Systems Biology Solutions for Biochemical Production Challenges

Highlights

Cost-effective production of chemicals in engineered organisms is challenging because there is a basic conflict between the needs of the cell for life, and our needs for commercial viability.
The synthetic biochemistry approach can free us from cellular limitations, opening up new possibilities for enhancing biobased chemical production parameters.
Cost-effective cell-free bioreactor construction requires designing simple, robust systems that can run efficiently for long periods of time. While considerable challenges remain before biomanufacturing is possible at the scale currently used in microbial systems, recent progress suggests that a cell-free approach has promise.

Metabolic engineering efforts that harness living organisms to produce natural products and other useful chemicals face inherent difficulties because the maintenance of life processes often runs counter to our desire to maximize important production metrics. These challenges are particularly problematic for commodity chemical manufacturing where cost is critical. A cell-free approach, where biochemical pathways are built by mixing desired enzyme activities outside of cells, can obviate problems associated with cell-based methods. Yet supplanting cell-based methods of chemical production will require the creation of self-sustaining, continuously operating systems where input biomass is converted into desired products at high yields, productivities, and titers. We call the field of designing and implementing reliable and efficient enzyme systems that replace cellular metabolism, synthetic biochemistry.

Keywords

metabolic engineering
enzyme cascade
cascade biocatalysis
multienzyme systems
biofuel
biomanufacturing
green chemistry
natural products
commodity chemical

Moving Biosynthesis Away from Living Organisms

Considerable efforts have been devoted to engineering living organisms to produce useful chemicals ranging from high-value natural products like cannabinoids to low-value products such as, fuels, plastics, and building block chemicals [

]. Microbial generation of natural products would free us from the problems with extraction from native sources, such as agricultural boom and bust cycles, as well as resource-intensive and expensive purification requirements. Moreover, microbial production can be more environmentally benign than chemical syntheses. Perhaps the greatest environmental impact of metabolically engineered microbes will be in replacing high-volume petroleum products like fuels and commodity chemicals [

]. There are numerous potential environmental benefits: the biomass starting materials are renewable; replacing petroleum-based starting materials can lower the release of global warming gases; and the products are generally biodegradable. However, replacing high-volume, low-value products is also the most economically challenging because cost is critical. To make bioderived products economically competitive will require high-efficiency conversion of the input biomass into useful compounds.

However, efficient conversion of biomass is difficult to achieve in biological organisms [

]. The complexity of cellular physiology makes it difficult to optimize volumetric productivity (see Glossary) because many regulatory mechanisms exist in the cell to control pathway flow rates. Life processes distribute resources away from the desired products, lowering yield. Moreover, product or intermediate toxicities can limit product titer. Although engineering of regulatory mechanisms and competing pathways have proven to be useful, the complexity of the problem is hard to overcome. Even successful efforts to engineer industrially scalable strains typically took large teams and hundreds of person years of effort [

Indeed there may be an effective 'speed limit' for biological production in microbes defined roughly by ethanol production. Ethanol production has had effectively thousands of years of optimization and is only a few enzymes away from the central glycolytic pathway. In contrast, next-generation biofuels and other commodity chemicals generally require more complex pathways. Ethanol can be produced at ~2 g/l/h productivity, ~100 g/l titer and ~90% yield [

]. Perhaps the greatest metabolic engineering success stories for commodity chemical production so far are 1,3 propanediol and 1,4-butanediol, both produced in engineered Escherichia coli. The reported production parameters for 1,3 propanediol are 3.5 g/l/h productivity, a final titer of 135 g/l, but at only 65% of the theoretical yield from glucose (0.51 g 1,3 propanediol/g glucose) [

]. For 1,4 butanediol the reported production parameters are 2.1 g/L/h, 99 g/L and a yield at 70% of theoretical [

]. Moving beyond the range of production parameters approximately defined by ethanol will likely require major technological innovations.

