Toward sustainable, cell-free biomanufacturing
Graphical abstract
Introduction
For decades, microbial hosts have been engineered as an economical and environmentally friendly approach to chemical production. Unfortunately, cellular platforms impose inherent limitations on the effective biosynthesis and release of chemicals [1]. For example, cellular survival reduces the carbon available for desired chemical products and limits the maximum titer to nontoxic concentrations. To avoid these constraints, synthetic biologists have developed cell-free systems for a wide variety of applications, including small molecule biosynthesis [2•]. The cell-free metabolic engineering (CFME) approach can be used to build metabolic pathways in vitro using purified enzymes and/or crude cell extracts, which enables fine tuning of reaction conditions and enzyme concentrations [3•]. Extract-based approaches employ the biological machinery harvested from cells through lysis to disrupt the membrane and centrifugation to remove cell wall fragments, genomic DNA, and insoluble components. The resulting cell extract contains ribosomes and cellular proteins that enable in vitro transcription and translation, as well as endogenous metabolism, to produce protein products or metabolic enzymes. Similarly, engineered cells expressing metabolic enzymes can produce enzyme-enriched extracts without the need for in vitro protein synthesis [4]. In addition, CFME platforms provide open reaction environments with flexibility for purification and varied reaction modes in the absence of cellular barriers. This presents unique opportunities (e.g. direct addition of substrates and cofactors, reaction monitoring, and simplified product purification) as well as disadvantages (e.g. loss of cofactor regulation) [5] (Table 1). Although many strategies for cell-free protein synthesis have been explored in extract-based systems, production of non-protein products has not been pursued extensively beyond laboratory-scale demonstrations [2•].
In this review, we highlight select advances in cell-free approaches for biomanufacturing and outline promising paths toward sustainable, cell-free chemical biomanufacturing (Figure 1). We first set the stage by describing seminal work using crude extract-based systems to prototype biochemical production pathways. Then, we discuss four core advances necessary to realize the potential of cell-free biomanufacturing platforms: addressing sustainable feedstocks, utilizing alternative energy sources, maintaining homeostasis in vitro, and increasing reaction scales for industrial biochemical production.
Section snippets
Cell-free metabolic engineering
CFME systems provide many advantages for building metabolic pathways, such as enhanced control over the chemical environment and rapid design-build-test cycles [2•,4,6]. Many groups have used kinetic characteristics from purified enzymes studies to better select enzymes for metabolic pathways of interest [7,8]. Compared to these purified in vitro systems, crude cell extracts contain native metabolic elements that can provide the added benefit of prototyping enzymes within the context of
Sustainable carbon utilization pathways
While many recent efforts have sought to expand the product portfolio of cell-free biomanufacturing platforms, feedstock utilization has received less attention. Most cell-free platforms rely on glycolysis with glucose as the starting substrate. However, there is significant potential for cell-free systems to move toward more efficient biochemical transformations using cheaper, more sustainable carbon sources. Biopolymers such as starch, whey permeate, and cellulose can be used as a feedstock
Efficient energy regeneration modules
Engineering sustainable energy regeneration systems can improve cell-free biomanufacturing, as ATP and NAD(P)H production are essential to power biosynthetic reactions and produce proteins. While most gene expression platforms regenerate ATP with sacrificial substrates (e.g. kinases and polyphosphate systems) to maximize yield, these molecules are expensive and result in inhibitory phosphate accumulation [2•,31]. In contrast, CFME platforms typically generate ATP from natural catabolic pathways
Maintaining homeostasis in vitro
Cells go to great lengths to maintain homeostasis and enable productive metabolism. To do this, cells degrade nonproductive enzymes, compartmentalize toxic intermediates, repair damaged cofactors, and recycle dead-end metabolites back into metabolism or export them out of the cell [42,43]. Integrating these components into the design of cell-free metabolisms and maintaining their proper equilibria may help to achieve more cell-like longevity for reactions in cell-free systems. In particular,
Scaling up cell-free reactions
Despite major advantages of cell-free systems, the transformation of these systems from benchtop to industrial-scale biomanufacturing remains limited by the expenses associated with biocatalyst generation [54]. Common strategies found acceptable for building and prototyping metabolic pathways, such as affinity tag purification, can become cost-prohibitive at large scales. One strategy to overcome this involves building metabolic pathways with enzymes from thermophiles and heat-treating crude
Outlook
Extract-based cell-free systems present appealing advantages for industrial biomanufacturing, including the presence of native-like metabolism and the ability to encode additional enzymes for catabolism, anabolism, and metabolic proofreading (Table 1). The biosynthetic potential of cell-free systems could be enhanced by replacing contemporary substrates with inexpensive carbon sources (e.g. cellulosic biomass, methanol or CO2) and ubiquitous energy sources (e.g. electricity or sunlight) to
Author contributions
The authors contributed to all aspects of the article.
Conflict of interest statement
M.C.J. has a financial interest in SwiftScale Biologics and Design Pharmaceuticals Inc. M.C.J.’s interests are reviewed and managed by Northwestern University in accordance with their conflict-of-interest policies. All other authors declare no conflicts of interest.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
M.C.J. acknowledges support from the Department of Energy Grant DE-SC0018249, the DOE Joint Genome Institute ETOP program, the Office of Energy Efficiency and Renewable Energy Grant DE-EE0008343, the David and Lucile Packard Foundation, and the Camille Dreyfus Teacher-Scholar Program. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No.
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