Developmental strategies and regulation of cell-free enzyme system for ethanol production: a molecular prospective.

Appl Microbiol Biotechnol (2014) 98:9561–9578 DOI 10.1007/s00253-014-6154-0

MINI-REVIEW

Developmental strategies and regulation of cell-free enzyme system for ethanol production: a molecular prospective Waleed Ahmad Khattak & Muhammad Wajid Ullah & Mazhar Ul-Islam & Shaukat Khan & Minah Kim & Yeji Kim & Joong Kon Park

Received: 17 July 2014 / Revised: 9 October 2014 / Accepted: 12 October 2014 / Published online: 31 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Most biomanufacturing systems developed for the production of biocommodities are based on whole-cell systems. However, with the advent of innovative technologies, the focus has shifted from whole-cell towards cell-free enzyme system. Since more than a century, researchers are using the cell-free extract containing the required enzymes and their respective cofactors in order to study the fundamental aspects of biological systems, particularly fermentation. Although yeast cell-free enzyme system is known since long ago, it is rarely been studied and characterized in detail. In this review, we hope to describe the major pitfalls encountered by wholecell system and introduce possible solutions to them using cell-free enzyme systems. We have discussed the glycolytic and fermentative pathways and their regulation at both transcription and translational levels. Moreover, several strategies employed for development of cell-free enzyme system have been described with their potential merits and shortcomings associated with these developmental approaches. We also described in detail the various developmental approaches of synthetic cell-free enzyme system such as compartmentalization, metabolic channeling, protein fusion, and coimmobilization strategies. Additionally, we portrayed the novel cell-free enzyme technologies based on encapsulation and immobilization techniques and their development and commercialization. Through this review, we have presented the basics of cell-free enzyme system, the strategies involved in development and operation, and the advantages over conventional processes. Finally, we have addressed some potential directions for the future development and industrialization of cell-free enzyme system. W. A. Khattak : M. W. Ullah : M. Ul-Islam : S. Khan : M. Kim : Y. Kim : J. K. Park (*) Department of Chemical Engineering, Kyungpook National University, Daegu 7020-701, Korea e-mail: [email protected]

Keywords Cell-free enzyme system . Whole-cell system . Glycolytic and fermentation enzymes . Ethanol . Conventional fermentation

Introduction Fossil fuels have remained the major energy source since last few centuries; however, the rapid depletion of their reservoirs has been brought under spotlight only recently (Khattak et al. 2014). Besides energy concerns, serious environmental complications have originated from fossil fuel depletion (Saptoro et al. 2014; Prasetyo and Park 2013; Wernick and Liao 2013). These concerns have been intensely debated on several platforms and demands have been put forward for strenuous efforts for an urgent resolution. In this scenario, renewable energy technology has been proposed as the only ultimate solution to overcome issues associated with fossil fuels (Johansson et al. 1993; Wang et al. 2013, Edwards and Doran-Peterson 2012). Considering the significance of renewable energy sources, a number of biofuels, including ethanol, have been produced from renewable sources through microbial fermentation (Shirsat et al. 2013; He et al. 2014; Ayeni et al. 2014). Among the fermentative microbiota, Saccharomyces cerevisiae is viewed as a common and attractive organism adopted for ethanol production and is highly valued in biochemical, genetic, pharmacological, and postgenomic studies (Mager and Winderickx 2005). Over the past few decades, ethanol has emerged as the most promising and attractive alternative energy resource, holding potential advantages both from ecological and environmental aspects (Prasad et al. 2007), despite past criticism of the use of food reservoirs for ethanol production as being responsible for possible future food crises. However, the eventual shift of ethanol production sources from food reservoirs to secondary food reservoirs and waste resources resolved the issue (Mai

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et al. 2013; Han et al. 2013; Ge et al. 2012). To date, conventional (microbial) fermentation process has encountered several serious limitations, including the inhibitory effect of high glucose and ethanol concentrations, thermal instability of yeast cell, and production of secondary metabolites, that negatively affect the efficacy and economics of the process (Khattak et al. 2014; Fan et al. 2013). Consecutively, most of these limitations are being surmounted through technical advancements in fermentation technology such as introduction of genetically modified microorganisms (GMOs), novel fed-batch and continuous fermentation processes with cell immobilization and recycling for more efficient substrate conversion into ethanol (Khattak et al. 2012; Lu 2011; Hasunuma and Akihiko 2012). However, the primary drawback of the microbial fermentation process is that the major fraction of energy supplied in the feed is utilized by cells for proliferation and maintaining their integrity rather than to produce the desired product (Rupp 2013). Therefore, novel approaches to engineer biological systems that can resolve all the complexities affiliated with microbial fermentation are crucial. The practical application of these methods will be principally dependent on the efficacy of the process to produce an economically viable fuel. In 1897, Buchner presented a novel concept of cell-free enzyme system, claiming that biological processes could be carried out without living cells (Buchner 1897). Currently, cell-free fermentation system is considered a possible solution for surmounting all complexities and shortcomings associated with conventional fermentation process (Khattak et al. 2014). The advantages include the following: well-regulated, continuous, and prolonged processing of substrate conversion; evaluation of the effect of additional cofactors; and the utilization of a substantial fraction of supplied energy for the synthesis of the desired products (Khattak et al. 2014; Welch and Scopes 1985). Moreover, the inhibitory effect of higher concentrations of glucose and ethanol can also be overcome through cell-free enzyme system (Khattak et al. 2014). The system comprises the whole machinery required for the cascade of biological reactions taking place inside the microorganism for the conversion of glucose into ethanol (Buchner 1897; Khattak et al. 2014). The proposed mathematical model of cell-free glycolytic system (Teusink et al. 2000) suggests that the rate of ethanol production is proportional to the concentration of all enzymes involved in the biochemical pathway provided that the concentration of each individual enzyme remains fairly constant. Moreover, the cell-free enzyme system offers the advantage that the concentration of each individual enzyme can be changed in a desired way. This has grabbed a tremendous interest for the production of various biocommodities, besides ethanol, such as recombinant proteins (Shimizu et al. 2001; Madin et al. 2000; Sheng, et al. 2014), proteinous antibiotics, vaccines, hormones (Khattak et al. 2014), and dihydrofolic acid reductase, etc. (Shimizu