While living organisms can clearly integrate complex inputs and perform remarkably sophisticated tasks, for engineering purposes it can make more sense to take inspiration from biology rather than try to harness life itself [

]. For example, we can see that birds fly and learn how they do it, but airplanes do not mimic bird flight mechanisms – and the result is more useful for our needs and more readily subject to engineering. The cell-free, synthetic biochemistry approach similarly seeks to remove the constraints of living organisms to develop simpler, more streamlined versions of biologically inspired systems that can be engineered efficiently for practical uses. Although considerable challenges remain for cell-free approaches, their potential has only begun to be explored. Here, we discuss the potential of cell-free production for biomanufacturing of high-volume chemicals. The possible advantages and challenges of the synthetic biochemistry approach are summarized in Table 1 and discussed in detail below. While there is considerable interest in using cell-free methods to prototype new metabolic pathways with the idea of putting the systems back into cells [

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], we concentrate on efforts that seek to move chemical manufacturing away from cells. Cell-free approaches for producing high-value chemicals like pharmaceuticals or for making biologics like proteins face different opportunities and challenges and are not the focus of this review [

Table 1 Potential Advantages and Challenges of the Synthetic Biochemistry Approach

Potential advantages	Challenges
High yields: no other pathways to divert input biomass	Enzyme production costs: there are added costs required for enzyme preparation not needed in cell-based methods
Facile optimization: precise control over all system components	Enzyme stability: not all enzymes can be made sufficiently stable to function long term outside the cellular environment
Rapid design–build–test cycles: simplified problem diagnosis and implementation of fixes	Enzyme inhibition, inactivation, or regulation by intermediates or products: the absence of compartmentalization or repair pathways can make enzyme systems more prone to problematic metabolites
Great pathway design flexibility: do not need to feed life processes	Cofactor costs: there are additional costs for obtaining cofactors that would otherwise be naturally present in cell-based systems [ATP, NAD(P)H, etc.]
No need to account for cell toxicity that might inhibit production
Simpler product purification: simpler metabolite mixture and no membranes to break open
High productivity: more precise pathway optimization and the possibility of higher enzyme concentrations
Simplified computational modeling: fewer and better defined reaction parameters
Flexible reaction conditions: option to use diverse, even nonphysiological conditions

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Approaches to Setting up Cell-Free Systems

Cell Lysate Method

In the cell lysate method (Figure 1), the pathway of interest is expressed in the cell as in any metabolically engineered microbe [

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Bechtold M.
et al.

Biotechnological development of a practical synthesis of ethyl (S)-2-ethoxy-3-(p-methoxyphenyl)propanoate (EEHP): over 100-fold productivity increase from yeast whole cells to recombinant isolated enzymes.

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]. Rather than using the cells as the biofactory, however, they are lysed, releasing the contents into a bioreactor. The lysate approach has a number of advantages. First, it is simple and potentially inexpensive. Second, the cytoplasmic contents may contain sufficient cofactors so that additional cofactors do not need to be added, further reducing cost [

]. A cell lysate approach is particularly effective for complex systems where it may be advantageous to use endogenous systems to make the desired product. Indeed the lysate approach is the one favored for commercial cell-free protein production [

A disadvantage of using lysates is that many metabolic pathways are maintained so that additional engineering may be required to remove unwanted pathways. While complex lysates may therefore appear to simply revisit many of the problems of metabolic engineering, in fact, it can be easier to eliminate pathways in a cell-free system because they are not needed to maintain life processes. For example, Swartz has proposed unleashing a protease upon lysis to eliminate certain target enzymes [

Purified Activity

In this approach, the enzyme activities are more extensively purified [

37.

Zhang L.
et al.

