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Energy demand is constantly growing, and, nowadays, fossil fuels still play a dominant role in global energy production, despite their negative effects on air pollution and the emission of greenhouse gases, which are the main contributors to global warming. An alternative clean source of energy is represented by the lignocellulose fraction of plant cell walls, the most abundant carbon source on Earth. To obtain biofuels, lignocellulose must be efficiently converted into fermentable sugars. In this regard, the exploitation of cell wall lytic enzymes (CWLEs) produced by lignocellulolytic fungi and bacteria may be considered as an eco-friendly alternative. These organisms evolved to produce a variety of highly specific CWLEs, even if in low amounts. For an industrial use, both the identification of novel CWLEs and the optimization of sustainable CWLE-expressing biofactories are crucial.
The plant cell wall is a complex and heterogeneous structure composed of polysaccharides and phenolic compounds assembled in two distinct layers called primary and secondary cell walls . Clusters of cell walls constitute lignocellulose, a heterogeneous material mainly composed by lignin, hemicellulose, and cellulose; in addition, to provide structural support to the plant, it represents a powerful barrier against both biotic and abiotic stresses . In order to open a breach and depolymerize the cell wall polysaccharides into simple sugars, microbes have evolved specialized enzymes, referred to as cell wall lytic enzymes (CWLEs). The conversion of lignocellulose into fermentable sugars by using CWLEs may be considered as an eco-friendly alternative compared to degradative methods based on chemical treatments. Moreover, the broad substrate specificity of many CWLEs makes these enzymes highly versatile and, thus, exploitable in other important fields such as agriculture, food processing, and medicine. However, the industrial use of CWLEs is characterized by a high cost and low efficiency. Here, recent advances in the heterologous expression of CWLEs as well as some of their possible biotechnological applications will be discussed. In order to support a sustainable biofactory of CWLEs, we will focus on the expression system in plants, since it is characterized by a high productivity/cost ratio and it consumes atmospheric CO2 through photosynthesis, thus positively impacting global warming.
Arrays of CWLEs are secreted by microbes to efficiently hydrolyze the variety of polysaccharides in plant cell walls into simple sugars . Ligninases, hemicellulases, and pectinases, together with cellulolytic enzymes, are required to efficiently hydrolyze the lignocellulosic biomass. CWLEs are classified in the CAZy database (www.cazy.org), where glycoside hydrolases, the most numerous class of carbohydrate active enzymes, are represented by more than 160 different families (Table 1).
Table 1. Distribution of conserved domains in major cell wall lytic enzymes (CWLE) families. Auxiliary activity (AA), polysaccharide lyase (PL), and glycoside-hydrolase (GH) domains are indicated. Numeration of conserved domains is in accordance with the CAZy Database (www.cazy.org).
Amongst the various organisms producing CWLEs, lignocellulolytic fungi and bacteria are the most relevant. These organisms are highly specialized in degrading the different components of lignocellulose since they use monosaccharides derived from the plant cell wall to survive and proliferate; thus, they represent a precious source of CWLEs with different substrate specificities and enzymatic characteristics. In general, these organisms evolved to produce a variety of CWLEs in sufficient amounts for their own subsistence. An overall distinction of such organisms could be done based on their lifestyle, for instance, specialized (i) in degrading the cell wall from living plants, as in the case of plant pathogens, or (ii) in degrading dead plant matter, as in the case of saprophytes. However, the exploitation of wild-type microbes as biofactories to obtain CWLEs has a main limitation represented by the low level of enzyme production. This strongly impacts the cost of CWLEs obtained from wild-type hosts, making the entire process of enzymatic degradation less sustainable. Heterologous expression of CWLEs using Escherichia coli, yeasts, and plants may be a valid alternative. To date, different bacteria and yeast species have been used as expression hosts for CWLE production. These microorganisms differ significantly in their cell wall structure and subcellular compartments, thus affecting both protein secretion and expression efficiency. The low levels of soluble proteins and the poor secretion ability represent the main limitations of Gram-negative bacterial hosts. Alternatively, different species belonging to Gram-positive bacteria were selected as expression hosts since they secrete the recombinant proteins more efficiently than Gram-negative bacteria . In eukaryotic organisms, yeasts are eligible hosts for overcoming the solubility and secretion limits that affect heterologous expression in the bacterial system. Nowadays, plant expression of microbial CWLEs is a major challenge that biotechnologists are facing. Plants are desirable expression hosts since they are characterized by low production costs and high productivity. Moreover, they consume atmospheric CO2 through photosynthesis, which positively impacts global warming and points to the plant expression system as a valuable green alternative. The in muro targeting of CWLEs may enhance the hydrolysis of cell wall polysaccharides, allowing an efficient conversion of fermentable sugars into biofuel-related compounds . However, expression of CWLEs using plants as a heterologous system may impart unwanted and undesired side effects. CWLEs are produced by microbial pathogens to open a breach in the cell wall, concomitantly supporting the infection process . Therefore, the uncontrolled in planta expression of CWLEs may result in impaired growth, reduced productivity, and lethality . In order to circumvent these undesired effects, different CWLEs expression strategies may be adopted, such as (i) compartmentalized expression/accumulation, (ii) inducible gene expression, (iii) inducible enzymatic activity, and (iv) use of plant hosts that are not sensitive to CWLE activity (Figure 1).
Figure 1. Plant production of microbial CWLEs. Production of CWLEs as obtained by (a) constitutive expression and compartmentalization or (b) inducible gene expression. Advantages and disadvantages of each strategy are indicated in black and red, respectively. In (a), the represented CWLEs were successfully produced upon compartmentalized expression. (c) Theoretical optimal temperature of XynA from the mesophilic fungus R. solani, from the thermophilic bacterium B. halodurans, and from the hyperthermophilic bacterium T. maritima, here reported as a representative set of CWL isoenzymes. Activity of XynA from T. maritima is strongly reduced in the temperature range of plant growth (green box) and increases the chance of preserving the productivity of the transgenic plant. AnPGII: endo-1,4-α-polyglacturonase II from A. niger; Cel6B: endo-1,4-β-glucanase from T. fusca; En-Cel E1: endo-1,4-β-cellulase from A. cellulolyticus; CBHI: cellobiohydrolase I from T. reseei.
Another attractive strategy resides in the use of microalgae as a platform for the expression of CWLEs. Microalgae are promising expression hosts since they are characterized by a relatively fast growth cycle, and their cultivation is less expensive compared to that of other microorganisms (e.g., bacteria and yeasts) . Moreover, differently from higher plants, microalgae do not require arable lands for their cultivation, thus avoiding the loss of areas that may be employed in the agri-food sector. Contrary to plant cells, some species of unicellular green algae, such as Chlamydomonas reinhardtii, possess a cell wall mainly constituted by proteins ; thus, the lack of polysaccharides in their cell wall circumvents the deleterious effects of expressing CWLEs in plants.