Construction of microorganisms for consolidated bioprocessings: economic motivation and strategies

Cellulose waste biomass is the most abundant and attractive substrate for "biorefinery strategies" that are aimed to produce high-value products (e.g. solvents, fuels, building blocks) by economically and environmentally sustainable fermentation processes. However, cellulose is highly recalcitrant to biodegradation and its conversion by biotechnological strategies currently requires economically inefficient multistep industrial processes. The need for dedicated cellulase production continues to be a major constraint to cost-effective processing of cellulosic biomass. Research efforts have been aimed at developing recombinant microorganisms with suitable characteristics for single step biomass fermentation (consolidated bioprocessing, CBP). Two paradigms have been applied for such, so far unsuccessful, attempts: a) “native cellulolytic strategies”, aimed at conferring high-value product properties to natural cellulolytic microorganisms; b) “recombinant cellulolytic strategies”, aimed to confer cellulolytic ability to microorganisms exhibiting high product yields and titers. By starting from the description of natural enzyme systems for plant biomass degradation and natural metabolic pathways for some of the most valuable product (i.e. butanol, ethanol, and hydrogen) biosynthesis, this review describes state-of-the-art bottlenecks and solutions for the development of recombinant microbial strains for cellulosic biofuel CBP by metabolic engineering. Complexed cellulases (i.e. cellulosomes) benefit from stronger proximity effects and show enhanced synergy on insoluble substrates (i.e. crystalline cellulose) with respect to free enzymes. For this reason, special attention was held on strategies involving cellulosome/designer cellulosome-bearing recombinant microorganisms. Mini Review Article Development of microorganisms for cellulose-biofuel consolidated bioprocessings: metabolic engineers’ tricks Roberto Mazzoli a,* Volume No: 3, Issue: 4, October 2012, e201210007, http://dx.doi. org/10.5936/csbj.201210007 aDepartment of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina 13, 10123 Torino, Italy * Corresponding author. E-mail address: roberto.mazzoli@unito.it (Roberto Mazzoli) 1 cutting edge solutions that were applied or could be employed in the construction of recombinant cellulolytic strains. Strain development via native cellulolytic strategies NCSs are currently hampered by some common limitations. Most native cellulolytic strains have been recently isolated from natural environments and are poorly characterized. Genetic manipulation tools have been set-up for relatively few of them. As far as cellulolytic fungi are concerned, most engineering efforts have been addressed to increasing cellulase production, although there is increasing interest in biofuel production engineering [6, 13-14]. C. thermocellum and C. cellulolyticum are the most established cellulosome-forming microorganisms [6, 15-17]. Furthermore, the metabolism of few strains has been investigated in depth, with C. cellulolyticum as by far the best characterized microorganism [7, 18]. Even in strains with fully sequenced genomes, many genes are annotated as hypothetical while others may have been improperly annotated since their function was deduced on the base of amino acid sequence homology only [7]. In addition, problems connected with the construction of recombinant strains for specific compound production occur. Biomass biorefinery potential for sustainable production of a large spectrum of high value products, such as building block chemicals (e.g. succinic acid, lactic acid, isoprene), higher alcohols, lipidic compounds, fine chemicals (e.g. vitamins, antibiotics), has been extensively reviewed [19-21]. Production of H2, ethanol and butanol has been targeted in this study because of their huge potential as biofuels [1, 2, 7]. Improving hydrogen production in cellulolytic microrganisms A number of anaerobic cellulolytic bacteria, including several Clostridia (e.g. C. cellulolyticum, C. cellulovorans, C. termitidis and C. thermocellum), Ruminococci (e.g. R. albus) and the extreme thermophile Caldicellulosiruptor saccharolyticus, are able to produce H2 [7, 22]. However, H2 yields obtained by direct cellulose fermentation are usually low because of other metabolic pathways (e.g. producing acids, alcohols and ketones) which compete with proton reduction to H2 for electron donors, i.e. reduced Ferredoxin (Fdred) and/or NAD(P)H [7, 23]. H2 yields of mesophilic cellulolytic bacteria generally range from 1 to 2 mol H2/mole hexose sugar, while values close to the theoretical maximum of 4 mol H2/ mole hexose sugar can be obtained by hyper thermophiles such as C. saccharolyticus [7, 23]. Fermentative pathways either promoting or competing with H2 biosynthesis have mostly been studied in Clostridium sp. [7, 24] and are depicted in Figure 2. H2ases are regarded as the most efficient enzymes catalyzing either proton reduction (i.e. H2-evolving H2ases) or H2 oxidation (i.e. “uptake” H2ases) [24]. Construction of strains with improved H2 production has been carried out by deletion of genes encoding “uptake” H2ases and/or overexpression of H2-evolving enzymes [2526]. Engineering more efficient H2-evolving proteins has also been proposed to increase H2 yield and/or productivity [27-28]. However, these strategies appear to have limited potential since all H2ases, although preferentially directed, are known to be reversible [7, 29]. Greater H2 yields could be obtained through repression of competing pathways (i.e. producing more reduced end-products than acetate) (Figure 2) [7, 29]. Deletion of ldhA in hemicellulolytic Thermoanaerobacterium sp. either had no effect or increased H2 production by 2-fold, depending on the strain, while repression of acetate production by ack and pta knockout reduced H2 yields by more than 25 fold [30-31]. Recent papers reported repression of lactate and/or acetate production in C. cellulolyticum and C. thermocellum by Ldh or Ack/Pta gene inactivation, respectively, but effects on H2 production were not studied [16, 32-34]. Suppression of butyrate production was recently obtained in C. acetobutylicum and Clostridium butyricum by inactivation of hdb gene encoding 3hydroxybutyryl-CoA dehydrogenase [35-36]. However, H2 production strongly decreased in both strains. Elimination of ethanol formation alone did not increase H2 production in C. butyricum [37]. Actually, the H2 metabolic network in strict anaerobes is very complicated. A paradigm for this is the high diversity of clostridial hydrogenases and the existence of multiple forms, likely involved in different functions (e.g. redox balancing, derivation of energy from H2 oxidation, proton respiration and/or proton-gradient build-up) within one species [24]. Recently, members of the so called bifurcating hydrogenases have been identified in several clostridia, including cellulolytic strains [24, 38]. Among them, butyryl-CoA dehydrogenases/EtfAB (Bcd/EtfAB) complex from Clostridium kluyveri couples NADH-dependent exergonic reduction of crotonylCoA to butyryl-CoA to endergonic reduction of Fd which can be used for H2 production [38]. Discovery of such complex in clostridia provides a clue to H2 yield decrease in strains in which butyrate production was suppressed by Hdb inactivation. More detailed understanding of the metabolic networks involved in H2 production is definitely essential for successful engineering of H2 hyper producing strains. Figure 1. Simplistic model of a cellulosome that includes only one anchoring scaffoldin. The scaffolding protein (blue) binds the enzymatic components through cohesin-dockerin interactions, enhances the cellulosome affinity for cellulose through the CBMs, and anchors the cellulosome complex to the cell surface through either non-covalent (by means of multiple S-layer homology domains) or covalent (mediated by sortases) bonds. Apart from the catalytic domains, cellulosomal enzymes include dockerin modules and, possibly, additional domains (e.g. CBM, SLH) (modified from [60]). Microorganism development for CBP