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]. This does not necessarily mean, however, that highly purified enzymes must be used. For example, with stable enzymes, activity purification could be as simple as just heating extracts [

], thereby enriching the desired enzyme activity and eliminating other problematic activities. The level of purification required depends on the system. Moreover, each enzyme does not necessarily need to be purified separately. Enzymes could be expressed together and purified together or expressed separately, then mixed and purified together. The advantages of the purified activity approach are a higher level of system control, the elimination of unwanted pathways, and the ability to express proteins in different organisms which affords considerable flexibility. To some extent the purified activity approach will involve additional complexity and preparation costs, however.

Hybrid

Another approach is to use a lysate for most of the enzymes, but then supplement the lysate with other enzymes expressed separately. This approach is of course the most flexible and could be particularly useful when most of the enzymes express in one organism, but others can only be expressed in a different organism.

Possible Advantages of the Cell-Free Approach

Potential for Better Production Parameters

There is potential for cell-free systems to more readily achieve high yields, titers, and productivity – key production parameters that in part define commercial viability. To obtain high yields, all of the input biomass (e.g., sugar) would ideally be converted into the desired product. In the cell-free approach, there is only one pathway in the bioreactor, so it is possible to achieve nearly 100% yields, limited only by enzyme specificity and thermodynamics [

]. In cells, there are myriad biochemical pathways diverting sugar into unwanted side products that are required for maintaining life processes. Indeed, much of metabolic engineering involves deleting these side pathways, which can be a difficult challenge if one wants to maintain cell viability. An alternative strategy, called dynamic metabolic control, seeks to decouple growth and production phases so that unwanted cellular pathways can be shut down during the production [

]. In many ways, the cell-free approach is the height of dynamic metabolic control because the growth phase (making enzymes) is completely separated from the production phase (bioreactor). There are indications that it will be possible to obtain higher productivity in cell-free systems. For example, Kay and Jewett reported a remarkable productivity of 11.3 ± 0.1 g/l/h for the cell-free production of 2,3 butanediol [

]. The ability to precisely optimize reaction conditions is likely one factor in obtaining high productivity (discussed below). Another factor that could favor cell-free productivity in the future is the ability to concentrate the relevant pathway enzymes to higher levels than would be possible when expressed in a cell culture. Finally, since there is no need to maintain life processes, there are no concerns about intermediates or products that are toxic to cells, a major benefit of cell-free systems. Thus, it may be possible to obtain much higher titers of toxic chemicals than is possible in living cells [

Great Flexibility in Design, Implementation, Optimization, and Re-engineering

When working with cell-free systems, there are few restrictions in pathway design. Even rebuilding central metabolism is straightforward [

]. While it is also possible to reconfigure central metabolism in living cells, it can be challenging [

]. Examples of pathway design that illustrate the flexibility of synthetic biochemistry are discussed below.

Pathway optimization in cell-free systems can be more facile than in microbial systems. In microbes, most enzyme levels can only be coarsely adjusted, while in a cell-free system, enzyme levels can be defined with precision. Moreover, even the levels of cofactors and input chemicals can be easily adjusted, which is largely impossible in cells. With a cell-free system, all the components can even be readily adjusted on the fly as the system runs. Even the reaction conditions such as temperature and pH can be controlled easily, opening up possibilities for using nonphysiological reaction conditions to the extent the enzymes tolerate them (or be engineered to tolerate them).

Diagnosing problems and fixing them is generally a more straightforward process in a cell-free system. Enzymes can be assayed directly so changes in enzyme activities are readily assessed. The mixture of metabolites is greatly simplified over cellular systems, so concentrations can be measured easily. Moreover, cofactor pools (e.g., redox states) can be monitored. Oftentimes, the solutions to any identified bottlenecks can also be implemented rapidly, by simply switching a regulated enzyme with an unregulated one or a more stable enzyme for one that loses activity rapidly. If enzyme engineering is required, it can be straightforward to introduce the newly remodeled enzyme and reoptimize the system.