Appl Microbiol Biotechnol (2014) 98:9561–9578

et al. 2001). Cell-free enzyme systems, therefore, hold great potential in the engineering of new biological systems for synthesis of novel biomolecules synthesis on large scales. However, these encounter several shortcomings such as reversibility, instability, leakage, and inactivation etc. Predominantly, these face limitations when a mixture of enzymes constituting a cascade of reactions is employed for production of a bioproduct. These limitations can be addressed through the development of synthetic cell-free enzyme systems. This can be achieved by reprogramming the existing and designing novel metabolic pathways using the synthetic biology principles. Reprogramming of existing or construction of entirely new metabolic pathway in vitro for the production of ethanol and other bioproducts is still at the early stages of development (Rupp 2013; Hodgman and Jewett 2012). This is probably due to the several factors that need to be considered such as cofactor balance, thermodynamics, reaction equilibrium, and product separation and purification, etc. (Zhang 2010). The developing strategies for constituting an in vitro synthetic metabolic pathway through recombining various enzymes and cofactors are indeed a promising avenue for future generation biotechnology. It is receiving increasing interest in order to achieve maximum yield and overcome the pitfalls of conventional one-pot multi-enzyme catalysis. A series of complex reactions is usually mediated in a one-pot by a group of enzymes, such as cellulose hydrolysis by the synergistic action of endoglucanase, cellobiohydrolase, and βglucosidase (Zhang and Lynd 2004). This approach offers several advantages, including fewer unit operations, smaller reactor volume, higher volumetric and space-time yields, reduced cycle duration, less waste generation, and by-product formation (Zhang 2010). The well-established reported approaches for development of synthetic cell-free enzyme pathways include micro-compartmentalization, ionic channeling, co-polymerization, and protein fusion (Jandt et al. 2013). To the date, several studies have been reported on the development of cell-free enzyme systems for production of various biocommodities. However, there is no comprehensive review that has extensively discussed the ethanol production using the synthetic cell-free enzyme pathway development employing the synthetic biology principles. Moreover, we have comprehensively analyzed the developmental approaches for regulation procedures and enzymatic reactions of cell-free versus whole-cell systems. Numerous advantageous features of cell-free enzyme systems in comparison to whole-cell systems have been summarized. The cell-free enzyme system for production of various biocommodities is an area of immense interest in the present timings for the industrial biochemical process development since it offers a powerful platform for energizing the development and optimization of synthetic metabolic and biochemical pathways. This review manuscript comprehensively discusses the biosystem


obtained from yeast cells, particularly S. cerevisiae, in detail and also provides fundamental and recent advancements in the area of cell-free enzyme system development. Particularly, it highlights the developmental strategies of cell-free enzyme system for ethanol production with their potential benefits and shortcomings. Furthermore, the cascade of reactions catalyzed by specific enzymes and their regulation at both transcriptional and translational levels has been elucidated in detail. Thus, it will provide a solid platform for both basic readers and researchers working in this area. In short, the inherent features of cell-free enzyme system make it an emerging platform for utilizing the capabilities of natural biological systems without the limitations associated with whole-cell-systems.

Progressive development of cell-free enzyme system The yeast cell, besides being an industrial workhouse for the production of a number of proteinous and non-proteinous bioproducts, is considered one of the most reliable and highly developed model organisms for different kinds of studies (Herrgård et al. 2008). Assimilatory and dissimilatory metabolism of any compound in a cell is initiated once the compound passes the membrane barrier either through active or facilitated diffusion. The yeast cell has the same mechanism for the metabolism of carbohydrates like other microorganisms. Polysaccharides are complex molecules that cannot pass directly through the cellular membrane. Their uptake by cells is possible only after extracellular hydrolysis by saccharification enzymes into simple sugars (Khattak et al. 2012). The cell-free biosystem is an in vitro tool for studying different biological reactions that usually take place inside the cell. By adopting this novel strategy, we cannot only reduce the tendency toward complex interactions involved in whole cells, but can also produce our desired product more efficiently using the same substrate. Furthermore, the approach can address problems associated with conventional whole cell processing. Cell-free biosystems obtained from single-cell organisms to multi-cellular organisms have already been reported for the production of numerous biocommodities [Khattak et al. 2014; Shimizu et al. 2001; Rollin et al. 2013). Immense research is still in progress in order to improve the system’s efficacy in terms of higher product yields, robust reaction rates, and reduced interference from toxic compounds. From cell to enzymes The utilization of microbial cells for the production of fundamental utilities of life including biofuels, biochemicals, and bioproducts has remained in practice since thousands of years. The processing technologies for the production of these commodities are largely based on the whole-cell fermentation

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system. The suspended or free whole microbial cells carrying out a metabolic pathway encounters several restrictions. Yeast cell, for example, faces the problems of growth inhibition and cell viability in presence of high substrate and ethanol concentration during fermentation (Khattak et al. 2012, 2014; Buchner 1897; Najafpour et al. 2004) resulting in distraction of membrane fluidity (Bischof et al. 1995; Singer and Lindquist 1998). Moreover, the difference in optimal temperature of saccharification enzymes and microbial cell growth is another limiting factor for effective development of SSF process development (Khattak et al. 2012; Kádár et al. 2004). Earlier attempts with thermotolerant strains and immobilized whole cell overcome these problems to some extent and imparted several advantages such as stability, resistance, lower cost, reusability, reduced labor, and provided products with high purity (Takamitsu et al. 1993; Kourkoutas et al. 2004; Melzoch et al. 1994; Norton and D’Amore 1994). Moreover, immobilization of microbial cells such as encapsulation helped prevent the cell injury, loss, and damage by physiological environmental factors such as variation in pH and elevated temperature to certain limits. Several studies have reported various other benefits of using the immobilized whole cells such as reduced incubation time (Prevost and Divies 1987, 1992), freeze-resistance (Sheu and Marshall 1993), protection against bacteriophages attack (Steenson et al. 1987), and high metabolic productivity (Arnaud et al. 1992), etc. Nonetheless, these immobilized whole cells encounter several problems such as thermal stability at further elevated temperature (Khattak et al. 2014), ineffective substrate utilization, byproduct formation, and downstream processing cost of an industrial product (Rupp 2013) that make this conventional immobilized whole cell system an ineffective approach for process industrialization. A revolution in microbiology began in the 1850s when Louis Pasteur stated that viable yeast cells play a key role as catalyst in wine fermentation (El-Mansi et al. 2006). Later, Moritz Traube described enzymes and proposed a theory of enzyme–substrate interaction to explain fermentation occurring inside the cells (Rollin et al. 2013). Moreover, with the introduction of new knowledge and recently developed techniques such as genetic engineering, peptide engineering, and metabolic engineering and areas of specialization such as system and synthetic biology, the whole-cell system has been successively modified in various ways for the improved production of various natural and newly synthesized non-natural bioproducts. Until the 1890s, the importance of the cell-free system could not get considerable attention, but thereafter, Buchner’s endeavor provided a strong base for the significance of cell-free enzyme system. Since then, the main limitations faced by the conventional microbial fermentation were overcome through this cell-free enzyme system (Buchner 1897). This system represents the state-of-art biotechnological conversion of substrate into the product (Zaks 2001).

