Metabolic engineering of synthetic cell-free systems

Biochemical Engineering Journal 105 (2016) 391–405

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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Review

Metabolic engineering of synthetic cell-free systems: Strategies and applications Muhammad Wajid Ullah a , Waleed Ahmad Khattak a , Mazhar Ul-Islam a,b , Shaukat Khan a , Joong Kon Park a,∗ a b

Department of Chemical Engineering, Kyungpook National University, Daegu 702-701, South Korea Department of Chemical Engineering, College of Engineering, Dhofar University, Salalah 211, Oman

a r t i c l e

i n f o

Article history: Received 1 June 2015 Received in revised form 11 October 2015 Accepted 26 October 2015 Available online 31 October 2015 Keywords: Fermentation Cell-free system Yeast Immobilization Enzyme biocatalysis Metabolic engineering

a b s t r a c t Recently, the focus of biotechnological research has shifted towards the improvement of biological processes. Among the various approaches to achieve this goal, the use of cell-free technology is receiving considerable attention because it offers several economic and technical benefits. This technology improves biological processes in several ways, leading to enhanced efficacy, stability, specificity, and selectivity, as well as allowing reconstruction of target metabolic pathways. However, the adaptation of this technology to large-scale industrial processes remains a major challenge. This review describes the major constrains of whole-cell-based biological processes and how cell-free systems have been used to overcome such limitations. Furthermore, the shortcomings of conventional cell-free systems and possible solutions involving developments in immobilization technology are discussed. Finally, we illustrate the development of synthetic metabolic systems using the principles of metabolic engineering and discuss their possible advantages in the production of biofuels and bioproducts. In short, this review describes the fundamentals of and provides new insights into cell-free and immobilization technologies for the improvement of synthetic metabolic systems. © 2015 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Thinking outside the cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Cell-free technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 3.1. The bare cell-free system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 3.2. The immobilized cell-free system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 3.2.1. Development of immobilized cell-free systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394 Metabolic engineering of cell-free systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 4.1. Development of synthetic metabolic systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 4.2. Self-sustainability of the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 4.3. Promising avenues of metabolic engineering research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 4.3.1. Enhanced specific activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 4.3.2. Improved substrate selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 4.3.3. Directed immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 4.3.4. Improved stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 4.3.5. Selective compartmentalization for a reconstituted multistep reaction process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 Cost analysis and industrialization of cell-free systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 Practical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 6.1. Biofuels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

∗ Corresponding author. Fax: +82 539506615. E-mail address: [email protected] (J.K. Park). http://dx.doi.org/10.1016/j.bej.2015.10.023 1369-703X/© 2015 Elsevier B.V. All rights reserved.

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6.1.1. Bio-ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 6.1.2. Bio-hydrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 6.1.3. Bio-butanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 6.2. Bioproducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401 6.2.1. Cell-free proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 6.2.2. Synthetic protein scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 Conclusions and prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403

1. Introduction The benefits of whole-cell fermentation are often overshadowed by various limitations, such as inhibition of microbial cell growth and loss of viability due to the disruption of membrane fluidity [1]. Besides, microbial cell systems have several drawbacks, such as low yield, formation of metabolic byproducts, and the need for maintaining optimal temperature [2,3]. Some of these shortcomings can be overcome by using thermotolerant microbial strains and immobilized microbial cell systems, which offer improved stability and resistance, lower production costs, and products with increased purity [4]. However, such systems encountered several other problems, including negative effects on the growth pattern and metabolism, ineffective substrate utilization, formation of byproducts, and high downstream processing cost [5–7]. The development of cell-free systems was a major breakthrough, which rectified many shortcomings and increased the efficiency of biological processes for production of low- and high-value biocommodities [8]. The concept of a cell-free system was first introduced by Buchner, who claimed that biological processes can be commenced in vitro without living cells [9]. He demonstrated bio-ethanol production by a yeast cell-free system. However, that system was not suitable for large-scale applications because of the adenosine triphosphate (ATP) imbalance. Many years later, Welch and Scopes developed a reconstituted cell-free system from various sources to solve this dilemma and to attain a high yield of bio-ethanol [10]. Their system, however, had two major restrictions: high cost of enzymes and instability at elevated temperatures. These problems were solved by the single-cell-based yeast cell-free system and the encapsulated cell-free system, which minimized the cost and provided high yield of bio-ethanol at elevated temperatures [5,11,12]. On the other hand, owing to incremental technological advances in synthetic biology, the development of synthetic cellfree systems is currently the most promising direction in this field [13]. A cell-free system represents a state-of-the-art biotechnological conversion of a substrate into a product by a mixture of enzymes and cofactors constituting a cascade of reactions [14]. The use of cell-free systems considerably increased selectivity, stability, durability, and efficiency of biochemical transformations. It also improved cost-effectiveness and significantly extended the range of practical biotechnological applications. Besides, cell-free systems offer several advantages, such as tolerance to inhibitors, maximal enzyme–substrate interactions, limited enzyme loss, extended and continuous production, controlled variables, and prevention of the abnormal accumulation of intermediary metabolites [5,15,16]. Recently, this technology has gained significant attention and was tested for production of such commodities as bioethanol, cell-free proteins, synthetic amino acids, and metabolites [5,17]. Nonetheless, this approach has several inherent problems, which include reversibility, instability, leakage, inactivation, complicated recycling of expensive enzymes, and the lack of stable enzymes, enzyme complexes and cofactors [11,12,18]. These lim-