Computational reaction modeling can be a more effective tool for optimization efforts in a cell-free system because modeling is more tractable [

]. Synthetic biochemistry systems are simpler and more well defined than whole cell systems, and it is often possible to measure many, if not most, of the enzyme kinetic parameters and the cofactor levels can be set. Moreover, with rapid metabolomics analysis [

], the evolution of the systems can be completely monitored over time, aiding in model construction and testing.

Easier Product Purification

Because the mixture of metabolites in a cell-free system is simpler than in a fermentation broth, downstream processing can be more straightforward, particularly for products that would normally be retained in cells [

]. Moreover, the use of organic overlays can be more flexible because there is less danger of emulsions and no need to worry about killing cells [

]. Indeed, product isolation can be as straightforward as simply extracting essentially pure product into an organic overlay [

Challenges of the Cell-Free Approach

Enzyme Production Costs

Clearly there will be an added cost to preparing a cell-free reactor compared to growing cells in a fermenter. How significant this cost is depends on many factors [

]. If a cell lysate approach is used, the added cost should be minimal as it is simply a matter of harvesting cells and lysing them after growth. If purified enzymes are used, the cost depends on expression levels, the host used, whether the enzyme is expressed intracellularly or into the medium, and stability.

High enzyme stability is a key factor, operating at many levels to ease the implementation of synthetic biochemistry systems. Purification of highly stable enzymes can be as simple as heating an extract [

]. Moreover, the longer the enzymes last, thereby increasing the total turnover number per unit enzyme, the lower the net cost contribution of preparing the enzymes [

]. Stability can also make additional process investments such as attaching enzymes to solid supports make sense [

For most enzymes, it can be straightforward to obtain stable variants by various means. In particular, genome mining for thermophilic enzymes can be effective. Because thermophilic enzymes are often most active at higher temperatures, they have often been used in cell-free systems at high temperature [

]. While high temperature can be useful for suppressing contaminating bacterial growth, it has the disadvantage that all the enzymes must be stable at high temperature and increases the degradation of cofactors (see below). Thus, an alternative is to screen for enzymes from thermophiles and hyperthermophiles that maintain sufficient activity at low temperatures [

]. Although the specific activities can be reduced, it is possible that the advantages of high stability can outweigh the loss of specific activity. Directed evolution is a particularly effective method for increasing thermostability; generally only requiring a rapid screen for activity [

]. Finally, effective design methods have now been developed for enzymes with a known structure or a close homolog of known structure [

There are, of course, enzymes that are more difficult to stabilize than others. For example, oxygen-sensitive enzymes that may have sensitive cofactors can be a challenge [

]. Redox enzymes with mechanisms involving radical intermediates have a tendency to self-immolate [

]. Although, membrane enzymes can be engineered to be highly stable [

], adding purified membrane proteins to a cell-free system is a challenge. Membrane proteins could be introduced in the presence of detergent, but the remaining components of the system would need to be compatible with the detergent. It might be possible to reconstitute membrane proteins into proteoliposomes or nanodiscs [

], or to use amphipols [

], but it may be hard to keep the costs low enough for high-volume chemicals. Lysate-based systems provide a way to introduce membrane proteins because they can retain intact cellular membrane vesicles with active membrane proteins [

Problem Metabolites

Reactive metabolites in a cell-free system may inactivate enzymes. For example, high concentrations of input-reducing sugars that may glycylate proteins [

] or aldehydes that are generated can react with nucleophiles on proteins [

]. These problems may be mitigated by engineering of enzymes to remove vulnerable sites, or using process or system optimization to keep sugar and aldehyde concentrations low. Cells often compartmentalize toxic intermediates [

] and it is possible that a similar bioinspired strategy could be used in vitro, perhaps by channeling toxic substrates to the next enzyme [

], thereby keeping its concentration low. Reactive oxygen species that are generated must be removed expeditiously; for example, by including catalases to eliminate peroxide [

]. Like cells, high titers of product compounds may destabilize and inactivate enzymes [

]. These problems can often be reduced by improving enzyme stability (see above) or through continuous product removal strategies such as an organic overlay. The advantage of cell-free systems is that problematic enzymes can generally be readily identified and the full panoply of engineering tools can be used to fix them.