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Extended and continuous product formation, controlled variables (such as pH, ionic strength), maintenance of cofactors concentration (ATP and NAD+, etc.), and prevention of the abnormal accumulation of intermediary metabolites (Welch and Scopes 1985) were the key features that made this system a preferable approach in a run with conventional microbial fermentation. The absence of membrane barrier between the substrate and biocatalyst in cell-free enzyme system favors high reaction rates per unit mass or volume (You and Zhang 2013; Zhu et al. 2013). Moreover, the system is more feasible approach in bioprocess technologies development since it remains more active under a broad pH range and elevated temperature. Furthermore, the system is less vulnerable to inhibitory effects of product (Khattak et al. 2014; Panke et al. 2004). Numerous useful applications using the cell-free enzyme system have been reported including discovery of genetic code and production of antibiotics, vaccines, hormones and biofuels (Khattak et al. 2014). Few major issues and limitations are, however, associated with the cell-free enzyme system such as the use of expensive enzymes and their non-reusability (Gong et al. 2013). Developmental strategies Cell-free enzyme system can be principally classified into two main categories on the basis of their preparation strategy (You and Zhang 2013). The first category includes the isolation of cellular extract from the whole-cell system by rupturing their outer envelope through different methods (Fig. 1; Khattak et al. 2014). More advanced strategies are based on the purified enzymes isolated from different sources and mixed together with their respective cofactors and energy sources (Welch and Scopes 1985). Table 1 lists the merits of cellfree enzyme system compared to the whole-cell system (Table 1). Several features such as process optimization, improved yield, product titer, reaction titer, operational conditions, system modification, possibility for SSF, downstream processing cost, and process control make the cell-free enzyme system a preferable approach for the development of an economically feasible system for industrial development. Moreover, for a desired product formation, the GMOs are


produced to obtain the enzymes or any other products with characteristics of interest (Le Borgne 2012). These approaches have led to the development of reconstituted cell-free enzymes systems that are expected to be far better than the whole-cell system or conventional cell-free enzyme systems (Alper et al. 2006; Gitai 2005). Complexity of cellular machinery and incomplete information about life have restricted the modification of the whole-cell system; besides, the strategy is timeconsuming and laborious (Zhu et al. 2013). Contrary, the novel and attractive features of cell-free enzyme systems, such as reusability and possibility to isolate, purify, and restore the system under optimum conditions, confer several advantages compared to whole-cell biosystems (Zhu et al. 2013). The yeast cellular envelop constitutes about 15 % of the total cell volume and is principally composed of three distinct layers from the inside out: the plasma membrane, the periplasmic space, and the cell wall (Fig. 1). An evaluation of the behavior of cell contents alone, without the barrier (envelope), was of great interest from early on in order to elucidate in detail the metabolic pathways responsible for the production of different bioproducts. The plasma envelope responsible for the exit and entry of substances in the intact cell was ruptured in order to obtain the cellular contents (Fig. 1). Development of the cell-free enzyme system from microbial cells has already been achieved by numerous researchers during different periods. Initially, attempts were made to rupture the enveloped cell by exposure to water for long periods followed by heating in glycerin solutions, but only fractions of cellular contents were isolated, most probably in altered states (Buchner 1966). Later on, endogenous cell contents were obtained through a number of developed approaches summarized here. Various approaches have been employed for the disruption of the cell wall in order to obtain cell-free enzyme systems. Table 2 summarizes the potential advantages and limitations of each approach used for the disruption of cell wall.

Ethanol production by cell-free enzyme system Ethanol, an attractive liquid fuel, is conventionally produced by a number of microorganisms, such as Escherichia coli, and

Fig. 1 Schematic representation of cell-free system produced from a single yeast cell through different rupturing techniques


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Table 1 Comparative analysis of cell-free enzyme systems versus whole-cell systems for ethanol production Features

Whole-cell system

Cell-free enzyme system

References

Temperature (°C)

25–30

30–40

Kádár et al. (2004), Won et al. (2012), Khattak et al. (2014), and Welch and Scopes (1985)

pH

Narrow (5.0)

Broad (5–7)

Ylitervo et al. (2011) and Khattak et al. (2014)

Maximum theoretical ethanol yield (%)

85–95

75–99

Bai et al. (2008), Khattak et al. (2014) and Welch and Scopes (1985)

Product titer (g/L)

1.2–4.46

3.37–3.83

Bai et al. (2008), Ullah et al. (2014), and Khattak et al. (2014)

Glucose effect (%)

10–12

None

Malilas et al. (2013) and Khattak et al. (2014)

Reusability (batch numbers)

1–5

15–20

Ylitervo et al. (2011), Ullah et al. (2014), and Elçin (1995)

Zymomonas mobilis, however, is more efficiently produced by the yeast S. cerevisiae through microbial anaerobic fermentation (Hildebrand et al. 2013; Jeon and Park 2010) with maximum theoretical yield as high as 85–95 % (Bai et al. 2008). However, microbial cells cannot bear more than 10–12 % ethanol concentrations in broth due to the growth inhibition effects on cells (Malilas et al. 2013). Similarly, high concentration of glucose in the culture media and elevated temperature initially retard but later stop the fermentation process (Khattak et al. 2014). Moreover, the SSF process for enhanced and cheap ethanol production encounters serious hurdles in whole-cell fermentation processes (Zhang 2010; Kádár et al. 2004). To overcome this dilemma, the cell-free enzyme system was developed since it bypasses the restrictions due to cellular growth and viability and is active even at elevated temperatures (Khattak et al. 2014). Similarly, the ethanol yield was higher than that obtained using a whole-cell system as there are no chances of energy consumption for processes such as the synthesis of bioproducts (Fig. 2a, b), maintenance

of cell integrity, and cell mass proliferation (Khattak et al. 2014; Zhu et al. 2013). Ethanol production using the cell-free enzyme system, discovered accidently by Buchner, has been suggested being more effective than conventional fermentation system for ethanol production (Khattak et al. 2014; Buchner 1897). Besides cell-free fermentation systems suitable for ethanol production, the additional ATP molecules produced during glycolysis prevent the complete conversion of hexose sugars into ethanol (Welch and Scopes 1985; Xu and Taylor 1993). The balance of ATP consumption and regeneration during fermentation is maintained by using reconstituted cell-free enzyme system with supplemented ATPase enzymes (Welch and Scopes 1985). Cell-free enzyme systems helped achieve the complete conversion of glucose (180 g/L) into ethanol (yield 99 %), and arsenate was used as an alternative to the ATPase enzyme in order to make the approach economically feasible (Welch and Scopes 1985). Welch and Scopes developed a reconstituted cell-free enzyme system that was,

