Engineering Ligninolytic Consortium for

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REVIEW ARTICLE

Engineering Ligninolytic Consortium for Bioconversion of Lignocelluloses to Ethanol and Chemicals Muhammad Bilal1*, Muhammad Zohaib Nawaz1,5,6, Hafiz M. N. Iqbal2, Jialin Hou1, Shahid Mahboob3,4, Khalid A. Al-Ghanim3 and Cheng Hairong1* 1

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China; 2Tecnologico de Monterrey, School of Engineering and Science, Campus Monterrey, Ave. Eugenio Garza Sada 2501, Monterrey, N.L., CP 64849, Mexico; 3Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia; 4Department of Zoology, Government College University Faisalabad, Faisalabad, 38000, Pakistan; 5Wuxi Metagene Science & Technology Co., Ltd, Wuxi, People’s Republic of China; 6Center for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad 38040, Pakistan

ARTICLE HISTORY Received: April 3, 2017 Revised: June 8, 2017 Accepted: December 15, 2017 DOI: 10.2174/0929866525666180122105835

Abstract: Background: Rising environmental concerns and recent global scenario of cleaner production and consumption are leading to the design of green industrial processes to produce alternative fuels and chemicals. Although bioethanol is one of the most promising and eco-friendly alternatives to fossil fuels yet its production from food and feed has received much negative criticism. Objectives: The main objective of this study was to present the noteworthy potentialities of lignocellulosic biomass as an enormous and renewable biological resource. The particular focus was also given on engineering ligninolytic consortium for bioconversion of lignocelluloses to ethanol and chemicals on sustainable and environmentally basis. Methods: Herein, an effort has been made to extensively review, analyze and compile salient information related to the topic of interest. Several authentic bibliographic databases including PubMed, Scopus, Elsevier, Springer, Bentham Science and other scientific databases were searched with utmost care, and inclusion/exclusion criterion was adopted to appraise the quality of retrieved peer-reviewed research literature. Results: Bioethanol production from lignocellulosic biomass can largely satisfy the possible inconsistency of first-generation ethanol since it utilizes inedible lignocellulosic feedstocks, primarily sourced from agriculture and forestry wastes. Two major polysaccharides in lignocellulosic biomass namely, cellulose and hemicellulose constitute a complex lignocellulosic network by connecting with lignin, which is highly recalcitrant to depolymerization. Several attempts have been made to reduce the cost involved in the process through improving the pretreatment process. While, the ligninolytic enzymes of white rot fungi (WRF) including laccase, lignin peroxidase (LiP), and manganese peroxidase (MnP) have appeared as versatile biocatalysts for delignification of several lignocellulosic residues. The first part of the review is mainly focused on engineering ligninolytic consortium. In the second part, WRF and its unique ligninolytic enzyme-based bio-delignification of lignocellulosic biomass, enzymatic hydrolysis, and fermentation of hydrolyzed feedstock are discussed. The metabolic engineering, enzymatic engineering, synthetic biology aspects for ethanol production and platform chemicals production are comprehensively reviewed in the third part. Towards the end information is also given on futuristic viewpoints. Conclusion: In conclusion, given the present unpredicted scenario of energy and fuel crisis accompanied by global warming, lignocellulosic bioethanol holds great promise as an alternative to petroleum. Apart from bioethanol, the simultaneous production of other value-added products may improve the economics of lignocellulosic bioethanol bioconversion process.

Keywords: Biofuel, lignocellulose valorization, ligninolytic engineering, delignification, metabolic engineering. 1. INTRODUCTION Modern industrial technologies have made it possible to synthesize versatile chemicals with desired characteristics *Address correspondence to these authors at the State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China; E-mail: [email protected]; [email protected] 0929-8665/18 $58.00+.00

and wide-ranging commercial applicability. Depending on today’s entreaty, industrial production of a variety of commodity chemicals profoundly depends on fossil-based resources. Diminishing of these resources accompanied by formidable environmental concerns, such as global warming and littering problems, has endangered the future of biofuel industry. The cost-effectiveness of fossil fuel has also disap© 2018 Bentham Science Publishers