Enzymes can make errors and some metabolites are prone to spontaneous chemical alteration [

]. Thus, unwanted, dead-end side products may build up, leading to a decrease in yield. Perhaps more thorny, the side products may inactivate enzymes in the system. In cells, there can be metabolite repair mechanisms to deal with these unwanted metabolites [

]. In a cell-free system it may be possible to adjust conditions to minimize side reactions or it is necessary to introduce repair systems, thereby adding to complexity. For example, Opgenorth and colleagues introduced a two-enzyme metabolite salvage pathway in a cell-free system to deal with the undesired products of a promiscuous enzyme [

Cofactors

Another important issue in cell-free systems is cofactor costs [ATP, NAD(P)H, CoA, etc.]. For low-cost production, it is essential that cofactors are regenerated in situ and used many times. A crude back of the envelope calculation illustrates the challenge in the example of converting isoprenol into limonene [

]. The pathway requires 4 ATP per limonene made. At a bulk ATP price of $1000/kg, the ATP cost for producing limonene is then ~$15 000/kg limonene/TTN, where TTN is the total turnover number for ATP. Thus, in this scenario, ATP must be recycled in the reaction at least 1500 times to lower the ATP cost contribution below $10/kg limonene. Lowering cofactor costs as well as effective cofactor utilization is a major challenge for low cost biomanufacturing by synthetic biochemistry.

Cofactor Generation and Recycling

Externally Powered Cofactor Generation

Most methods for recycling cofactors involve the use of a sacrificial substrate in coupled enzyme systems [

]. For example, many industrial enzyme processes utilize dehydrogenases to regenerate NAD(P)H [

]. ATP can be supplied by conversions of other high-energy phosphate molecules such as creatine phosphate, polyphosphate, or acetyl phosphate [

]. One drawback of using sacrificial substrates, however, is that the substrate is consumed in the process, which can add to the cost. A second problem is that a side product may build up in the system that can ultimately inhibit enzymes. The relative importance of these limitations depends on the specific system and the value of the final product. For low-value chemicals like biofuels, it is likely necessary to move away from sacrificial substrate methods.

A potentially exciting approach would be to use electrical power to energize biochemical systems [

]. Cheap solar electricity would provide a carbon free way to drive biochemical manufacturing. It is conceptually straightforward to convert electrical power into reducing equivalents in the form of NAD(P)H and both enzyme-mediated and chemically mediated methods are under development [

]. Cell-free conversion of electrical power into ATP free energy is harder to envision and we are not aware of any methods. If it were possible to efficiently power both NAD(P)H and ATP regeneration electrically, it could provide a general solution to the cofactor regeneration problem. Membrane vesicles used in lysate-based systems that retain active oxidative phosphorylation might provide a pathway for the complex conversion of reducing equivalents into ATP [

In Situ Cofactor Generation and Recycling

Until electrically driven methods come on board, the cheapest method for generating the necessary high-energy cofactors may be to develop systems in which biomass degradation and new synthesis are coupled in a continuous system. In a generic synthetic biochemistry system illustrated in Figure 2, sugar (or other input biomass) is typically fed into a catabolic pathway such as glycolysis, that breaks the sugar down into simpler 2- or 3-carbon building blocks (e.g., pyruvate, acetyl-CoA), simultaneously generating high-energy cofactors (e.g., ATP, NADH). We refer to the catabolic portion of the sugar processing as a breakdown module. The carbon building blocks and high-energy cofactors can then enter an anabolic pathway to make our desired product. We refer to the anabolic portion of the conversion as a build module. The key is to design systems that can run for as long as possible without intervention, which requires highly efficient recycling of the cofactors so that as much product as possible is generated with a minimum of input cofactors.

Figure 2 Basic Components of a Synthetic Biochemistry System That Operates Like Cellular Metabolism.