Table 2 Comparative study of different cell-lysis methods to produce a cell-free enzyme system Method of cell lysis Limitations

Advantages

References

Hydraulic press

Expensive equipment required, difficulty in maintenance,

Fast and ideal for large scale lysis, effective for all cells lysis

Buchner (1966) and Goldberg (2008)

Organic solvent lysis Enzymatic lysis

Proteins secreted are susceptible to organic Economical as no specialized equipment Breddam and Beenfeldt (1991) solvent, difficult in purification required and fast process Can cause alteration in target protein structure, Required non specialized equipment, highly Lam and Wassink (1990) required expensive exogenous enzymes specialized approach Comparatively slow process, chances of Economical, obtained proteins with original Taskova et al. (2006) and Yeng contamination activity et al. (2013)

Freeze grinding Centrifugation

Applied for cell with weak cell wall, slow process

Economical

Cell bomb

Only applicable to specialized cells

Ultrasonification

Increase in temperature may cause protein denaturation High energy required, difficulty in target proteins purification, heat produces can cause alteration in target proteins structure

Fast, inexpensive equipment required, obtain Goldberg (2008) and Simpson the protein stability and activity (2011) Really effective for large scale cell disruption Liu et al. (2013)

Beads beating

Effective for all kind of cells, economical, sample, fast

Martz (1966) and Lodish et al. (2000)

Khattak et al. (2014) and Griffiths et al. (2006)

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Fig. 2 A schematic representation of whole-cell systems (a) versus cell-free enzyme systems (b) for ethanol production using glucose as the substrate

however, a relatively expensive approach considering the cost of isolation and purification of different enzymes and the cofactors that needed to be obtained from different sources. In addition, the supplementation of cofactors and energy in the form of ATP required for enzymatic reactions further enhanced the overall cost of the fermentation system for ethanol production (Welch and Scopes 1985). Most recently, Khattak et al. (2014) developed a single-cell based enriched cell-free enzyme system consisting of all the glycolytic and fermentation enzymes with appropriate concentrations of essential cofactors, ATP, and ATPase (Khattak et al. 2014). The system was evaluated for ethanol production at broad range of temperatures (30–60 °C) using a pure glucose solution (10 g/L) as substrate. The interesting features of this study were that it made it possible to obtain significantly higher levels of ethanol production at elevated temperature (40 °C) at which conventional fermentation is not possible since wild microbial cells lose their viability (Khattak et al. 2013a, b). Considering the overall description, a conclusion can be drawn that, by using cell-free enzyme systems obtained from thermophilic strains, one may be able to overcome several limitations associated with ethanol fermentation through simultaneous saccharification and fermentation (SSF). The production of ethanol and other alcoholic biofuels through novel cell-free system has been briefly described in Fig. 3. Cell-free enzyme system for ethanol production consist of both glycolytic and fermentation enzymes with relevant cofactors and ATP. A cell-free fermentation system for ethanol production is a series of enzymatic reactions linked together that begin with the transport of glucose into metabolic pathways and the metabolism of glucose into ethanol in carefully

defined fashion, as shown in Fig. 4. Yeast cell-free lysate produced through several lysis methods consists of cellular machinery, which includes the translational system required for encoding genetic information for endogenous proteins synthesis, which are essential to metabolic pathways, including glycolysis and fermentation (Calhoun and Swartz 2005), oxidative phosphorylation (Jewett et al. 2008), and amino acid synthesis (Calhoun and Swartz 2006). Glucose molecules play a pivotal role in the initiation of cell-free enzyme system for ethanol production. In comparison to phosphate base compounds, usage of glucose has been reported to be an attractive and economical approach for the initiation of cellfree system and for the synthesis of additional enzymes expressed via cell-free expression systems (Jewett et al. 2008; Kim et al. 2011). First stage of glycolysis Three different enzymes are involved in the first enzymatic reaction of glycolysis, depending on the nature of the sugar molecule available for metabolism into ethanol. Hexose sugars (glucose, mannose, and fructose) are the substrates of hexokinase (HXK) I and II encoded by two different genes, HXK1 and HXK2, respectively. Alternatively, the glucokinase (GLK) enzyme encoded by GLK1 shows high specificity for glucose and mannose only (Walsh et al. 1983; Lobo and Maitra 1977). All these three enzymes have different affinities to glucose in the order HXKII (0.25 mM) > HXKI (0.1 mM) > GLK (0.03 mM) (Entian 1997). Different factors such as high ATP concentrations, trehalose-6-phosphate, etc., abolish the HXK activity. Alternatively, these factors also manage the


Fig. 3 Schematic representation of a novel cell-free metabolic pathway for the production of ethanol and isoburtanol from glucose precursor. Yellow box represents the conversion of glucose into two molecule of pyruvate by the action of four enzymes. Pyruvate can either be metabolized into ethanol (light blue box) or isobutanol (light orange box) in the second part of the reaction cascade with the action of different enzymes associated with each product formation. The figure has been reproduced from “Adv Biochem Eng Biotechnol 131:89–119” with the permission from “Springer Science and Business media”

entry of glucose molecule to glycolytic pathway (Dickinson and Schweizer 2004). Both genes, HXK1 and GLK1 are transcriptionally repressed in the presence of high glucose concentrations that in contrast induce the expression of HXK2 (Herrero et al. 1995; Rodriguez et al. 2001). Considering the other two enzymes, the role of HXK2 in glycolysis is particularly interesting as it has been reported that the enzyme has both regulatory as well as catalytic functions (Dickinson and Schweizer 2004). Considerable accumulation of the product of the first glycolytic reaction, glucose 6phosphate, inhibits the catalytic activity of the HXK enzymes and indicates that no more glucose is required by the cell for the metabolic pathway (Berg et al. 2010). The second chemical reaction of the glycolytic process is the isomerization reaction catalyzed by the enzyme glucose-6phosphate isomerase (PGI) encoded by PGI1 (Aguilera and Zimmermann 1986; Tekamp-Olson et al. 1988). The genes coding for this dimeric enzyme belong to the GPI family, whose members encode multifunctional phosphoglucose isomerase proteins involved in energy pathways. With respect to growth conditions, particularly the carbon source, the expression of the PGI1 gene is essentially constitutive (Green et al. 1988). The expression of the enzyme is considered to be strongly linked with glucose as it has been reported that several mutants with altered phosphoglucose isomerase