itations require the assembly of enzymes and cofactors on most compatible carriers by means of efficient immobilization strategies. An immobilized cell-free system confines multiple enzymes and cofactors catalyzing a cascade of biochemical reactions onto or within a support material (i.e., the carrier) to reduce or prevent their mobility [19,20]. Compared to free or soluble enzymes, immobilized enzymes are distinguished by their higher stability, lower cost, reusability, lower labor intensity, and simplicity of recovery and purification [4,21]. The carrier should be inert, biocompatible, biodegradable, mechanically resistant, and insoluble to provide a suitable environment for maximum efficacy of the enzymes and cofactors [22]. The efficiency and durability of immobilized systems can be enhanced further via cofactor engineering and using more cost-effective and stable biomimetic cofactors [23]. Moreover, careful selection of the immobilization strategy, which takes into account characteristics of enzymes, biochemical reactions, substrates, and desired products, will help to design more efficient biochemical pathways [24]. Nevertheless, this method has several limitations with regards to the multi-enzyme reactions, such as feedback inhibition, degeneration, loss of reaction intermediates, and consumption of cofactors [6,25]. Therefore, further research is needed to ascertain new design strategies for the development of improved and stable synthetic enzymatic pathways for industrial scale applications. Synthetic biology is currently focused on the construction and implementation of synthetic cell-free metabolic systems based on metabolic engineering-driven approaches. A synthetic cell-free system is composed of a number of purified enzymes and cofactors for the selective synthesis of a desired product through complex biochemical reactions [13]. These systems generally rely on multi-enzyme reactions and aim to provide more selective product formation, fewer unit operations, smaller reactor volume, higher volumetric yields, and reduced cycle duration [13]. They have been tested for production of several useful substances, such as biohydrogen, protein scaffolds, synthetic amino acids, and cell-free proteins [26–28]. The lack of comprehensive information on the durability, daunting complexity of cellular processes, and unintended interference between in vivo (native microbial) and in vitro (synthetic) systems prompted metabolic engineers to reprogram the existing systems and to design novel biological systems [26,29]. Moreover, cell engineering is often complex, laborious, and costly. In addition, it has several limitations, such as difficulties in maintaining cellular viability and physiology and the presence of an external barrier in the form of the cell wall or membrane [30]. These limitations have been resolved by synthetic cell-free systems through such strategies as compartmentalization, metabolic channeling, and co-immobilization [13,31]. Although the use of synthetic cell-free systems is still largely unexplored, its potential benefits are very clear. The method follows the traditional guiding principle of synthetic biology, namely the use of existing small building blocks, for example, those used for DNA synthesis during PCR, RNA synthesis through in vitro transcription, and production of polypeptides by in vitro transcription/translation [32].


This review is intended to address the needs for the development of cell-free and immobilization technologies. It emphasizes the need to improve the existing systems and construction of novel metabolic systems by applying fundamental principles and tools of synthetic biology. Furthermore, it discusses the requirements, characteristic features, and design of compatible materials for a more stable immobilization of enzymes and cofactors as well as immobilization strategies for future development of cell-free technology. This review shows that in contrast to cell engineering, in vitro cell-free systems are more attractive candidates for the construction of synthetic metabolic pathways via metabolic engineering approaches. Such approaches will not only facilitate the process control but also lead to the extension of the already long list of industrial applications. In addition, this review describes the strategies and recent achievements in the field of cell-free synthetic metabolic systems and their applications in the production of biofuels and bioproducts. We hope that this review will provide new insights into the future development of metabolic engineering of cell-free systems and serve as a solid platform for both novice readers and experienced researchers. 2. Thinking outside the cell In contrast to “red” or medical biotechnology that is concerned with the production of high-value medical biocommodities, “white” or industrial biotechnology is focused on the production of low-value bio-commodities through fermentation [33]. From the biochemical perspective, this fermentation approach is important because it is used to recycle NADH into NAD+ to sustain anaerobic glycolysis until the complete utilization of a substrate [34]. In biotechnological processes, carbohydrate metabolism is generally carried out by microbial cells. However, microbial fermentation has several constrains: the use of yeast, for example, poses the problems of growth inhibition and cell viability in the presence of high concentrations of the substrate and ethanol during fermentation. In addition, these conditions disrupt membrane fluidity [1–3,5]. Moreover, the need for maintaining optimal temperature is another limiting factor for the efficient process development [2]. It should be noted that in addition to being used for the synthesis of the desired product, the energy supplied in the form of growth medium nutrients is partly utilized as a material for cell growth, survival, and proliferation. Furthermore, the substrate may undergo biochemical side reactions resulting in the formation of metabolic byproducts or native cell components, which are impurities retarding the growth rate due to limited space [3]. Earlier attempts to use thermotolerant strains and immobilized whole cells alleviated these constrains to some extent and provided products with much higher purity [4]. Nonetheless, the use of immobilized cells posed several additional problems, such as thermal instability at elevated temperatures, ineffective substrate utilization, byproduct formation, and high cost of in vivo downstream processing of the industrial product [5,6]. Besides, this method affects the growth pattern and metabolism of microbes and, therefore, limits effective utilization of components in a repeated batch operation [7]. The shortcomings of immobilized whole-cell systems required the development of an alternative technology, such as cell-free systems. 3. Cell-free technology Although the concept of the cell-free system was presented a long time ago [9], it received substantial attention only in the recent years. The use of such systems has shown promising results because it enabled to considerably enhance the selectivity, stability, durability, efficacy, and cost-effectiveness in a variety of practical