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Sugar is broken down into carbon building blocks and high energy cofactors (breakdown module). The carbon building blocks and high energy cofactors are employed to make new molecules (build module). In this process the spent cofactors must be recycled back into the breakdown module.

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How can we ensure long-lived recycling of cofactors? The simplest way to envision a cell-free system is to design it with perfect stoichiometric recycling. For example, a system using standard glycolysis in the breakdown module will generate 2 ATP per glucose that enters the pathway. Thus, in perfect stoichiometric recycling, one would want to design the build module to consume 2 ATP, so that ADP is completely regenerated for a new breakdown/build cycle. If fewer than 2 ATP are used in the build phase, then ATP would build up and ADP would be depleted, causing the pathway to shut down. If the build module requires more ATP than is generated in the breakdown phase, then ATP is rapidly depleted and the system shuts down. Thus, on the face of it, stoichiometric recycling might seem like a good solution.

For biomanufacturing, however, we do not just want systems to run, we want them to run for a long time. For sustainable operation, stoichiometric recycling is not ideal because there will always be loss of ATP, either spontaneously or by contaminating ATPase activity. Similarly, NAD(P)H can be oxidized over time. Thus, stoichiometric recycling is not necessarily the best solution in the real world. Ideally, the process would generate an excess of the high-energy cofactors to create additional capacity in the system, allowing losses to be tolerated. However, what then is the best way to manage the excess to avoid building up cofactors and shutting the pathways down?

Generation and Maintenance of Surplus Cofactor Supply

Two systems have been designed to manage excess cofactor generation in the breakdown phase [

] to allow for improved performance over stoichiometrically balanced systems [

]. In both systems two pathway options are introduced: one that generates more of a cofactor and one that does not. The flow down the two pathways is dependent on how much of the high energy cofactor is present.

In the ATP rheostat system [

], shown in Figure 3, one pathway uses enzymes from standard glycolysis. In glycolysis, low-energy phosphate (Pi) becomes high-energy phosphate at the glyceraldehyde dehydrogenase (Gap) step and then gets converted into ATP at the phosphoglycerate kinase (Pgk) step. To create the rheostat system, we introduce a nonphosphorylating Gap enzyme, GapN, bypassing ATP formation in a parallel route to 3-phosphoglycerate. The flow down the ATP generating pathway is controlled by the levels of P_i and ADP. If there are high levels of P_i and ADP in the system, it indicates that ATP has been hydrolyzed and more should be generated. In contrast, if there is plenty of high-energy phosphate in the system, free P_i and ADP are depleted, shutting off flow down the ATP-generating pathway and increasing flow down the GapN pathway.

Figure 3 Systems That Autoregulate Cofactor Levels.

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(A) The rheostat concept for regulating ATP levels. Two alternative pathway are used. One pathway generates ATP and the other does not. The flow down the two pathways is adjusted by the ATP concentration status of the system. At low ATP levels, the free phosphate levels rise, thereby increasing flux down the ATP generating pathway. At high ATP levels, the free phosphate concentration is depleted, choking off flux down the ATP generating pathway and thereby increasing flux down the non-ATP generating pathway. (B) The purge valve concept for regulating reducing equivalents. Two pathways are installed. One pathway generates reducing equivalents (in this case NADPH), while the other does not, due to the presence of an NADH-oxidizing enzyme NoxE. When NADPH levels are low, the NADP⁺ levels rise, thereby increasing flux down the NADPH-generating pathway. When NADPH levels are high and NADP⁺ levels low, flow down the NAD+ utilizing pathway becomes favored and no net reducing equivalents are produced because of the presence of NoxE.