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activity were unable to grow on glucose as the sole carbon source in growth medium (Maitra and Lobo 1977; Clifton et al. 1978; Ciriacy and Breitenbach 1979). Phosphofructokinase (PFK) is the third enzyme of the chain reaction involved in the phosphorylation of fructose 6phosphate to fructose 1, 6-biphosphate at the expense of ATP. This enzyme is composed of a total of 300 amino acids, is an octamer, and is composed of α (4 molecules) and β subunits (4 molecules) encoded by PFK1 and PFK2, respectively. The activity of the enzyme is strongly linked with the ATP concentration in the medium. At higher concentrations, the allosteric site of the enzyme is occupied by ATP molecules and this brings significant changes in the catalytic site of the enzymes. The conformational changes in enzyme structure lead to sluggish enzyme affinity toward the substrate, fructose 6-phosphate (Selwood and Jaffe 2011). In contrast, the higher AMP concentrations nullify the inhibitory effect of ATP on enzyme activity, and thus, boost up the reaction rate by phosphorylating fructose 6-phosphate into fructose-1, 6biphosphate (Berg et al. 2010). Similarly, a decrease in pH of the medium has an inhibitory effect on enzyme activity (Berg et al. 2010), whereas the presence of fructose-2, 6bisphosphate allosterically activates enzyme activity (Bartrons et al. 1982; Kopperschläger and Heinisch 1997). The fourth step of the glycolytic process is the cleavage of fructose-1, 6-biphosphate into three-carbon compounds (G3P and DHAP), which are readily inter convertible. The cleavage reaction is catalyzed by aldolase enzyme encoded by FBA1 (Lobo 1984), which is, in a similar manner, transcribed constitutively with respect to the carbon source (Compagno et al. 2001). Aldolase enzymes from all organisms are classified into two categories depending on whether they are inhibited by metal-chelating agents (Rutter 1960). It is a well-known fact that the produced G3P is directly consumed in glycolysis while the other three-carbon compound, DHAP, should be converted to another G3P. This isomerization process is catalyzed by another enzyme called triosephosphate isomerase (TPI) encoded by TPI1 gene, whose expression is essentially constitutive (Scott and Baker 1993). Mutation in TPI1 leads to increase in DHAP, as at equilibrium 96 % of triose sugar is DHAP, which is the substrate for glycerol production (Fig. 4), and therefore, has no importance in ethanol production (Berg et al. 2010; Compagno et al. 2001). Second stage of glycolysis The second phase of the glycolysis begins with the oxidation of G3P into 1,3-bisphosphoglycerate and the reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (TDH), one of the most abundant proteins in yeast, existing in three different isoforms encoded by their respective genes TDH1, TDH2, and TDH3 (Holland and Holland 1980; McAlister and Holland 1985). These three genes show high sequence

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Fig. 4 Ethanol production: chain of reactions for ethanol production occurring in yeast catalyzed by their respective enzymes, including both glycolytic and fermentation enzymes

homology and are expressed coordinately. Depending on the carbon source, gene expression in glucose medium has been found to be around twice that in an ethanol-containing medium (McAlister and Holland 1985). The next reaction of the glycolytic pathway is the transfer of high-energy phosphate from 1, 3-bisphosphoglycerate to an ADP molecule for the synthesis of another ATP molecule and the concomitant formation of 3-phosphoglycerate. The reaction is catalyzed by the enzyme phosphoglycerate kinase encoded by PGK1 (Hitzeman et al. 1982). The PGK1 gene is an intriguing example of a gene that is expressed with all carbon sources but with different expression levels (Dickinson and Schweizer 2004). In comparison to the findings for other gluconeogenic carbon sources, approximately five times higher upregulation of the PGK1 gene was noted in glucose medium at the transcriptional level (Chambers et al. 1989). The product of the phosphoglycerate kinase reaction, 3-phosphoglycerate, is the substrate of the enzyme phosphoglycerate mutase, which is involved in the formation of 2-phosglycerate (Dickinson and Schweizer 2004). In contrast to GPM2 and GPM3 homologues, the GPM1 homologue is the only functional homologue and encodes for phosphoglycerate mutase (GPM; Heinisch et al. 1991, 1998). For exhibiting activity, the enzyme GPM requires 2, 3-bisphosphateglycerate as a cofactor,

which is suggested to be the product of the reaction catalyzed by the same enzyme, GPM (Fothergill-Gilmore and Watson 1990). The subsequent conversion of the 2-phosphoglycerate into phosphoenolpyruvate (PEP) is catalyzed by the enzyme enolase (ENO), a metalloenzyme encoded by ENO1 and ENO2 (Holland et al. 1981). The expressions of both the homologues differ as ENO1 is constitutively expressed during glycolysis while ENO2 expression is stimulated by glucose only (McAlister and Holland 1982). The enzyme has the ability to catalyze the reaction in both directions, depending on substrate availability (Pancholi 2001). For catalytic activity, ENO mostly requires metal ions as cofactors, and hence, it is classified as a metal-activated metalloenzyme. The highenergy phosphate group possessed by PEP in the subsequent reaction needs to be released to form another ATP molecule with concomitant formation of pyruvate, the final product of glycolysis. The enzyme pyruvate kinase (PYK) encoded by PYK1 catalyzes this substrate-level phosphorylation process (Maitra and Lobo 1977; Kawasaki and Fraenkel 1982). The upregulation of the PYK1 gene is strongly dependent upon the availability of its substrate PEP, fructose-1, 6-biphosphate, and ADP in generous amounts in the medium (Hess et al. 1966; Morris et al. 1986). In contrast, the availability of citric acid and ATP at an elevated concentration abolishes the