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applications. Besides, it offers several advantages, such as improved inhibitor tolerance, maximized enzyme–substrate interaction, limited enzyme loss, extended and continuous production, controlled experimental variables, and reduced accumulation of abnormal intermediary metabolites [5,15,16]. A conventional cell-free system obtained through cell lysis contains a variety of enzymes and cofactors in addition to those constituting the target metabolic pathway [6,36]. Thus, it produces several byproducts in addition to the desired product [6,36]. Depending on the approach to the development, operation, and reusability, conventional cell-free systems can be categorized into bare and immobilized cell-free systems. 3.1. The bare cell-free system Since the advent of cell-free technology, bare cell-free systems were developed to manage the complexities and limitations of conventional biotechnological processes, such as fermentation [5,11]. According to Buchner, a biological process can be established in vitro through a series of enzymatic reactions constituting a biochemical pathway [9]. This system possesses several characteristic features, such as continuous product formation, controlled experimental variables (such as pH and ionic strength), regeneration of cofactors (e.g., ATP and NADH), and ways to prevent accumulation of unwanted intermediary metabolites. These properties make such systems a preferred choice for industrial applications, and they have already yielded promising results [10,36,37]. For example, a mathematical model of bio-ethanol production using this approach showed considerably improved efficiency [38]. This model suggested that the rate of bio-ethanol production would be proportional to the concentration of all enzymes involved in the biochemical pathway, provided that the concentration of each enzyme remains fairly constant. In addition to industrial production, this system can be used as an in vitro tool for studies of various biological processes, which usually occur inside a living cell. This approach not only reverses the trend towards more complex interactions in a living cell, but also provides a platform for more efficient production of the desired product. To date, several bare cell-free systems have been developed to manufacture various biocommodities and biological tools [5,39]. Nevertheless, a widespread practical application of conventional bare cell-free systems has been limited by a number of factors, such as reversibility, instability, and inactivation of constitutive enzymes. Moreover, the use of expensive enzymes without a possibility of their recycling restricts applications of cell-free systems on the industrial scale [18]. Some of these constraints were addressed by the immobilization of cell-free systems. 3.2. The immobilized cell-free system Cell-free system capability can be substantially enhanced by ensuring the effective use and reusability of enzyme(s). These aims led to the development of immobilized cell-free systems. Immobilization can be defined as the attachment or confinement of a single or multiple enzymes and/or cofactor(s) catalyzing a single biochemical reaction (or a cascade thereof) to the surface of or within a support material to reduce or prevent their mobility. The enzymes and their respective cofactors are attached onto or within a biologically inert, biodegradable, mechanically resistant, insoluble, and biocompatible material by such methods as encapsulation, adsorption, confinement, entrapment, or cross-linking [19]. The development of immobilized cell-free systems has attracted a lot of interest due to several advantages and potentially widespread applications of this technology in comparison with conventional bare cell-free systems. Immobilized cell-free systems overcome major limitations of bare cell-free systems, particularly the high cost and poor reusability of enzymes [11,12].

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Fig. 1. Schematic representation of the mechanism of formation of an alginate–chitosan capsule; (a) dropwise addition of a sodium alginate solution to the stirred CaCl2 solution, (b) interaction of Ca+2 ions with alginate, (c) formation of calcium–alginate beads, (d) liquefaction of the beads by sodium tripolyphosphate (Na-TPP) and formation of a chitosan layer, and (e) the alginate–chitosan core–shell capsule.

Moreover, this system can offer thermal stability at elevated temperatures and provide protection from consequences of physiological environment variability. Furthermore, immobilized systems may potentially achieve more efficient substrate utilization [40]. These remarkable features make immobilized cell-free systems a preferred approach for cost-effective industrial-scale product manufacturing. 3.2.1. Development of immobilized cell-free systems Immobilization of cell-free enzymes on a solid substrate, entrapment in a gel matrix, or confinement within a capsular membrane structure are possible strategies that make immobilized cell-free systems an effective approach to manufacturing various biological products. Immobilization of a single enzyme is a simple process; however, it may be limited by such factors as leakage, inactivation due to feedback inhibition, degeneration of support matrix, loss of reaction intermediates. The consumption of costly cofactors, when it comes to the use of multi-enzymes to drive reaction cascades should also be taken into account [6,25]. Therefore, extensive further research is needed to develop more efficient enzyme immobilization strategies, which would allow selection and design of more appropriate carrier substances as well as optimization of immobilization and operation conditions. These improvements are expected to make immobilized cell-free systems a better choice for product manufacturing on an industrial scale. 3.2.1.1. Carrier design. Effective enzyme use often requires immobilization onto or within a solid support material referred to as carrier. At present, there is no universal carrier that can support

immobilization of all types of enzymes for various applications [41]. However, there are certain characteristic features that should be considered during the selection of an appropriate carrier, such as high affinity for enzymes, availability of reactive functional groups, mechanical stability, rigidity, regeneration capacity, non-toxicity, hydrophilicity, insolubility, biocompatibility, and biodegradability [42]. In addition, several other parameters, for example, the inertness towards irrelevant enzymes, resistance to microbial attack, anti-compression stability, and availability at low cost must also be taken into account [41,43]. Overall, the selection of the appropriate carrier material is determined by the type of material to be immobilized, immobilization method, nature of the substrate, and desired product [24]. Porous materials are often preferred candidates because they offer free contact of the substrate with the immobilized enzyme. Utility of various materials for immobilization has been explored. In general, the support material used as an excipient may often have certain limitations, such as leakage, instability, dissolution, liquefaction, degradation, swelling, and toxicity [44]. Therefore, extensive research is needed to develop more appropriate biocompatible synthetic materials for immobilization of single or multiple enzymes and/or cofactors. Several approaches have been developed and utilized to improve the stability and characteristics of various materials that are used as carriers for enzyme immobilization. Earlier attempts involved the incorporation of polymer or fiber reinforcement into gel beads [45], coating of gel beads with a thin polymeric film [46], and the use of heavier ionic cross-linkers, such as Zn2+ , Sr2+ , or Ba2+ ions [47]. Later on, natural biosilicate formation has inspired innovative ways to design novel carriers for enzyme immobiliza-