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peared in the last few decades since the declining fossil reserves will further increase the oil price [1]. A consistent and high-level utilization of petrochemicals has caused overwhelming environmental concerns, hence demanding for sustainable and cleaner production alternatives. In this context, the employment of a bio-based economy is an indispensable choice for the development of any civilization. The European Union has already approved rules for reducing environmentally-abusive materials and rekindled their attention in exploring eco-friendlier materials based on renewable natural resources. Therefore, alternative ways to develop sustainable and natural resources-based biopolymers are of high importance for declining the contemporary reliance on fossil resources [2]. Inarguably, biomass has been projected to be the only sustainable and impeccable consonant to petroleum for producing biofuels [3] and other industrially-pertinent chemicals. Lignocellulosic biomass feedstocks have prominent advantages over starch and sugar crops since they do not interfere with food and feed chain supplies. The agricultural, forestry and agro-based lignocellulosic wastes are gathered to high magnitudes which cause serious ecological problems. They might be exploited for the development of an invaluable array of added value products [4]. Numerous studies have corroborated that lignocellulosic-based biomass demonstrates the massive potential for producing biomolecules, biomaterials, and biofuels. Furthermore, it is a carbonneutral bio-renewable substrate abundantly available around the globe, with zero net carbon emission and thus atmospheric pollution. The principal constituent of lignocellulosic biomass; i.e., cellulose, is manifested as a potential candidate for the replacement of petroleum-based polymers due to its environmentally-friendlier properties such as biocompatibility, bio-renewability, and biodegradability [5-6]. Notwithstanding immense benefits, the transformation of lignocellulosic waste to fine chemicals and biopolymers, on the other hand, remains a great biotechnological challenge. Multifarious nature of lignocelluloses renders them particularly recalcitrant to physicochemical and enzymatic degradation. Extensive research is currently underway across the world to address this dilemma. Increasing number of biorefinery and biofuel-related technologies have been developed aiming to upgrade biomass for producing advanced biofuels, biochemicals, and biomaterials from biomass feedstocks [7]. In this review, engineering ligninolytic consortium and their potential exploitation for biodelignification of lignocelluloses are discussed. A detailed literature is given on the enzymatic hydrolysis and fermentation of hydrolyzed feedstock. Finally, the recent trends in the arena of lignocellulosic biomass to produce biofuels and highadded platform chemicals are discussed. 2. ENGINEERING THE LIGNINOLYTIC ARMORY WRF exhibit a high-redox-potential multi-enzymatic cascade with wider substrate acceptability and the aptitude for carrying out the complete degradation of lignin. The main ligninolytic enzymes are peroxidases [lignin peroxidase (LiP, E.C. 1.11.1.14), manganese peroxidase (MnP, E.C. 1.11.1.13), and versatile peroxidases (VP, E.C. 1.11.1.14)] and laccases (E.C. 1.10.3.2). Other known auxiliary lignin-

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modifying enzymes are unspecific peroxygenases (UPO); glyoxal oxidases (GLX), H2O2-supplying oxidases, glucose oxidases (GO), quinone reductase (QR), P-450 monooxygenase and ferric-ion reducing enzymes. In fact, ligninolytic consortia could offer potential alternatives in various industrial sectors. Potential applications include the generation of fine chemicals, fuels, organic syntheses, bioremediation, food industry, and pulp biobleaching, among others [8]. Nevertheless, to fulfill increasing market demands, these multipurpose biocatalysts should be engineered to improve their bio-catalytic potentiality so that their properties can be modified to unfavorable industrial settings [9]. The ligninolytic armory works efficiently in a wet and slightly acidic environment due to the release of organic acids by the WRF. The environmental conditions found during biological delignification are far from those required in each biotechnological application. Worth mentioning is that the ligninolytic enzymes can be adopted to unnatural environments through directed evolution approaches. One of the most interesting applications for high-redox-potential laccases (HRPLs) is the development of three-dimensional nanodevices for myriads of impressing biomedical and biotechnological purposes [10]. The mutant laccase has shown the potential capability to oxidize H2O to O2 during water splitting under an alkaline environment, a concern that surpasses the necessities for large-scale production of H2 and O2 [11]. The use of non-conventional media is required for many bio-transformations, such as in remediating xenobiotics, and syntheses of lignin intermediates, biopolymers, and pharmaceuticals. In such scenario of furnishing the final variant with co-solvent promiscuity, directed evolution was performed in the presence of elevated concentrations of co-solvents of different polarity and chemical nature. Outstandingly, the final mutant exhibited moderate activity and stability at elevated levels of the co-solvents (50% v/v). Alkalophilic fungal laccases are relevant to the textile and paper industries. After fifteen evolutionary generations, a pH shift for activity towards the alkaline region was noticed. After five additional series of evolution, the engineered laccase presented exceptional catalytic performance and improved kinetic parameters at alkaline pH profiles against both phenolic and non-phenolic substrates [12]. Of most recently, the installation of consensus mutations into existing enzymes consequences diverse activities in contrasted with laboratory revived enzymes [13]. A new emerging field of contemporary research frame-up by tailoring fully autonomously consolidated bioprocessing microbes (CBM). The reported example of co-expression of two ligninolytic genes (HRPL and VP) in yeast was the result of a laboratory-evolved S. cerevisiae at high titers without interrupting cellular metabolism [14]. Nevertheless, the engineering of other ligninases would answer the uncertainties as to whether S. cerevisiae can withstand the secretion of lignin-degrading enzymes without undergoing unnecessary metabolic liability and thereby expression restraints. 3. ENZYMATIC HYDROLYSIS AND FERMENTATION Enzymatic hydrolysis of lignocellulosic biomass is usually carried out either by microorganisms that secrete en-