enzyme activity as they are considered important inhibitors (Hess et al. 1966; Haeckel et al. 1968). In the case of cells growing on glucose medium, an increment in kinase activity was observed as glucose induces the transcriptional activation of PYK1 gene (Nishizawa et al. 1989; Moore et al. 1991). Regulation of pyruvate fermentation Pyruvate formed at the end of glycolysis is the central point and is either fermented to ethanol or passes through the citric acid cycle, which depends partly on the kinetic properties of the respective route enzymes, pyruvate dehydrogenase and pyruvate decarboxylase (PDC). Moreover, the distribution of the pyruvate is mainly controlled at transcription level as glucose favors the expression of PDC and represses the respiration enzymes (Dickinson and Schweizer 2004). Due to the Crabtree effect associated with S. cerevisiae, and besides the presence of oxygen, generous amounts of the pyruvate formed during glycolysis enter the fermentative route rather than the oxidative route (van Dijken et al. 1993). As described earlier, the fermentative route consists of two reactions catalyzed by their respective enzymes. The first irreversible reaction is the decarboxylation of pyruvate to produce acetaldehyde catalyzed by the enzyme PDC that exists in different isoforms encoded by PDC1, PDC5, and PDC6, respectively (Dickinson and Schweizer 2004). In contrast to PDC1 and PDC5 genes, the importance of PDC6 gene in glycolysis is unknown; however, it plays an important role in the catabolism of amino acids to accept the α-ketoacids required for “fusel” alcohol production (Dickinson et al. 2000; ter Schure et al. 1998). Compared to ethanol, glucose medium strongly stimulates the transcription regulation of both PDC1 and PDC5 genes (Kellermann et al. 1986; Seeboth et al. 1990). The activity of the enzyme strongly depends on the concentration of the substrate “pyruvate” as it allosterically activates the active site (Sergienko and Jordan 2002; Lu et al. 2000). The last reversible reaction of the fermentation route is the reduction of acetaldehyde to ethanol with concomitant oxidation of NADH. The reaction is catalyzed by alcohol dehydrogenase, 20 different types of which are found in S. cerevisiae; however, only 13 of them have known functions (Dickinson and Schweizer 2004; Delneri et al. 1999). Among the 13 alcohol dehydrogenases (ADH) reported in literature, Adh1 encoded by ADH1 is the main cytosolic enzyme involved in the production of ethanol from acetaldehyde (Tekamp-Olson et al. 1988). The availability of zinc ion as a cofactor polarizes the carbonyl group of the acetaldehyde in order to favor the transfer of hydride from NADH, as described earlier. Like most of the glycolytic and the fermentation enzymes, the transcription of ADH1 is upregulated in the presence of glucose (Denis et al. 1983), which triggers the expression of Rap1 and Gcr1 transcription factors (Santangelo and Tornow 1990).

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Synthetic cell-free enzyme system for ethanol production Recently, the reconstruction of entire metabolic pathways in vitro including multi-enzyme reactions comprised of various constituents (Fig. 5) has received tremendous attention (Bai et al. 2008) Advances in bioprocess and industrial technology and metabolic engineering have open new gateways for production of novel products as well as improving the existing bioproducts. The technology offers promising avenues for future-generation biotechnology for production of various biocommodities including ethanol even though a cascade of enzymatic reactions in a cell-free environment constituting a metabolic pathway is at early developmental stages. The constitution of enzymes cascade whether in living cell, cell compartments, or organism is greatly dependent upon the well-organized regulatory mechanism, controlling feature information, energy, and mass transfer. These include exchange of signals between components of the system, movement of messenger molecules between the components, and metabolic channeling within temporary or permanently fashioned complexes and micro or nano-scale compartments. The technology is limited by several factors such as incomplete knowledge about how life works, the daunting complexity of cells, and the unintended interference between native and synthetic parts of a metabolic pathway. Moreover, the economic development of this technology is limited by high cost and unstable cofactors. The rupturing of cell through various strategies as described above exposes the inner cellular machinery to the

Fig. 5 The development of cycle of cell-free synthetic pathway biotransformation, which is composed of five parts: (a) pathway reconstruction, (b) enzyme selection, (c) enzyme engineering, (d) enzyme production, and (e) process engineering. The figure has been reproduced from “Biotechnol Bioeng 105:663–677” with the permission from “Springer Science and Business media”

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external environment as well as removes the genomic DNA and hence undesirable genetic regulation no more exists. Moreover, removal of external barrier allows easier enzyme– substrate interaction; product separation, isolation, purification, and reduced downstream processing cost; and easy system monitoring. This decrease dependency of the metabolic system over cellular regulatory system results in increased engineering flexibility (Forster and Church 2007). These findings led to the development of idea of reconstructing the entire metabolic pathway for production of important biofuels such as ethanol. Despite the Buchner reports (Buchner 1897, 1966) for the production of ethanol using cell-free enzyme system without live cells, the ATP imbalance prevents the complete conversion of glucose into ethanol. The reconstituted cell-free enzyme system supplemented with ATPase and exogenous cofactors was more efficient in producing high yield ethanol (Welch and Scopes 1985). The synthetic cell-free enzyme systems are believed to be much more efficient due to the several reasons including; absence of external barrier that favors maximum enzyme–substrate interaction, utilization of substrate only for product formation instead of being used for growth, proliferation and transport as in case of whole-cell biosystems, and higher concentration of biocatalyst. Allain (2007) predicted a kinetic model for ethanol production using the synthetic cell-free enzyme systems that could produce much higher yield than microbial fermentation when the same concentration of proteins is used (Allain 2007). Compartmentalization Compartmentalization, involving physical separation of biological reactions, has been evolved naturally. The arrangement of inner mitochondria membrane and membrane bound organelles in eukaryotic cells, such as peroxisomes encapsulating oxidative reactions for consumption or generation of toxic hydrogen peroxide (Gehrmann and Elsner 2011), and bacterial micro compartments, such as carboxysomes encapsulating Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and carbonic anhydrase (Bonacci et al. 2012), are the common examples of natural compartmentalization (Chen and Silver 2012; Chen et al. 2013). Besides encapsulation, these membranes or protein envelops function to protect the biocatalyst from external environment such as toxic intermediates of a biochemical reaction (Sampson and Bobik 2008). Besides the naturally occurring phenomenon of compartmentalization, these could also be introduced synthetically that displays a versatile approach for confining enzymes to a specific geometry. Precisely, in vitro compartmentalization involves the immobilization of enzymes in water droplets suspended in an oil emulsion (Griffiths and Tawfik 2003). Despite the encapsulation of cell-free enzymes in phospholipid membrane (Murtas et al. 2007), novel functions can be assigned to the system by introducing synthetic biocompatible