tion [48]. These new methods facilitated synthesis of alginate–silica (ALG–SiO2 ) composites [49]. Taqieddin and Amiji developed a novel alginate–chitosan core shell capsule by cross-linking alginate and chitosan through sodium tripolyphosphate (Na-TPP) to avoid alginate liquefaction [44]. This procedure is briefly described in Fig. 1. Subsequently, synthetic monomers were grafted onto the surface of alginate that resulted in an accelerated diffusion of water and nutrients into polymer beads [50]. However, the use of such materials as enzyme immobilization carriers is associated with high cost of materials and technology required to apply fixation methods, which ultimately result in high biocatalyst costs. Recently, several advanced approaches and biomaterial-based carriers have been explored for their capacity to immobilize single or multiple enzymes. Most common materials used as excipients include cellulose, starch, collagen, modified sepharose, ion exchange resins, clay, hydroxyapatite, treated porous glass, certain polymers, and active charcoal [41,51–54]. Zhang et al. developed a novel approach for entrapment and encapsulation of enzymes using carboxymethyl cellulose (CMC) and CaCl2 , which formed a layered support material and provided an environment more compatible with immobilized enzymes [23]. Similarly, regenerated amorphous cellulose (RAC) has also been used for the immobilization of large enzyme molecules since it has a large external surface area [55]. Several enzyme complexes, such as triosephosphate isomerase (TIM), aldolase (ALD), and fructose 1,6-bisphosphatase (FBP) have been immobilized on RAC through a CBM-containing scaffold that has showed improved activity compared to the simple enzyme complex linked by scaffolding [56]. Currently, the nanomaterials, such as nanoparticles, nanofibers, nanotubes, and nanocomposites are preferred candidates for enzyme immobilization and stabilization [41,57]. Such materials possess ideal characteristic features, such as inherent large surface area, high biocompatibility, and excellent mechanical properties that balance key aspects determining the efficacy of biocatalysts [58,59]. These materials allow effective enzyme loading, high enzyme volumes, and have minimal diffusion limitations [58–60]. Nanoparticles-based enzyme immobilization has received a particular attention compared to the conventional enzyme immobilization. This can be explained by several important attributes of nanoparticles, such as their simple synthesis, relative easiness of bulk manufacturing, possible tailoring of their size, and homogeneous preparation with a well-defined core shell and a thick enzyme shell [61]. Furthermore, nanotubes also allow easy separation or reusability of the enzyme through simple filtration [62] or by using a magnetic field [63]. Immobilization of multi-enzyme, synthetic cell-free systems and co-immobilization of enzymes on these nanomaterials can also be accomplished [41]. However, further efforts are required for the development of more compatible materials that may support immobilization of enzymes and cofactors for the construction of novel synthetic cell-free systems. 3.2.1.2. Microenvironment for enzyme immobilization. Immobilization is a complex process that readily affects enzyme stability by changing the surface microenvironment and the degree of multipoint attachment. Therefore, it is mandatory to obtain the information about the enzyme microstructure and its interaction with the carrier and other enzymes in a metabolic pathway. Characterization of enzymes and carriers at micro- and nano-scales allows for obtaining valuable quantitative and qualitative information, including visualization of the morphology of the immobilized enzymes. High costs of enzymes prompt their efficient use, since unwanted and harsh conditions in industrial processes are likely to destabilize enzymes and shorten their lifespan. These limitations can be dealt with through the development of an optimized microenvironment for enzyme immobilization. Nonetheless, the

395

Fig. 2. Illustration of various methods for immobilization of cell-free enzymes: (a) cell-free enzymes, (b) the physical detention approach, (c) covalent linking of an enzyme, (d) single-ligand assisted immobilization, (e) cross-linking or copolymerization, and (f) confinement to a semipermeable membrane.

establishment of optimized microenvironment is a complex process that involves kinetic modeling, metabolic flux and control analysis, optimized ratios of substrate and relevant enzymes, elevated reaction temperature, variable pH and ionic strength values, etc. [5,64,65]. Careful design and optimized microenvironment in conjunction with effective enzyme loading, cofactor engineering, compartmentalization, metabolic channeling, and enzyme copolymerization will improve output of biotechnological processes [13,25]. These factors are crucial for assessing the efficacy of the immobilization approach and they are likely to guide the development of future immobilization strategies. 3.2.1.3. Research and development strategies. The choice of the immobilization strategy is largely determined by the number and type of enzymes, characteristics of the support material, operational conditions, and the desired product. These features are briefly described in Table 1. This notion has resulted in a progressive development of immobilization approaches to respond to challenges faced by cell-free systems. Here, we summarized key immobilization strategies for single and multi-enzymes and cofactors (Fig. 2). 3.2.1.3.1. Single ligand-assisted immobilization . In this approach, single or multiple enzymes and cofactors are allowed to bind to a carrier or a support, which may be a mineral (e.g., aluminum oxide or clay), organic matter (e.g., activated charcoal), modified sepharose, or a natural ion exchange resin [66]. The binding takes place on an external or internal surface through ionic interactions, salt linkage, hydrophobic bonds, hydrogen bonds, or van der Waals forces [66]. This binding may be achieved statically, dynamically, through reactor loading, or via an electrodeposition process. Thus, the immobilized enzymes and cofactors are protected from abrasion, leakage, and inhibitory effects of the bulk solution, and hence, stably retained. This type of immobilization resolves the problem of substrate diffusion, as it allows the substrate to interact freely with enzymes and cofactors. Furthermore, this method minimizes the chances of enzyme inactivation. This method is simple and cost-effective but suffers from several drawbacks, such as weak and reversible binding between the carrier and the enzyme and low probability of co-immobilization of the enzyme and the relevant cofactor [66,67]. Moreover, the process design and downstream processing are limited by enzyme leaching, especially in aqueous solvents [67]. In

396


Table 1 Illustration of characteristic features of constituents of an immobilized cell-free enzyme system. Constituent

Feature

Characteristics

Reference

Enzyme

Biochemical properties

Known molecular mass and prosthetic groups, active functional groups, high purity Conformation, active site Specific activity, known pH and temperature profiles, kinetics of activity and inhibition, stability against pH, temperature, solvents, contaminants, and impurities

[4,10]

Porous, insoluble, permeable Known chemical base and composition, functional groups, maximum enzyme-substrate interaction, accessible volume of matrix and pore size, chemical stability of carrier Mechanical resistance, gelling and swelling behavior, flow resistance (for fixed bed application), sedimentation velocity (for fluidized bed), abrasion (for stirred tanks) Biocompatible, biodegradable, inert, non-toxic,

[22,40] [23]

