. STCH565-A was not able to grow on lignin as a sole carbon source in the evaluated conditions. It is known that phenolic compounds derived from lignin inhibit lignocellulosic bioconversion (Qin et al., 2016), which might be alleviated by the concurrent action of the enzymatic activity of a community in an aquatic free-living context, such as the original environment from which Streptomyces sp. STCH565-A was retrieved. Enzymatic activity in vitro can further be experimented, optimizing growth media and conditions (Kumar & Chandra, 2020).

A broad study of the evolution of cellulolytic activity in the genus Streptomyces (Book et al., 2016) grouped highly cellulolytic organisms in two clades that related to host-associated origin. A third clade of less cellulolytic organisms grouped free-living Streptomyces. Authors demonstrated that highly cellulolytic (mostly host-associated) Streptomyces encompassed a particular combination of important enzyme families to deconstruct cellulose: (GH5, GH6, GH9, GH12, GH48, and AA10), and cellulose-targeting carbohydrate-binding module (CBM) families 2 and 3. However, a cellulolytic complex codified in the genome of our aquatic free-living Streptomyces sp. STCH565-A is also composed of cellulases (GH5, GH6, GH12, GH51), and a very robust complex of domains such as CBM2, CBM2+GH6, CBM2+AA10, AA10 and CBM6. It is notable that the codified CAZy inventory of STCH565-A seems closer to what is expected for a host-associated organism.

Previous findings show that free-living soil strains, which have evolved within diverse communities, have likely experienced limited selection for rapid cellulose-degrading activity in isolation (Book et al., 2016); our results, however, strongly contrast to high cellulolytic activity of our aquatic isolated strain Streptomyces sp. STCH565-A Considering stoichiometry imbalance and oligotrophy found in ponds of Cuatro Ciénegas (Souza & Eguiarte, 2018; Souza et al., 2018), it is likely that this selection pressure and high endemism, particularly in the Churince ponds whose water content had a deep ancient magmatic influence (Wolaver et al., 2012) might have exerted a selective pressure on the Actinomycete, and that highly cellulolytic Streptomyces sp. STCH565 A is probably related to its evolutionary origin.

Conclusions

A systematic bioprospecting strategy to obtain highly cellulolytic actinomycetes was implemented in Cuatro Ciénegas Basing, producing the isolation and selection of an aquatic Streptomyces sp. STCH565-A, whose endoglucanse-specific activity stands out among other highly cellulolytic Streptomyces. Genomic analysis of this protruding strain reveals a robust CAZyome of singular simultaneous presence of valuable GH, AA and CBM domains, making our gifted strain a promising candidate for lignocellulosic bioconversion applications. Furthermore, we propose that this systematic approach could also be applied to other environmental contexts or to evaluate bacterial collections to retrieve highly cellulolytic microorganisms.

Supplemental Information