Engineering Ligninolytic Consortium

zymes during their growth in the media or commercially available enzyme system. Enzymatic hydrolysis is the method of choice for future lignocellulose-to-ethanol processes. Nonetheless, enzymes cost is a major challenge for cost-effective production of ethanol on a commercial scale [15]. Furthermore, enzymatic hydrolysis of cellulose or hemicellulose is inadequate, and yield is relatively lower. The major enzyme systems used in hydrolyzing lignocellulosic biomass including cellulose and hemicellulose. Cellulolytic enzymes or cellulase is a combination of enzymes acting synergistically on cellulose polymer converting it into glucose monomers. Adequately, the collaborated action of at least three enzymes including endo-1-4-β-glucanase (EC 3.2.1.4), cellobiohydrolase (CBH, EC 3.2.1.91) and βglucosidase (EC 3.2.1.21) are believed to be involved in cellulose hydrolysis [16-17]. Most favorable traits of cellulases for lignocellulosic biomass biotransformation are the complete saccharifying machinery, high catalytic activity, thermos-stability, diminishing vulnerability to product feedback inhibition, and unique ability to resist wear and shear forces. These inclinations could be achieved through developing optimal enzyme cocktails, overexpression techniques, and protein engineering approaches. The simple sugars generated because of biomass delignification and cellulose hydrolysis are converted into ethanol by the pertinent strain used. A good number of microorganisms including yeast, bacteria and filamentous fungi can ferment soluble sugars under anaerobic conditions. The most widely exploited and well-suited microorganism for producing large-scale ethanol from lignocellulosic hydrolysates is S. cerevisiae [18]. It can proficiently metabolize hexoses, but scarcely pentoses due to the deficiency of enzymes involved in converting xylose to xylulose. 4. IMPROVING THE LIGNOCELLULOSIC ETHANOL PRODUCTION 4.1. Bio-Delignification of Lignocellulosic Biomass Biological pretreatment exploits wood-degrading microbes, particularly white rot, soft rot, brown rot fungi to alter the chemical composition of the lignocellulosic network. In general, soft and brown rot fungi essentially attack cellulose polymer while leaving the lignin largely unaffected and WRF are most efficient decomposers of the lignin constituent. The biological conversion routes of different ligninbased feedstocks offer immense benefits, but technoeconomic barriers still hamper the scale-up implementation. Undeniably, biological pretreatments are of low cost and operated under milder conditions, the poor hydrolysis rates and elongated contact times are the associated drawbacks [19]. In recent years, ligninolytic enzymes have been advocated as a promising alternative pretreatment approach for biomass deconstruction. More importantly, ligninasesmediated pretreatment technology is valuable in delignifying as well as detoxifying processes. In delignification, ligninolytic enzymes have been successfully employed to remove the lignin content in a wide-range of agro-based feedstock. Even more, these enzymes have also shown potential in removing toxic compounds from sugar hydrolysates.