Type of material immobilized, operation conditions such as temperature and pH, yield of active enzyme Lower mass transfer resistance; compartmentalization, diffusion properties Thermal, operational and storage stability Continuous and extended production, high yield, easier product recovery and purification Maintenance of cofactors, controlled variables such as pH, ionic strength, lower capital cost

[134–135]

Structural properties Kinetic parameters

Carrier

Physical properties Chemical properties

Mechanical properties

Biological properties Immobilized enzyme system

Strategy Mass transfer effects Stability Efficacy Feasibility

[133] [15,134]

[77]

[22]

[15,40] [18,135] [41,36] [4,10,136]

Table 2 Illustration of various strategies for immobilization on several carriers. The immobilized enzymes have been used for various practically important functions and applications. Enzyme

Carrier

Technique

Function/Application

Reference

Cal-B Fluorescens lipase Alcohol dehydrogenase Candida antarctica lipase

Adsorption Adsorption Covalent binding Entrapment

Production of specialty chemicals Resolution of menthol Thermal stability, pharmaceutics Biofuels

[137] [138] [73] [79,139]

Entrapment

Biomedicine, biosensors

[140]

Entrapment

Probes, biosensors

[141]

Phospholipase

VP OC1600 (Bayer) Celite Attapulgite nanofibers Polysiloxane (POS)-polyvinyl alcohol (PVA) hybrid matrices Co-polymer: poly 2-hydroxyethyl acrylate and poly (dimethylsiloxane) Co-polymers N-isopropylacrylamide (NIPAAm), glycidyl methacrylate (GMA) and N,N-dimethyl acrylamide Liposome

Encapsulation

[142]

Invertase Urease Urease DNA repair enzyme

Lectins Porous glass beads Cotton threads Liposome

Affinity Entrapment Adhesion Encapsulation

Hydrolysis of phospholipids (control hypercholesterolemia) Sucrose hydrolysis Sucrose hydrolysis Urea hydrolysis (biosensor) Repairing DNA damage (skin aging and cancer)

Horseradish peroxidise and chloroperoxidase Trypsin,

Table 2, we provide several examples of enzyme immobilization using this strategy for various practical applications. 3.2.1.3.2. Covalent enzyme linkage. This method is routinely used for immobilizing a single enzyme or a group of enzymes. A covalent link is formed between functional groups of a support material (such as amino, tyrosyl, hydroxyl, imidazole, indolyl, phenolic, or sulfhydryl groups) and the enzyme molecule containing amino acid residues like arginine, aspartic acid, and histidine [66,68]. Chemical bonding is achieved through diazotization, peptide bond formation, group activation, or polyfunctional reagent interaction. The material used as an excipient may be polyacrylamide, porous glass, agarose, and porous silica, which form strong links with the enzyme [69]. Commonly, the -amino group of lysine, frequently located on the surface of proteins,serves as a common reactive group [70]. In more advanced strategies of enzyme immobilization through covalent linkages, the carriers are engineered by introducing epoxide groups to enhance the affinity for lysine under mild conditions [71]. The development of an immobilized cell-free system may be carried out in two steps: (1) activation of the carrier by the addi-

[143] [144] [145] [146]

tion of reactive compounds to produce electrophilic groups on its surface, which ultimately react with strong nucleophilic groups on the enzymes and cofactors to be immobilized [72], (2) chemical modifications into the polymer backbone [66,72]. This process is influenced by pH, ionic strength, and other environmental variables. The later step justifies the suitability of this strategy for the development of a cell-free system that can withstand such varying conditions [5]. Although this strategy has shown promising results, conformational changes occurring at the active site of the enzyme and cofactor may limit its widespread practical application. Nevertheless, such limitations may be overcome if immobilization is performed in the presence of a specific enzyme’s substrate, a competitive inhibitor, or a protease. Enhanced longevity, operational stability, and reusability enabled numerous practical applications of enzymes immobilized on magnetic nanoclusters obtained through covalent linking (e.g., in pharmaceutical industry) [73]. Several examples of enzymes immobilized in this way are described in Table 2.


397

Fig. 3. Illustration of development of an immobilized (encapsulated) cell-free system; (a) cell lysis via different methods, (b) isolation and concentration of the cellular lysate, (c) preparation of a cell-free suspension in a calcium chloride solution and loading into a syringe, (d) dropwise addition of the cell-free suspension into a stirred sodium alginate solution, (e) encapsulation of cell-free enzymes, and (f) an encapsulated cell-free system.

3.2.1.3.3. A cross-linking or copolymerization scheme. Copolymerization involves covalent linking of various molecules of an enzyme or a group of enzymes catalyzing a cascade of reactions via a polyfunctional reagent under different physiological conditions [55]. Generally, this procedure is carried out in two ways: (i) either the support matrix is precoupled to an affinity ligand for the target enzyme(s) to be immobilized or (ii) the enzyme(s) is conjugated to another reagent that acquires affinity for the carrier [74]. Commonly used reagents for covalent cross-linking include glutaraldehyde, diazonium salt, dimethyl adipimidate, hexamethylene diisocyanate, aliphatic diamines, and N-N -ethylene bismaleimide [75]. The most recent improvement of this strategy was the utilization of a bioaffinity layer, which increased enzyme-binding capacity and reusability by Coulomb forces, hydrogen bonds, and van der Waals forces [76]. This strategy cannot be used for routine synthetic-pathway development due to lower immobilization efficacy and risk of enzyme denaturation. Nevertheless, this approach is preferable for industrial applications, where downstream processing of the product is required (Table 2). 3.2.1.3.4. Physical detention approach. This method can be used for immobilization of a single enzyme or a group of enzymes and cofactors. Immobilization is carried out inside the matrix of a water-soluble polymer, such as cellulose triacetate, agar, gelatin, carrageenan, or alginate, which constitutes a gel, fiber, or a microbead [51,66]. The pore size of the polymer is adjusted by varying its concentration in order to avoid leakage of the biocatalyst from the core matrix. This strategy offers several advantages, such as a decreased risk of protease attack or contamination by microbial and other enzymes, as well as beneficial effects of gas bubbles, mechanical sheer, and hydrophobic solvents that ensure larger surface area [66,77]. Besides, this strategy is inexpensive, robust and requires only mild conditions for a cascade of reactions. However, it carries a risk of enzyme inactivation and has limitations on mass transfer, low probability of colocalization of enzymes and relevant cofactors, and low enzyme loading [77,78]. Moreover, this strategy is of limited practical utility with substrates and/or products of high molecular weight, because large molecules are less likely to reach active catalytic sites of entrapped enzyme molecules due to steric hindrances. The physical detention approach has a wide range of applications in the fields of fine chemistry, biomedicine, and biofuels. It is commonly used for the development of probes and biosensors

