Toward sustainable, cell-free biomanufacturing

https://doi.org/10.1016/j.copbio.2020.12.012Get rights and content

Highlights

  • Cell-free systems enable biochemical reactions in the absence of cell viability constraints.

  • Cell-free metabolic engineering presents opportunities for biomanufacturing diverse chemical products.

  • Native metabolism in cell extracts can be coupled with exogenous or artificial pathways to use renewable carbon sources.

  • The open, cell-free reaction environment is amenable to electrochemical and photosynthetic energy regeneration modules.

  • Increasing cell-free reactions beyond the prototyping scale will enable sustainable biomanufacturing.

Industrial biotechnology is an attractive approach to address the need for low-cost fuels and products from sustainable resources. Unfortunately, cells impose inherent limitations on the effective synthesis and release of target products. One key constraint is that cellular survival objectives often work against the production objectives of biochemical engineers. Additionally, industrial strains release CO2 and struggle to utilize sustainable, potentially profitable feedstocks. Cell-free biotechnology, which uses biological machinery harvested from cells, can address these challenges with advantages including: (i) shorter development times, (ii) higher volumetric production rates, and (iii) tolerance to otherwise toxic molecules. In this review, we highlight recent advances in cell-free technologies toward the production of non-protein products beyond lab-scale demonstrations and describe guiding principles for designing cell-free systems. Specifically, we discuss carbon and energy sources, reaction homeostasis, and scale-up. Expanding the scope of cell-free biomanufacturing practice could enable innovative approaches for the industrial production of green chemicals.

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.

References (63)

  • S. Berhanu et al.

    Artificial photosynthetic cell producing energy for protein synthesis

    Nat Commun

    (2019)
  • E. Van Schaftingen et al.

    Metabolite proofreading, a neglected aspect of intermediary metabolism

    J Inherit Metab Dis

    (2013)
  • C.F. Megarity et al.

    Electrocatalytic volleyball: rapid nanoconfined nicotinamide cycling for organic synthesis in electrode pores

    Angew Chem Int Ed Engl

    (2019)
  • K. Petroll et al.

    A novel framework for the cell-free enzymatic production of glucaric acid

    Metab Eng

    (2020)
  • K. Cheng et al.

    Upgrade of wood sugar d-xylose to a value-added nutraceutical by in vitro metabolic engineering

    Metab Eng

    (2019)
  • H. Taniguchi et al.

    Developing a single strain for in vitro salvage synthesis of NAD(+) at high temperatures and its potential for bioconversion

    Microb Cell Fact

    (2019)
  • A.D. Silverman et al.

    Cell-free gene expression: an expanded repertoire of applications

    Nat Rev Genet

    (2020)
  • J.U. Bowie et al.

    Synthetic biochemistry: the bio-inspired cell-free approach to commodity chemical production

    Trends Biotechnol

    (2020)
  • T.P. Korman et al.

    A synthetic biochemistry platform for cell free production of monoterpenes from glucose

    Nat Commun

    (2017)
  • G. Morgado et al.

    Synthetic biology for cell-free biosynthesis: fundamentals of designing novel in vitro multi-enzyme reaction networks

    Adv Biochem Eng Biotechnol

    (2018)
  • I.W. Bogorad et al.

    Synthetic non-oxidative glycolysis enables complete carbon conservation

    Nature

    (2013)
  • F. Zhu et al.

    In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli

    Biotechnol Bioeng

    (2014)
  • A.S. Karim et al.

    A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery

    Metab Eng

    (2016)
  • Q.M. Dudley et al.

    Cell-free mixing of Escherichia coli crude extracts to prototype and rationally engineer high-titer mevalonate synthesis

    ACS Synth Biol

    (2016)
  • R. Kelwick et al.

    Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

    Synth Biol

    (2018)
  • J.E. Kay et al.

    Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol

    Metab Eng

    (2015)
  • T. Yi et al.

    Synthesis of (R,R)-2,3-butanediol from starch in a hybrid cell-free reaction system

    J Ind Eng Chem

    (2018)
  • W.S. Grubbe et al.

    Cell-free biosynthesis of styrene at high titers

    Metab Eng

    (2020)
  • A.S. Karim et al.

    In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design

    Nat Chem Biol

    (2020)
  • J.E. Kay et al.

    A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds

    Metab Eng Commun

    (2020)
  • C. You et al.

    An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch

    Biotechnol Bioeng

    (2017)
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