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Enzymatic delignification is a process of lignin removal by crude enzyme consortia and semi-purified or purified form of ligninolytic enzymes (either native or commercial enzyme) [20]. Literature survey revealed that laccase is the most studied enzyme followed by MnP and LiP; nonetheless, some investigations also reported the biomass deconstruction using combinations of two or three ligninolytic enzymes [21]. In these cases, the concerted acquaintance amongst the ligninolytic enzymes substantially enhanced lignin removal efficiency. The enzymatic processes achieved the same delignification percentages as a microbial pretreatment in a short duration of 24 to 96 h [22]. The lignin removal profile of various lignocellulosic feedstocks by ligninolytic enzymes in the last few years is summarized in Table 1. Despite the advantages of minimum delignification timespan, the enzyme-based delignification cannot meet the existing pretreatment technologies in terms of time and costs [23]. Enzymatic delignification can be promoted by manipulating enzymatic catalysis using some advanced molecular and protein engineering strategies. Rational, semi-rational approaches and directed evolution approaches are proposed to modify lignin-degrading enzymes [11]. Rational approaches (site direct mutagenesis) have been used successfully to enhance the laccases capacity for oxidizing large phenolic molecules and to widen its degradation potential even for nonphenolic substrates [24]. In semi-rational approach, any selected amino acid is substituted with the desired one by changing its codon [11], and has been used to tailor enzymes with higher catalytic efficiencies [25]. 4.2. Process Development and Fermentation Strategies Despite the evidence that intensive research efforts have been concentrated in upgrading the conversion process of bioethanol from lignocelluloses, some technical and economic challenges remain to be executed. Cellulose hydrolysis and fermentation of simple monomers are frequently carried out individually, a process known as separate hydrolysis and fermentation (SHF). A unique advantage of this technique is the possibility of conduct hydrolysis and fermentation in their optimal conditions; while the risk of feedback inhibition due to the excessive sugars accumulation in the hydrolysates is a major drawback [26]. Subsequently, following a minor modification in SHF, a process explicitly separates hydrolysis and co-fermentation (SHCF) develops where both hexose and pentose sugars are generated separately, but fermented concomitantly. The most favorable and extensively used process integration in both laboratory and industrial scales bioethanol production is the simultaneous saccharification and fermentation (SSF). In this process configuration, the cellulolytic-based hydrolysis and fermentation are performed in a single reactor. SSF involving cofermentation of hexoses and pentoses is referred as simultaneous saccharification and co-fermentation (SSCF). SSF is considered as a cost-effective solution for its reduced process duration and fewer equipment requirements, even though some bottlenecks encountered by this technique. Recently, new process integration, i.e., simultaneous saccharification, filtration and fermentation (SSFF) that combine both SHF and SSF has been developed [27]. In this technique, pretreated lignocellulosic biomass is hydrolyzed in a reactor, while the suspension is continuously propelled through a


Table 1.

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Ligninolytic enzymes mediated lignin removal of various lignocellulosic biomasses.

Lignocellulosic biomass

Microbial culture

Percent delignification

Reference

Corn stover

Ganoderma lucidum

50.23

[81]

Cotton stalk

63.01

Sorghum stover

64.1

Sugarcane bagasse

52.94

Wheat bran

61.25

Newspaper

Ganoderma lucidum

42.3

[82]

Wheat straw

Ganoderma lucidum

58.5

[83]

Sugarcane bagasse

Trametes versicolor

46

Rice straw

Pleurotus sapidus

52

Banana stalk

Shizophyllum commune

61.7

Corncobs

47.5

Sugarcane bagasse

63.6

Wheat straw

58.6

Corn cobs

Pleurotus eryngii

48.05

Corn stover

49.52

Rice straw

43.45

Banana stalk

39.15

Sugarcane bagasse

56.9

Rice straw

Pleurotus sapidus

46.72

[21]

[84]

[85]

53.1 Sugarcane bagasse

33.97 51.08

Corn cobs

45.01 61.16

Wheat straw

45.6 57.4

Saccharum spontaneum

Pleurotus sp.,

Corn cobs Sugarcane bagasse

84.67

[86]

62.8 Trametes villosa

35.04

[87]

35.68 37.81 Coconut shell

Trametes villosa

39.61

[87]

39.452 40.201 Sisal fiber

Trametes villosa

63.04

[87]

63.14 63.15 Sugarcane bagasse

Pleurotus ostreatus

33.6

[22]

Wheat straw

Ganoderma lucidum

39.6

[88]