[79]. This strategy has been used for industrial scale manufacturing of various products. Several enzymes immobilized using this strategy for industrial applications are listed in Table 2. 3.2.1.3.5. Confinement in a semipermeable membrane. This method involves confinement of a group of enzymes and cofactors within a semipermeable membrane [16]. The development of an encapsulated cell-free system is shown in Fig. 3. The process is usually carried out by the liquid-droplet formation method [11,12]. The gel or membrane formation around the enzymes and cofactors is instantaneous and irreversible and involves diffusion of these two components. The catalytic activity of the encapsulated enzyme is strongly dependent on its tolerance to variations in the microenvironment, such as changes in pH, temperature, and ionic strength regardless of the enzyme being effectively retained within the capsule. The capsular liquid core provides sufficient space for metabolic reactions and brings other advantages, such as improved stability and inhibitor tolerance, maximal enzyme–cofactor and enzyme–substrate interactions, limited leakage, continuous diffusion, controlled release, and lower mass transfer resistance [15,16,40]. This strategy, however, is not effective for larger substrates or products because of mass transfer limitations [77]. Applications of encapsulated biocatalysts have been reported in food industry [80], biomedical research [44], and many areas of industrial importance as summarized in Table 2.

4. Metabolic engineering of cell-free systems Engineering of an artificial cell is a highly complex, timeconsuming, and costly approach associated with viability and sustainability problems, physiological deficiencies, and low success rate [30]. Improving the characteristic features of a desired product and the development of novel biocommodities through conventional cell-free systems still remain major challenges for biotechnologists. With incremental improvements in the fields of synthetic biology, metabolic engineering, and cell-free systems, the focus is shifting toward reprogramming of existing and construction of novel metabolic pathways [13,31]. Metabolic engineering of cell-free systems may be a solution to the above-mentioned serious problems. Although this technology is still at the early developmental stage and is largely unexplored, creation of synthetic cell-free systems on the basis of recent synthetic biology advancements is rapidly gaining pace.

398


4.1. Development of synthetic metabolic systems A simple biochemical reaction results in the conversion of a substrate into a product by a single catalyst. According to the old-school paradigm, the simplicity of such biochemical reactions is conducive to large-scale industrial applications involving manufacturing of the desired product [14]. In contrast, real-life complex metabolic pathways mediate biological processes by reaction cascades, which are catalyzed by a group of enzymes and cofactors [14]. Formation of the desired bioproduct with improved characteristics requires in vitro reprogramming of such metabolic pathways. Currently, synthetic biology research is focused on novel strategies for the development of synthetic metabolic systems. For example, the assembly of several purified enzymes and their respective cofactors can be utilized for the formation of a desired product through a series of complex biochemical reactions [13]. The two principal preparations commonly used for cell-free production of bio-commodities are crude cell-free extracts and purified cell-free enzymes. The conventional approach for bioconversion by means of a synthetic cell-free system involves a two-step procedure. During the first step, metabolic engineering of an organism of interest is done in order to block target routes of the relevant metabolic pathways and to insulate the product from further processing. During the second step, the substrate is transformed into a desired product using the crude cell-free extract [81]. This approach obviates the laborious and costly step of the purification of target enzymes participating in the utilized metabolic pathway. The second approach involves reconstitution of a complete metabolic pathway from purified components [39]. This approach is a growing field in industrial biotechnology. The development of efficient synthetic pathways comprising multiple enzymes is of immense interest because it can accomplish a maximal yield and curtail the risks and shortcomings associated with one-pot multienzyme catalysis. Several possible limitations of such systems, which include feedback inhibition, degeneration, a loss of reaction intermediates, and excessive consumption of cofactors [25] can be dealt with by applying compartmentalization, metabolic channeling, and co-immobilization of multi-enzymes. Compartmentalization involves a physical separation of biological reactions to arrange cascade enzymes close to each other in the appropriate sequence [16,25]. Microcompartments encapsulating synthetic metabolic pathways, enzymes, and substrates have been heterologously expressed. Typical examples of microcompartments are membrane-bound organelles, such as peroxisomes [82], and bacterial microcompartments, such as carboxysomes [83]. The function of these microcompartments is to protect cells from toxic intermediates [84]. The cascade of enzymes can be brought together via so-called metabolic channeling [16,25]. Common examples of metabolic channeling are tryptophan synthase [85], polyketide synthase [86], pyruvate dehydrogenase complex [87], ketoglutarate dehydrogenase complex [88], and cellulosomes [89]. During co-immobilization, multi-enzymes catalyzing a cascade of reactions are either immobilized on a common solid scaffold [90,91] or cross-linked directly [25]. This solid support can be a synthetic organic polymer, such as Amberlite XAD-7, an inorganic polymer, such as silica or zeolite, or a natural polymer, such as cellulose, agarose, or chitosan [25]. The enzymes can be distributed randomly, positionally assembled, or placed opposite each other [25]. Co-immobilization can result in a loss of enzymatic activity. Zhang reported a five-step developmental cycle for a cellfree synthetic pathway including; (a) pathway reconstruction, (b) enzyme selection, (c) enzyme engineering, (d) enzyme production, and (e) process engineering (Fig. 4) [13]. Several key factors are given a significant consideration during the development of a synthetic pathway: the balance and maintenance of cofactors, thermodynamics, reaction equilibrium, developmental cost, feasibility,