compartments, such as DNA hydrogels called “P-gells” for activating high yield cell-free synthesis (Park et al. 2009) or lipid disks for membrane bound protein expression (Shimono et al. 2009). Moreover, micro compartments can be heterologously expressed in various organisms to encapsulate a foreign pathway, including enzymes and substrate, which is otherwise not common in the particular organism. Common examples include the expression of carboxysomes from cyanobacteria in E. coli to encapsulate RuBisCo (Bonacci et al. 2012), expression of ethanolamine and 1,2-propanediol utilizing micro compartments from Salmonella in E. coli (Parsons et al. 2010). The approach is effective in desired product formation since these heterologously expressed micro compartments have proved to be potent in increasing the metabolic flux in enzymatic biosynthesis. However, some challenges still exist such as targeting of more exogenous enzymes to micro compartments and their architecture besides the stoichiometry of enzymes. Another limiting factor is the limited knowledge of transport mechanism of substrate and product excretion across the micro compartments (Klein et al. 2009). These issues need to be addressed in the future process design for more effective synthetic micro compartments development. Metabolic channeling Metabolic channeling involves the linkage of a group of enzymes together constituting a metabolic pathway. Several attempts have been made to exclusively elaborate the designing and action mechanism of metabolic channeling within the enzymes and between enzyme complexes. These studies were based on the structural modeling methods. “Channeling by proximity” that involves bringing the active sites of the corresponding enzymes close to each other to facilitate the metabolic channeling (Bauler et al. 2010). This optimal orientation approach has been quantitatively employed in successful development of partial channeling between the subsequent enzymes of glycolytic pathway (Hakobyan and Nazaryan 2010). Common example of naturally occurring linkage of enzymes includes tryptophan synthase (Dunn 2012), polyketide synthase (Tran et al. 2010), pyruvate dehydrogenase complex (Vijayakrishnan et al. 2010), ketoglutarate dehydrogenase complex (McLain et al. 2011), and cellulosomes (Doi and Kosugi 2004). Other commonly studied examples of metabolic channeling include aldehyde channeling (Carere et al. 2011), glutamate, and aspartate channeling (Gu et al. 2009). Metabolic channeling is effective both in long-range and short-range interactions. Tryptophan synthase metabolic channeling has been extensively studied and represents the long-range synchronization (Dunn 2012). In this enzyme complex, allosteric interaction is responsible for activity switching of the site depending on the stage of subunit


catalytic cycle and conformational changes that prevent the escape of substrate. Proton channels in thiamine enzymes represent a short-range interaction (Frank et al. 2004). Protein fusion via cross linker Literally, fusion proteins are created of parts from different sources. At DNA level, these can be described as the fusion of two or more genes that originally code for separate proteins. The fusion genes code for single or multiple polypeptide that exhibit the properties of their original proteins. Recombinant fusion proteins, important tool in biological research and therapeutics, could also be produced through recombinant DNA technology. At protein level, these are comprised of two or more cascade enzymes that are combined by a linker to form a multifunctional single polypeptide (Conrado et al. 2008). These fusion proteins are believed to have several useful applications in industry since these are pretty useful in directing metabolic flux to a preferred pathway (Bulow et al. 1985). These usually give high yield, concentration and sustainable productivity of a desired product. Their efficacy is greatly dependant on the designing strategy and composition of fusion protein especially the length of linker molecule. Silver and coworkers studied the effect of different linker lengths of a fusion dehydrogenase and ferrodoxin for hydrogen production and found that 14 amino acids linker was optimal in vivo with an enhanced factor of more than four and no substrate channeling was observed by in vitro tests (Agapakis et al. 2010). About three decades ago, Bulow et al. (1985) reported a bifunctional enzyme containing cascade reactions mediated by E. coli β-galactosidase (LacZ) and galactokinase (GalK) genes (Bulow et al. 1985). Their protein product showed the enzymatic activity of both gene products. Later, they reported the fusion of LacZ and galactose dehydrogenase genes from Pseudomonas fluorescens whose product fusion protein displayed the sequential hydrolysis of lactose followed by the oxidation of galactose to form the corresponding lactone. Their kinetic proficiency was several folds higher than the free enzyme mixtures (Ljungcrantz et al. 1989). A striking example of industrially important fusion proteins is glycerol 3phophate dehydrogenase and glycerol 3-phosphatase hybrid protein encoded by the fusion of their respective genes and linked by a four amino acid linker (Meynial et al. 2007). Co-immobilization Co-immobilization, involving the tethering of enzymes on a solid support or direct cross-linking, is another strategy to facilitate the multienzymatic biotransformation (Norton and D’Amore 1994). It can be carried out on the surface of solid support (van Dongen et al. 2009) or without support (Moehlenbrock et al. 2010). Different types of materials have

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been reported for the immobilization of multi enzymes such as; organic polymers (e.g., Amberlite XAD-7 and Eupergit®C), inorganic polyers (e.g., silica, zeolites, and mesoporous silica), and natural polymers (e.g., cellulose, starch, agarose, and chitosan; Jandt et al. 2013). Besides, regenerated amorphous cellulose (RAC) made from Avicel has also served as an excellent solid support material as it offers a large surface area that is externally accessible to the large-size enzymes, and hence, enzymes rarely lose their apparent activity (You et al. 2012). During co-immobilization, multienzymes are precipitated by adding salts, organic solvents, or nonionic polymers followed by cross-linking that result in cross-linked enzyme aggregates (CLEAs). The components of system can be randomly distributed (Betancor et al. 2006), positionally assembled (Wilner et al. 2009), or the active site of the enzyme can be adjusted to that of another enzyme (Mansson et al. 1983). This strategy offers several advantages in multi enzyme mediated biocatalysis such as fewer unit operations, less reactor volume, higher volumetric and space-time yields, shorter cycle times, less waste generation, and by-product formation (Betancor et al. 2006). Moreover, pairing various steps of a synthetic metabolic pathway can drive un-favorable equilibrium of reaction towards a desired product formation (Mateo et al. 2006). The position of different enzymes on the support matrix has significant role in the degree of substrate channeling. For example, precisely controlled distance between glucose oxidase and horseradish peroxidase through scaffolds, the distance of 13 and 33 nm between two enzymes result in 25-fold and 22-fold enhanced reaction rate, respectively, compared to free enzymes mixture (Wilner et al. 2009). You and Zhang, reported an enzyme complex immobilized on RAC, comprised of TIM, ALD, and FB. The complex displayed higher activity towards the simple enzyme complex linked by the scaffolding (You and Zhang 2012). Similarly, direct linkage of Kreb’s cycle enzymes from mitochondria of S. cerevisea show static in vitro metabolons and displayed 38–49 % enhanced reaction rate compared to free enzyme mixtures (Moehlenbrock et al. 2010).