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Figure 1. Consolidated biomass processing of lignocellulose biomass for enzyme and bioethanol production.

cross-flow membrane. Another emerging and alternative to SSF technique is the consolidated bioprocessing (CBP), which integrates not only cellulose hydrolysis and fermentation of sugars but also involves the production of hydrolyzing enzymes for converting biomass to ethanol in a single step. CBP aims to directly produce bioethanol from lignocellulosic biomass in a sustainable way that could ultimately lead to economically more competitive technologies for bringing bioethanol vision into reality [26]. Figure 1 illustrates a consolidated biomass processing of lignocellulose biomass for enzyme and bioethanol production.

4.3. Engineering the Saccharomyces cerevisiae for Enhanced Ethanol Production Lignocellulose hydrolysates often contain mixtures of hexoses and pentoses, particularly glucose and xylose, respectively. Among naturally occurring microorganisms, S. cerevisiae has been widely exploited over the past several years in fermenting glucose to ethanol with high yield. There are, however, several limitations with this yeast in producing lignocellulosic-based ethanol. One of the major hindrances is the lack of capacity to metabolize xylose, which is produced concomitantly with glucose following the hydrolysis of cel-


Table 2.

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Different reported strategies for co-utilizing glucose and other pentoses by Saccharomyces cerevisiae.

Carbohydrate

Strategy

Reference

Cellobiose, xylose, and acetic acid

Integration of the fermentation pathways of cellobiose and xylose and an acetic acid reduction pathway

[89]

Glucose and Xylose

Directed Evolution to Improve Xylose Transportation of AN25; mutational Analysis and Combinatorial Engineering

[90]

Glucose and xylose

Deletion of D-ribulose-5-phosphate 3-epimerase

[36]

Glucose and xylose

Construction of a growth-based screening system for mutant hexose transporters

[35]

Glucose and xylose

Expression of xylose reductase, xylitol dehydrogenase, and xylulokinase; Engineering of hexose transporters

[34]

Cellobiose and xylose

Expression of a cellodextrin transporter, intracellular β-glucosidase and xylose reductase and optimization of the expression

[38]

Glucose and xylose

Maintaining glucose in the useful concentration range in fed-batch reaction

[91]

Xylose, and arabinose

Evolutionary engineering via continuous culture using xylose and arabinose as limiting carbon sources

[39]

Glucose, xylose, and arabinose

Evolutionary engineering strategy based on repeated batch cultivation with repeated cycles of consecutive growth

[40]

lulose and hemicelluloses. The hydrolysates of agricultural residues comprise 5-20% xylose, which cannot be metabolized by S. cerevisiae [28]. On the other hand, several xylose-fermenting yeasts, such as Candida shehatae, Pichia stipitis, Spathaspora arborariae and Spathaspora passalidarum have also been reported. Low conversion efficiency has rendered them unappealing for large-scale lignocellulosic ethanol production. As efforts to ferment both glucose and xylose, a co-culture system has been investigated using glucose (S. cerevisiae) and xylose (P. stipites) fermenting microorganism in either single or two bioreactors. Considerable research efforts have been made in recent years to co-metabolize glucose and xylose through recombinant DNA technology or adaptation and evolution techniques [29]. The genetic modifications have been widely carried out in S. cerevisiae, Z. mobilis and E. coli [30-31]. Table 2 demonstrates the different genetic engineering strategies for co-utilizing glucose and other pentoses from lignocellulosic biomass in S. cerevisiae. Despite the fact that engineered strains contain glucose and xylose-assimilating pathways, glucose is preferentially consumed by these strains in a mixture of sugars evidently due to the lack of a xylose transporter protein. The subsequent fermentation of mixed sugars results in diminished ethanol titer and final ethanol productivity [32]. To overcome this bottleneck and instantaneously consuming both sugars it is advised to maintain a low glucose level through adding a controlled level of cellulolytic enzymes. Insertion and expression of the xylose transporter genes in a metabolically engineered strain could be a choice for simultaneously metabolizing both sugars during fermentation [33]. Integrating xylose reductase, xylitol dehydrogenase, and xylulokinase in S. cerevisiae confer the strain co-fermenting glucose and xylose after fine-tuning the expression of glucose transporter permeases. Moreover, yeast with transporter overexpression encoded by HXT1 could achieve the maximum ethanol production co-utilizing glucose and xylose. Remarkably, the ethanol titer was more pronounced than either with individual glucose or xylose. It cannot assimilate