broad impact, and the downstream processing cost of the product. Compared to simple biochemical reactions, complex reactions are mediated by multiple enzymes in one pot. This method offers several advantages: fewer unit operations, smaller reactor volume, higher volumetric and space-time yields, reduced cycle duration, and minimal formation of byproducts [13]. Compared to reactions in conventional cell-free systems, metabolic reactions catalyzed by synthetic pathways produce only the desired product because of the presence of essential selected enzymes, whereas thousands of irrelevant proteins are present in crude cell-free extract that results in the formation of various byproducts. Thus, in contrast to typical cell-free systems, the synthetic pathway holds promise of maximal substrate utilization. 4.2. Self-sustainability of the system The continuous energy supply to a cell-free system in the form of cofactors (e.g., ATP) is important. This arrangement will ensure higher productivity and a longer lasting process resulting in the enhanced efficiency and economic feasibility of the system on an industrial scale. To achieve these goals, the development of immobilized cell-free systems allowing cofactor regeneration has been thoroughly analyzed in the past few years [92]. Several attempts have been made to develop a system that continuously regenerates ATP as a constant energy supply for biotechnological processes. ATP regeneration was mimicked by the liberation of inorganic phosphate in the medium that sequesters Mg2+ ions and thus inhibits ATP regeneration [17]. Crude cell-free extracts obtained from microbial cells contain enzymes able to maintain basic intrinsic cell functions and reactions, such as glycolysis and fermentation, central carbon atom metabolism, tricarboxylic acid cycle, and oxidative phosphorylation [6,31,36]. These metabolic pathways work simultaneously and recycle inorganic phosphate for the regeneration of ATP [6]. Addition of a small amount of cofactors (ATP and NAD) to the crude cell-free extract will initiate efficient metabolic reactions because the ATP concentration is relatively low during the initial phase of the incubation [36]. These pathways then continuously regenerate these cofactors through metabolism of various intermediates, such as pyruvate, phosphoenolpyruvate, glucose-6-phosphate (G6-P), creatine phosphate, and acetylphosphate [36,37]. In addition, in the presence of glucose, ATP is regenerated during metabolism, but this phenomenon is readily controlled by the decrease in pH [93]. Moreover, pyruvate is partly utilized for ATP regeneration and recycling of NADH to NAD [26]. This two-stage utilization of pyruvate ensures a constantly elevated ATP concentration, which results in improved metabolic functioning of the system [37]. Similarly, during anaerobic fermentation, the accumulation of pyruvate at the intersection of glycolysis and fermentation will also favor enhanced ATP production and, therefore, improve metabolic processes [37]. An efficient dual-energy system consisting of creatine kinase and glucose has been recently reported that readily generates ATP molecules through the glycolytic pathway [94]. Creatine phosphate is rapidly hydrolyzed by creatine kinase to jump-start the metabolism by providing ATP at the beginning of the process [6]. In conclusion, cofactors are supplied only for the effective initiation of metabolic functions of cell-free systems. Further processes are triggered by a continuous regeneration of cofactors through the metabolic pathways driven by all metabolic enzymes present in the cell-free extract. Thus, such immobilized cell-free systems may turn out to be effective for repeated batch operations [95]. The process can be further improved by means of ATPases that rapidly hydrolyze ATP, thereby releasing the energy harnessed by the enzymes, which are implicated in metabolic functions of the system [11,12]. The use of inverted membrane vesicles from


399

Fig. 4. Schematic representation of development of a synthetic cell-free system; (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 permission from Springer Science and Business Media.

Escherichia coli or membrane modules containing free or embedded ATPase, respectively, may be a promising avenue of future research and development efforts to improve the efficacy of such systems via a more sophisticated and effective ATP regeneration [6]. 4.3. Promising avenues of metabolic engineering research Recent advances in the development of synthetic pathways involving immobilized multiple enzymes highlighted numerous advantages of this technology. Rapid technological developments opened up a wide range of practical applications and new technical possibilities for enhancing the efficacy and specificity of synthetic metabolic systems. Here, we describe some of the possibilities that emerged following recent developments in this field. 4.3.1. Enhanced specific activity Enzymes are generally specific in their activity and are characterized by high degrees of absolute, group, relative, linkage, and/or stereochemical specificity. Free enzymes, however, are susceptible to the formation of aggregates in organic solvents, an effect, which limits enzyme access to the substrate. Immobilization of multi-enzyme complexes on the appropriate matrix to catalyze a cascade of reactions constituting a synthetic pathway led to ∼100-fold improvement of the activity of stand-alone enzymes or multi-enzyme complexes in such solvents [96]. A high density of immobilized enzymes may increase specificity, as was reported for cellulase [97], due to cooperative effects on the substrate. This improved feature of immobilized enzymes has made them good candidates for practical applications in diagnostics and research. 4.3.2. Improved substrate selection Advances in the efficiency of enzyme immobilization in the recent years came about because of the development of molecular biological approaches, such as directed evolution or site-directed

mutagenesis, which permitted incorporation of additional binding residues [71] or adaptation to a particular scaffold [98]. The extent of enzyme–substrate selectivity and the degree of prevention of catalyst inhibition can be optimized by selecting the appropriate immobilization approach [99]. Substrate imprinting can modify substrate preference by the enzyme during the immobilization. This process is known as cross-linked imprinting (CLIP) [67].

4.3.3. Directed immobilization Directed immobilization relies on selecting a specific location for the attachment via interactions between functional groups of the substrate and immobilized enzyme(s). Modulation of enzyme enantioselectivity has received considerable attention in recent years due to advancements in biotechnology and novel genetic engineering methods successfully utilized to achieve this goal [100]. The efficiency of this process has been improved by immobilization and it can even be reversed if required [101]. Such achievements are possible only when researchers have detailed knowledge of the structure and conformation of relevant enzymes. The attachment to the mechanical support facilitates manipulations of the catalyst’s orientation and hence, its activity [101].