Benchmark future prospects The production of ethanol as an alternative to fossil fuel significantly decreases the dependency on the availability of fossil fuels, which is drastically decreasing day by day (Ravikumar et al. 2013; He et al. 2014). Statistical data show that in 2011 the global ethanol production was about 88.7 billion liter, which was counted enough to replenish about one million barrels crude oil per day (Khattak et al. 2012). Although success in ethanol production has overcome the fuel demand to certain extent, there still exist certain serious issues

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regarding their lower production (Kádár et al. 2004; Zhang 2010). The success in ethanol production through cell-free enzyme system at elevated temperature has been suggested as an attractive approach to overcome the limitations associated with conventional fermentation, particularly SSF (Khattak et al. 2014). Cell-free enzyme system is a novel approach for significantly higher ethanol yield, although the recovery process of the system is expensive (Ratanapongleka 2010). The development of the relatively stable and reusable enzymatic system is currently under investigation in order to make the cell-free system feasible for ethanol production on industrial scale. The concepts of enzyme immobilization and synthetic cell-free pathway development are suggested as attractive and reliable approaches as these insure the recovery and reuse of cell-free enzyme system for several consecutive batches. The requirement that enzymes and substrates is kept in close proximity to enhance the reaction rate can be fulfilled through immobilization of the cell-free enzyme system (Panesar et al. 2010). In comparison to wild yeast strains, genetically engineered thermostable strains have the advantage of maintaining stability at elevated temperature and under stress conditions (Hohmann and Mager 2003). The immobilized reconstituted cell-free enzyme system obtained from a thermotolerant strain with an appropriate quantity of cofactors is suggested to be a great contribution toward commercialization of the cell-free enzyme system for ethanol production. Similarly, with the addition of saccharification enzymes, ethanol production can be achieved from cheap and abundantly available carbon sources such as lignocellulosic biomass and different waste materials by using a cell-free enzyme system. Heat shock proteins (HSPs) also contribute significantly in maintaining the activity of the enzymes at the elevated temperature (Trott and Morano 2003) essential for conducting SSF. Thus, it can be expected that with the addition of HSPs to cell-free systems, a significant difference in ethanol yield can be achieved. Cell-free enzyme system has received a great deal of attention since last few decades and has emerged as a powerful technology capable of complementing success in cellular in vivo systems. This can be attributed to the advanced control level and surprising diversity of strategic approaches developed for constructing synthetic biosystems. However, cellfree enzyme system still faces several challenges since it is still unclear to what extent this system can serve as test-beds for accelerating the design of synthetic programs or as a commercially relevant factories for producing biofuels (e.g., ethanol, bio-methnaol, biodiesel, and bio-hydrogen, etc.), therapeutics, metabolites, and non-natural products. Major challenges encountered by synthetic cell-free enzyme system include; integration and activation of highly efficient enzyme networks at low cost and large scale, bridging the in vitro and in vivo functionality, and scale-up of an industrial process for production of biocommodities. These challenges are quite


complex, daunting, and exciting, and by addressing them, the potential of cell-free systems could be explored and justified in a more profound way. Moreover, it will open the gateways to control and tune the cellular ensembles for biomanufacturing any bioproduct of material from renewable resources and waste materials in a more advanced way. Perhaps in the near future, we will see the industrial scale cell-free biorefineries for the production of multitude of products. The potential of cell-free ethanol production combined with high interest in renewable fuels emphasize that this idea deserves a serious consideration for future research. Technological advancements Genetic engineering strategies to modify the plant genome at commercially accepted level is believed to be a promising approach for improvement of ethanol production at costeffective level. These strategies could eventually enhance the expression level of all hydrolyzing enzymes, thereby reducing the need of exogenous enzyme supplementation in bioreactors for production of ethanol (Chapple et al. 2007). Genome modification has resulted in development of strains that are thermally stable and adaptable to other environmental stresses to certain limits, however, more advancement are still required for process optimization. Recent developments in cell-free enzyme strategies such as the biotransformation via a cell-free synthetic pathway are equally potent for advancement in this technology. We believe that combining the biotransformation of plant genome and cell-free synthetic enzyme pathway will have a significant impact on global ethanol production in the near future. Moreover, the progressive development in the field of cellfree enzyme system development is believed to ultimately result in breakthroughs in several sectors including research and development of novel and powerful algorithms, development of multi-scale modeling approaches, computational design of new biomolecules, designing novel metabolic channeling and micro-compartments, novel synthetic pathways designing, novel material synthesis, and discovery of novel scaffolds or development of scaffold-free technologies of multi-enzyme immobilization. Cost-effective process development During the early years of industrial scale ethanol production, high energy input compared to potential output was a major issue. Moreover, the economical cost of bioprocessing technology is readily affected by the relative cost of enzymes and cofactors. Later on, progressive developments in the bioprocessing technologies has lowered this cost to several extent by improving the efficacy of the process through several strategies, most recently the cell-free enzyme technology. According to the United States Department of Agriculture, the


ration of input to output for ethanol is about 1:1.34 (Shapouri et al. 2002). Enzyme immobilization could be a most fascinating and feasible approach for minimizing the economic cost of a biochemical reaction. The enzymes immobilized on a solid support could be eventually recovered after batch ethanol production, or more effectively, utilized in a continuous process. The efficacy of the technology can be further improved by bringing in action the latest developmental strategies in cell-free enzyme technology such as micro compartmentalization, metabolic channeling, protein fusion, and copolymerization etc. Control process design An important aspect of further development of synthetic cellfree enzyme system is the controlled process design and operation for productivity improved to enhance the efficacy of system. This task could be achieved via optimization of components of synthetic metabolic pathway mainly enzyme components through kinetic modeling, metabolic flux analysis, metabolic control analysis, high substrate concentration, high enzyme loading, and elevated temperatures by using thermophilic or even hyperthermophilic enzymes (Zhang 2009). The most striking advantage of cell-free enzyme system comes from the fact that the process conditions would no longer be needed to constrain the cell viability. This could enable the researcher develop enzyme isoforms that could withstand elevated temperature. From ethanol perspective, if one could find or engineer glycolytic enzymes that could bear thermal variation, it could enable ethanol production in a continuous fashion. This could solve the problems of enzyme recycling and high production rate. It could also simplify the hydrolysis of recalcitrant feedstocks such as lignocellulosic biomass. Moreover, the structure of enzyme could be altered in a desired way without worrying about the consequences affecting the viability of cell or organisms. This could eventually increase the enzyme flux, positively affecting the efficacy of metabolic pathway.

Conclusion The concept of cell-free enzyme system has given rise to an alternative strategy that helped resolved the several major barriers associated with conventional bioprocess development, precisely microbial fermentation. It could open new doors for similar processes for production of other valuable products such as chemical intermediates and pharmaceuticals from renewable resources. Designing the novel synthetic pathways, biotransformation of natural pathways, and development of reconstituted cell-free enzyme systems have

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significantly influenced the global ethanol production in the near future. The information provided on the cascade of reactions catalyzed by the respective enzymes and their regulation at both transcriptional and translational levels in this study would amount valuable for the development of such systems in the future. We anticipate that this review will provide answers to many queries regarding cell-free enzyme systems and will add a new dimension to the future study and production of ethanol and other biocommodities. Acknowledgments The research was supported by the Basic Research Program through the National Research Foundation (NRF) of Korea founded by the Ministry of Education, Science and Technology (no. 2011-0016965). The research was additionally supported by the BK21 plus (2014–2019) Korea (21A.2013-1800001)

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