xylose efficiently in a xylose-enriched medium, whereas the HXT7 permease would be a superior approach allowing the xylose uptake rate to be equivalent as that of glucose [34]. The mutant Gal2-N376F can transport xylose at the highest affinity, while not allowing transporting any hexose [35]. Furthermore, the inactivation of ᴅ-ribulose-5-phosphate 3epimerase (RPE1) is another effective approach to coutilizing C5 and C6 sugars for ethanol production [36]. To circumvent the inhibitory effect of glucose and inadequate supply of NAD(P)H for xylose reductase, Oh and coworkers, [37] chosen a cellobiose to co-utilize with xylose. A similar strategy was adopted by Zha et al. [38] to attain the cofermentation of cellobiose and xylose. The evolved strain harboring arabinose, and xylose pathways were able to generate ethanol with a final yield of 0.29 g/ per g xylose consumption [39]. Similarly, a novel evolutionary engineering strategy involving series of successively repeated growth cycles in three media was used by Wisselink et al. [40] to improve the strain. Strikingly, the engineered strain exhibited an elevated ethanol yield of 0.44 g/g of total sugars. 4.4. Immobilization of Microbial Cells and Enzymes The prerequisite of fermentative microorganisms and enzymes is considered as a significant and cost increasing facet for lignocellulosic ethanol. Microbes are usually incorporated in the fermentation media as free cells, where they proliferate and perform their metabolic activities. Similarly, enzymes are exploited as free form in the media. Recycling microbes and biocatalysts with pronounced efficacy are often challenging in their native state. These hindrances could be circumvented by immobilizing cells and enzymes on different solid supports that provide several technoeconomic benefits over the free system [41]. Immobilization of cells or biocatalysts enables its separation and improves catalytic activity as well as thermal stability characteristics. A great variety of techniques have been envisioned for encapsulating both individual cells/enzymes and a multicomponent enzymatic complex. These techniques include ad-


sorption, entrapment, covalent linking, affinity interactions, crosslinking of enzyme crystals (CLECs) and crosslinking of enzyme aggregates (CLEAs) [42-44]. It is demonstrated that immobilized cells have prolonged cellular stability and greater toleration to elevated substrate concentration. Prominent advantages of carrier-attachment are accelerated ethanol titer, better volumetric productivity, easier product retrieval, minimum end-product impediment, reduced microbial contamination, recyclability in repeated batch fermentations, and less vulnerability of cells against toxic substances. Reportedly, several immobilizing carriers including alumina, kcarrageenan, polyacrylamide, chrysotile, Ca-alginate, apple and sugarcane pieces, orange peel and banana leaf sheath have been attempted for cell immobilization [41]. Two basic approaches, such as immobilization on supporting material and immobilization by self-flocculation are most commonly endeavored for immobilizing the bakers yeast. The cells growing at log phase in enriched culture media are harvested and immobilized on any suitable support to develop beads with entrapped cells. Self-flocculation of yeast cells is another nonsexual and reversible cell aggregation approach, where yeast cells adhere to each other establishing a floc. In contrast to immobilization, self-flocculation is a more robust, techno-economically competitive, and contamination free technique offering the cells to propagate without being affected by environmental perturbations [45]. 4.5. Use of Synthetic Tools to Achieve Robustness Unlike high-value products, fuels are eminently costsensitive materials and can merely be competitive if biosynthesis costs are lesser than drilling and refining petroleum. Novel technologies that enable rapid prototyping, testing and optimization of pathways and the host organisms are crucial to trim-down the biofuel production cost. Synthetic biologists are striving to reduce the time necessary to construct genetic devices and expanding their consistency and predictability. For instance, BioBricks [46] and ligation-free assembly [47] techniques are assisting rapid construction of operons and pathways from existing DNA fragments. These novel methods are ripe for automation, enabling the development of large permutation libraries quickly. Another success of synthetic biology is the development of a diverse array of expression systems [48] that can be exploited for fine-tuning expression purposes in engineering metabolic pathways. The development of switch-based environmental responsive technologies has considerable prospects in the biofuel industry [49]. It may be likely to construct bacteria that switch from a cellulose assimilation mode to biofuel production mode by intuiting the environmental fluctuations. Such cutting-edge and decision-making competencies have already been proven in the tumor sensing bacteria that can sense and particularly invade anaerobic environments in a tumor [50]. Looking forward, the development of a ‘chassis’ organism for synthetic biology applications is a challenging and ongoing endeavor. Shifting an operational metabolic pathway from one organism to another is difficult for metabolic engineers, but sometimes indispensable maneuver for enhanced productivity. Synthetic biologists are now putting effort to either manipulate existing organisms or construct microorganisms with minimal targeted genomes and thereby