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A purge valve system has also been designed to regulate reducing equivalents [

]. A basic purge valve is shown in Figure 3 and consists of the following components: (i) an NAD⁺-dependent dehydrogenase; (ii) an NADP⁺-dependent dehydrogenase for the same substrate; and (iii) an enzyme to oxidize any generated NADH, in this case a water-forming NADH specific oxidase, NoxE, to oxidize NADH. In the pathway shown in Figure 3, if the NADP⁺-dependent dehydrogenase is used, both product and NADPH is generated. If the NAD⁺-dependent dehydrogenase is used, however, only product is generated because the NADH is reoxidized back to NAD⁺. Thus, there is a NADPH-forming pathway and a parallel one that generates no additional reducing equivalents. When NADPH levels are high (NADP⁺ levels low), the non-NADPH-forming pathway is preferred. When NADPH becomes depleted, raising NADP⁺ levels, flow down the NADPH forming pathway increases proportionately.

The rheostat and purge valve systems not only allow building systems with surplus high-energy cofactor capacity, but also they are completely autoregulated so that they are simple to implement, requiring no additional intervention as the systems run. Moreover, they make synthetic biochemistry systems more modular because it is not necessary to precisely match cofactor generation and usage so that new build modules can be tacked on to an already constructed breakdown module, without any need to redesign the breakdown module.

Examples of Cell-Free System Design

In this section, rather than provide a comprehensive overview of prior work, we simply highlight a trio of published cell-free systems that illustrate how freedom from cellular constraints can unleash creative and effective pathway designs.

Biohydrogen beyond Biological Limits

Biohydrogen production provides a dramatic illustration of the power of cell-free approaches to move us beyond what is possible in cells. Natural cellular metabolic pathways are only capable of generating up to 4 H₂ per molecule of glucose consumed [

]. Yet in theory, it should be possible to extract 12 H₂ per molecule of glucose. In 2000, Woodward and coworkers showed that it was possible to couple enzymes of the pentose phosphate pathway to nearly quantitatively convert glucose-6-phosphate to hydrogen and CO₂ [

]. Zhang and colleagues advanced the practicality of cell-free H₂ production by developing systems to feed low cost sugar feed stocks into the pathway [

]. The fist system using low-cost starch as an input is illustrated in Figure 4 [

]. The scheme converts starch nearly quantitatively into H₂ and CO₂, with the following overall stoichiometry (ignoring the sugar units that are not subject to the action of starch phosphorylase):

C₆H₁₀O₅ + 7H₂O = 12H₂ + 6CO₂

Figure 4 Examples of Synthetic Biochemistry Systems.

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In these subway map depictions, the breakdown modules are shown as purple lines and build modules as green lines. (A) Complete metabolism of sugar for hydrogen production. (B) An elegantly designed sugar breakdown module and application to isobutanol synthesis. (C) A terpene production system that employs a purge valve to regulate NADPH levels.

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It is hard to imagine that a system like this could be introduced into cells. The basic chassis was also creatively used to develop a sugar powered biobattery with an energy density an order of magnitude higher than a lithium ion battery [

Simplified Sugar Breakdown

The Sieber group developed a creative way to break glucose down into pyruvate using only four enzymes (Figure 4) [

]. The remarkable economy of enzymes is aided not only by a reduction in the number of enzymes relative to glycolysis, but also by using the same enzyme for two steps in the pathway. The breakdown module is coupled to both an ethanol and an isobutanol module. We only illustrate the isobutanol module in Figure 4, which again shows the impressive economy of enzymes by using a breakdown module enzyme in the build module as well. The production parameters obtained are low, and the breakdown module is somewhat limited because it generates only NADH and no ATP, so ATP-dependent build modules cannot be used. However, the elegance of the pathway suggests that it may be worth pursuing further in synthetic biochemistry systems that only require reducing equivalents. The construction of a system like this illustrates the creativity possible when there is no concern about having to compete with other cellular metabolic pathways. If it can be dreamt up conceptually, the system can be readily tested in a cell-free approach.