4.3.4. Improved stability The optimal temperature and pH of the enzyme-containing medium can be modified upon immobilization. Immobilization can enhance enzyme thermal stability [11,12]. However, even slight deviations from optimum pH may significantly affect the activity of immobilized molecules. The exact mechanism of this phenomenon is not clear and it is assumed that it can be caused by the changes in the enzyme microenvironment after immobilization [102].

400


Table 3 Illustration of production of various biofuels and bioproducts via synthetic cell-free approaches and their practical applications. Product

Category

Strategy

Applications

Reference

Bio-hydrogen Bio-ethanol

Biofuel Biofuel

Synthetic enzymatic pathway Cell-free fermentation

[13,116] [5,10–12]

Bio-butanol

Biofuel

Bio-electricity

Biofuel

Fuel cells, electrocardiogram,

[147–148]

Fusion proteins

Bioproduct

Synthetic pathway biotransformation (SyPaB) Surface immobilization of multi-enzymes Enzyme cross-linking

Petroleum and chemical industries Beverages, vehicle fuel, household heating, feedstock, antiseptic, solvent, pharmacology Vehicle fuel, solvent, paint thinner, perfumes

[65]

Protein scaffolds

Bioproduct

Metabolic channeling

Synthetic amino acid

Bioproduct

Cell-free system

Cell-free proteins

Bioproduct

Bio-cellulose

Bioproduct

Synthetic cell-free system; PURE (protein synthesis using recombinant elements) system Single cell-based cell-free system

Metabolic flux regulation, Industrial product formation e.g., lactone Development of artificial photosynthesis system, Mevalonate and glucaric acid synthesis Expanding the genetic code, recombinant protein synthesis, engineering the synthetic metabolic pathways Production of enzymes and enzyme variants

[150–153]

Metabolites

Bioproduct

Crude extract cell-free system (CECF)

Minimal cells

Bioproduct

Synthetic cell-free system

Nanocomposite development, Biomedical applications, drug delivery, enzyme immobilization, scaffold synthesis Vicinal diols synthesis, dihydroxyacetone phosphate synthesis (DHAP) Understanding the basis of life, membrane bound protein synthesis

4.3.5. Selective compartmentalization for a reconstituted multistep reaction process Examples of reconstitution of an entire metabolic pathway via multistep reactions have been recently published [103,104]. Immobilization favors multistep enzymatic reactions, synthetic metabolic pathways, and chemoenzymatic cascade reactions through compartmentalization of individual catalysts [96]. Betancor et al. reported that co-immobilization of coupled enzyme systems enhances the activity of individual enzymes [90], for example, when nitrobenzene nitroreductase and G-6-P dehydrogenase are co-encapsulated in silica particles. This system is sufficiently reliable to retain the cofactors, such as cross-linked enzyme crystals (CELE), for redox reactions [105]. van Dongen et al. reported co-immobilization of three enzymes in a copolymer: horseradish peroxidase (anchored to the membrane), hydrophobic candida antarctica lipase B (Cal-B) (located in the bilayer membrane), and hydrophilic glucose oxidase (immobilized in polymersome chain). This system catalyzed three sequential steps [106]. 5. Cost analysis and industrialization of cell-free systems The economic feasibility of any industrial metabolic process is principally dependent on the relative cost of its constituents, mainly enzymes and cofactors [12]. Enzymes and cofactors are usually prepared by expensive biotechnological methods that present serious limitations to the industrialization of cell-free system processes. Therefore, it is believed that the production of low-value biocommodities by cell-free systems is a costly approach due to the lack of stable standardized building blocks (enzymes or their complexes), costly labile cofactors, and substitution of enzymes and cofactors [13]. Thus, the cost of enzymes and cofactors acts as an economic bottleneck for the development of desired metabolic pathways using synthetic cell-free systems based on de novo assembly of its constituents. Considering the high price of cofactors, the industrialization of cell-free systems faces the question of economic feasibility. Several attempts have been made to resolve this issue. Two approaches have been used: (1) recycling of costly cofactors through immobilization and (2) replacement with more stable and low-cost artificial biomimetic analogs [107–109]. Recycling of cofactors is crucial for an immobilized cell-free system, because it is imprac-

[13,30]

[27] [6,28]

[28,39,149]

[27,81] [32]

tical to add exogenous cofactors, for example, to an encapsulated cell-free system [12]. Methods of regeneration of essential cofactors via metabolism of various intermediates, such as glucose, glucose6-phosphate (G6P), pyruvate, phosphoenolpyruvate (PEP), creatine phosphate, glycerol, and acetylphosphate from various metabolic pathways have been well established [37,95]. Peng et al. have reported that metabolism of glycerol effectively regenerates cofactors for subsequent repeated batches in an immobilized cell-free system [95]. Glucose has been reported to effectively regenerate ATP in a cell-free enzyme-mediated reaction [93]. Similarly, Kim and Swartz have found that besides ATP regeneration, a small fraction of pyruvate is also utilized to recycle NAD from NADH [37]. This two-stage utilization of pyruvate for the regeneration of essential cofactors (ATP and NAD) ensures subsequent batch operation of a metabolic process by an immobilized cell-free system. Some recent studies employed more cost-effective and stable biomimetic cofactors, which replaced unstable and more costly analogs to improve existing protocols and to develop novel products via fermentation technology [109]. From the economic perspective, it is suggested that the development of cheaper and more stable biomimetic cofactor analogs can lower overall production expenditures in comparison with costs of the conventional whole-cell fermentation-based technology. The above discussion leads to the conclusion that the development of immobilized cell-free systems can be a step towards the industrialization of the system. Immobilization of enzymes and autocatalytic regeneration of cofactors in repeated batches is expected to improve the productivity and lower the cost of bioproduct manufacturing. In addition to regeneration, replacement of costly cofactors with more stable and cost-effective artificial biomimetic compounds can be a promising avenue for future applied research. Besides, the utilization of raw feedstock, such as industrial waste (e.g., waste from beer fermentation broth) [35,110], which accounts for