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with the desired set of metabolic pathways [51]. It is worth mentioning that many synthetic biology techniques are readily malleable to microbial metabolic engineering for fuels biosynthesis. Quick implementation of the ever-expanding synthetic biology tools would be immensely beneficial in endeavoring exciting metabolic engineering ventures, such as biofuels production [52]. 5. PLATFORM CHEMICALS Platform chemicals are building blocks of various valueadded chemicals and composed of 2-6 carbon atoms, which can be made from sugars either by microbial fermentation or synthetically using petroleum-based resources. Further, these building blocks can subsequently be converted to some highvalue bio-based chemicals. The industrial production of various chemicals for multipurpose applications heavily relies on petroleum resources which are deteriorating together with their frightening ecological consequences e.g. global warming [53-54]. In this context, the divergence from nonrenewable (petroleum-based resources) to renewable materials (lignocellulose-based resources) is becoming the center of interest for research in industrial communities, worldwide [55-57]. The fact is that petroleum resources are finite and becoming increasingly costly. A consistent depletion of petrochemical resources has pushed up prices in essential sectors, worldwide, including energy, materials, and medical [56, 58]. One of the biggest challenges of the modern world is in decreasing the dependency on such petrochemical resources based products. Owing to the threatening concerns regarding environmental, eco-friendlier production strategies, and petroleum limitations, researchers have directed or redirecting their interests in developing green technologies from a sustainable perspective to produce industrially relevant chemicals. Herein, the biomass-based biorefinery approach should ease disputes on eco-pollution and reliance on fossil resources, thus can be considered as an evolution of concepts like “Green Chemistry” [56, 59]. In this context, the generation of high value-added platform chemicals from a naturally abundant lignocellulose-based biomass has considerable advantages from energy and environmental concerns. Figure 2 illustrates a simplified scheme to produce various value-added biochemicals using lignocellulose biomass [60]. A plethora of search and research have already been undertaken for the conversion of lignocellulosic materials, and hydrolysate derived carbohydrates, into valueadded products and industrially relevant platform chemicals [53-54, 56-57, 60]. Figure 3 shows a simplified stepwise processing of lignocellulosic biomass to platform chemicals. The polymeric fractionation of the agricultural residues such as corn stover, rice straw, sugarcane bagasse, eucalyptus, spent grain, and corncob to obtain a variety of marketable and renewable biochemicals, including the xylitol, phenols, guaiacols, syringols, eugenol, catechols, vanillin, vanillic acid, syringaldehyde, benzene, toluene, xylene, styrene, biphenyls, and cyclohexane has gained a particular interest (56, 60-63). Despite much advances in lignocellulose biotechnology, the efficient and cost-effective biosynthesis of various platform chemical types using lignocellulose-based biomass remains a significant challenge [54, 64]. Around the globe, research scientists and research-based organizations are investigating novel approaches to address these efficacy


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Figure 2. Generalized scheme for the production of various value-added bio-chemical products (Reproduced in color with permission from Iqbal et al., [60].

Figure 3. A simplified illustration for the production of platform chemicals using lignocellulosic biomass via multistep processing approach.

and cost-effective ratio issues among others [54, 56]. In this context, Wettstein and co-workers, [65] have presented different prospects for lignocellulose biomass biotransformation into platform chemicals including sugars, fuels, and polymers.