Terpene Production with Purge Valve Regulatory System

The system outlined in Figure 4 produces monoterpenes from glucose [

]. The system is complex, involving dozens of enzymes and a purge valve for regulating reducing equivalents. Nevertheless, the system functions for up to 7 days without intervention, producing monoterpenes at up to 16 g/l. To our knowledge, the titers are an order of magnitude higher than those produced in cells so far, perhaps because monoterpenes are toxic [

81.

Brennan T.C.R.
et al.

Alleviating monoterpene toxicity using a two-phase extractive fermentation for the bioproduction of jet fuel mixtures in Saccharomyces cerevisiae.

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]. These results indicate that even highly complex cell-free systems can be robust. It is possible that longevity can be improved with highly stabilized enzymes, but that remains to be seen.

Concluding Remarks

No doubt extensive technical developments will be required before synthetic biochemistry systems involving more than a handful of enzymes are ready for large scale commercial manufacturing (see Outstanding Questions). Enzyme stabilization and large-scale enzyme production are likely surmountable challenges in most cases, but some enzyme classes will require additional fundamental work to become useful for biomanufacturing. There has been little experience in scaling of complex enzyme systems at the levels needed for commodity chemical production, although You and coworkers showed xylitol production using a five-enzyme system at the 20 000-l scale [

]. No doubt much will need to be learned as we attempt to scale more complex systems. Is it better to work at high temperature despite the cofactor instability engendered, or at low temperature where microbial growth mitigation becomes necessary? Can aerobic systems that require oxygen dispersion be scaled effectively? At what point does it make sense to attach enzymes to a solid support? Perhaps the greatest need for cell-free biomanufacturing is further developments in cofactor technology. Research on ways to produce cofactors more cheaply, more effective cofactor recycling methods, or the development of stable nonnatural cofactors should be encouraged.

Despite the technical unknowns, the promise of synthetic biochemistry is hard to ignore. Already, cell-free production parameters can match or exceed those achieved in cells, which is particularly notable given the tiny resources that have been devoted to it compared to cell-based metabolic engineering. Clearly, building large scale bioreactors with complex enzyme systems is a major challenge, but the same can be said for fermentation platforms. Indeed, we already know that biomanufacturing with engineered cells is tough. We believe it is time to see what synthetic biochemistry can do.

Outstanding Questions

How long can cell-free systems run at high productivity? How can we make cell-free reactions run longer?

How well do complex cell-free systems scale? Are there any unexpected problems that arise when going from laboratory scale to extremely large reactors?

Will microbial contamination be a major problem and how can it be mitigated?

How best to deal with cofactor requirements? Can cofactors be recycled effectively enough? Can biological cofactors be replaced with cheaper, more robust nonnatural alternatives?

Acknowledgments

This work was funded by the US Department of Energy (DOE)-ARPAe grant DE-AR0000556 and DOE grant DE-FC02-02ER63421 to J.U.B.

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Glossary

Breakdown module

catabolic portion of a synthetic biochemistry system that can generate carbon molecule building blocks and high energy cofactors. A simple example is standard glycolysis that generates pyruvate, ATP, and NADH.

Build module

anabolic portion of a synthetic biochemistry system that generally takes carbon molecule building blocks and high energy cofactors to make new desired chemicals.

Synthetic biochemistry

design of reliable and efficient enzyme systems to replace cell-based metabolism for the production of chemicals, including all elements required for practical implementation (e.g., pathway designs, enzyme engineering, and process engineering).

Theoretical yield

amount of product that would be obtained if there was complete conversion of the input chemical.

Titer

concentration.

Volumetric productivity (or just productivity)

rate of product formation per unit volume, often expressed as g/l/h.

Yield

amount of input material converted to product.

Article Info

Publication History

Published online: January 23, 2020

Accepted: December 20, 2019

Received in revised form: December 19, 2019

Received: October 28, 2019

Identification

DOI: https://doi.org/10.1016/j.tibtech.2019.12.024

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Systems Biology Solutions for Biochemical Production Challenges

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