6. LIGNOCELLULOSE-DERIVED CHEMICALS

PLATFORM

As discussed earlier, various types of platform chemical can be produced from lignocellulose-based biomass though using different methodologies including chemical or biologi-



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Figure 4. Lignin derived chemicals (Reproduced from Isikgor and Becer, [54], an open-access article distributed under the terms and conditions of the Creative Commons Attribution 3.0 Unported Licence (https://creativecommons.org/licenses/by/3.0/).

cal. Nonetheless, a comparatively a broader and in-depth processing approaches for the conversion of lignocellulose biomass are of principal interest. Indeed, many of the inpractice approaches are still at the pre-commercial level except the generation of bioethanol and lactic acid which are well established and commercially available [7, 66-67]. Based on lignocellulose-based C5 and C6 sugars and lignin derivable chemicals, 16 different platforms have been established so far. Among them, major includes 1,4-diacid platform, 5-HMF platform, 2,5-FDCA platform, 3-HPA platform, aspartic acid platform, glutamic acid platform, glucaric acid platform, itaconic acid platform, glycerol platform, sorbitol platform, 3-hydroxybutyrolactone platform, levulinic acid platform, lactic acid platform, ABE platform, xylose–furfural–arabinitol platforms and others. The production mechanism and potential routes of the platforms mentioned above have been reviewed elsewhere [54]. Herein, just to highlight, Figure 4 illustrates the conversion or transformation of lignin to different types of platform chemicals. Among potential platform chemicals, succinic acid has been considered significant in food-based nutraceuticals or medicine-related pharmaceuticals. Using lignocellulosebased biomass, the current global market of 15,000 tons per year could easily grow enormously with some efficient biobased production strategies [68]. In an earlier study, Niimi

and co-workers, [69] recorded 1,2-propanediol (1,2-PD), another important chemical compound, production using a metabolically engineered Corynebacterium glutamicum, in comparison to the US market of around 500,000 tons, currently satisfied by petroleum-based production. Owing to the excellently applied potentialities e.g. as a solvent, building block, liquid fuel and important precursor for different synthetic polymers, 2,3-butylene glycol is also considered a noteworthy chemical feedstock [70]. Interestingly, almost all of the sugar molecules e.g. glucose, xylose, arabinose, mannose, galactose, and cellobiose available in cellulose or hemicellulose based hydrolysates can be converted to butanediol [71]. The maximal theoretical yield of butanediol from sugar is 0.50 kg per kg. With a heating value of 27,200 J/g, 2, 3-butylene glycol compares favorably with ethanol (29,100 J/g) and methanol (22,100 J/g) for use as a liquid fuel and fuel additive [72]. Hexose and pentose can be converted to 2,3-butylene glycol by several microorganisms including Aeromonas [73], Bacillus [74], Paenibacillus [75], Serratia, Aerobacter [76], Enterobacter [77] and Klebsiella [78]. From the fermentation point of view, various fermentative substrates including glucose, xylose, starch, molasses, and Jerusalem artichoke tubers among others have been investigated for 2, 3-butylene glycol production via fermentation [79-80].


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CONCLUDING REMARKS AND FUTURE EXPECTATIONS

[6]

Given the present unpredicted scenario of energy and fuel crisis accompanied by global warming, lignocellulosic ethanol holds great promise as an alternative to petroleum, but it faces daunting challenges on multiple fronts. An efficient fermentation of hexose, as well as pentose sugars, is a major technical barrier to commercial-scale ethanol production from lignocellulosic wastes. To develop cost-effective lignocellulose-based ethanol bio-refinery, genetically engineered S. cerevisiae strains with potential ability to equally ferment both C6 and C5 sugars is crucial to today’s scientific world. Genetic engineering of yeast might include introducing pentose assimilation pathway, overexpressing existent pentose phosphate, and ethanol producing pathways, inactivating byproduct generation pathways, as well as expression of detoxifying enzymes. Apart from bioethanol, the simultaneous production of other value-added products may improve the economics of lignocellulosic ethanol bioconversion process. Moreover, using the advanced, sophisticated synthetic biology based approaches, it is now plausible to produce biofuel from unnatural candidates that allowing exploiting contemporary transportation infrastructure rather than replacing it with natural bioethanol. In any milieu, synthetic biology and metabolic engineering will surely be the vital players in revolutionizing the biofuel industry.

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CONSENT FOR PUBLICATION Not applicable.

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CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

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ACKNOWLEDGEMENTS

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The authors are grateful to the Shanghai Jiao Tong University, Shanghai 200240, China, and Tecnologico de Monterrey, Mexico for providing literature facilities. The authors (SM and KAAG) would like to express their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding through the Research Group Project No. RG- 1436-011. REFERENCES [1] [2] [3]

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