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Cell-free Synthetic Biology and Biomanufacturing for sustainable production of Biological Materials

We live in a material world. In today’s society, virtually every segment of our everyday life is influenced by the limitations, availability, and economic considerations of the materials used. There are more than five trillion plastic pieces in the world’s oceans, an estimated 15 billion trees are cut down each year and global natural fiber production is estimated to have already exceeded 30 million tons per annum.


Whilst we continue to approach irreversible climate change, it is of increasing importance that positive advances in the materials sciences are balanced against any negative environmental consequences that relate to material consumption. Arguably, these challenges warrant significant shifts in the manufacturing paradigm, from global mass production to local, sustainable and personalized manufacturing strategies. Likewise, instead of the chemical industries, the next generation of materials may come through developments in synthetic biology, sustainable biotechnology and the burgeoning bioeconomy.


The synthetic biology approach is geared toward the rational engineering or repurposing of biological parts, devices and systems into solutions that can help address societal challenges. Synthetic biology has wide application space from medical technologies  (e.g., biosensors and therapeutics), food security and sustainable energy (e.g., biofuels and synthetic photosynthesis), bioremediation  (e.g., pollution monitoring and sequestration), education (e.g., iGEM competition), and biomanufacturing (e.g., fine chemicals and materials production).


However, biological systems are highly complex, and initial attempts at engineering biological systems to fulfill specific application goals, are often only partially successful. To help overcome these challenges, synthetic biology employs the concept of the design cycle, through which biotechnologies are iteratively designed, built and tested.


Naturally, metabolic engineering, driven by the desire to arrive at new drugs, materials, and energy in technologically useful quantities, has been the prime goal of in vivo –based synthetic biology. It distinguishes itself from traditional biotechnology mainly by the degree of complexity of genetic manipulations, where whole gene networks, rather than simple genes, are being transferred, to the point of actually
“reprogramming” cells. Among the most noticeable breakthroughs of this kind of synthetic biology are the production of Artemisinin, an anti-Malarial drug, the alteration of photosynthetic pathways, the production of biofuels with engineered bacteria, and genetic manipulations in biopharmaceutical manufacturing, gene therapy and tissue engineering applications.


There are, however, fundamental limitations to an in vivo synbio approach. These limitations are innate to the living organism that hosts the synthetic pathway and include: Control of the gene circuits and chassis behavior; Monitoring of synthetic pathway dynamics; Automation and high-throughput optimization; Economic expansion into low margin applications; and Efficient and broadly applicable chassis systems.


The exact opposite of this approach of using and reprogramming existing organisms is the concept of truly designing functional biological systems from scratch. Technically speaking, this is a bottom-up concept. The starting point is here obviously not the cell, sometimes not even complex biomolecules such as nucleic acids and proteins, but molecules that do have the potential of assuming relevant biological functions, such as carrying information, self-assembling, self-organizing, or entertaining metabolism, when brought together purposefully. In contrast to the in vivo-based top-down approach discussed above, which belongs mainly to the realm of biotechnology, molecular and cell biology, many different research disciplines contribute with their specific techniques and interests to this purely in vitro-based synthetic biology


Learning how to improve a biological system may require multiple attempts, which could be made easier by more rapid and systematic workflows. One potential solution is to utilize cell-free synthetic biology for rapid prototyping. Typically, cell-free reactions make use of isolated cellular components and machinery (e.g., ribosomes and recombinant proteins), rather than live whole-cells.


Cell-Free systems

A distinctive feature of cell-free systems is the absence of cellular growth and maintenance, thereby allowing the direct allocation of carbon and energy resources toward a product of interest. Moreover, cell-free systems are more amenable than living systems to observation and manipulation, hence allowing rapid tuning of the reaction conditions. Recent advances in cell-free extract preparation and energy regeneration mechanisms have increased the versatility and range of applications that can be produced. Thus, the cell-free platform has evolved from merely an investigative research tool to a promising alternative to traditionally used living systems for biomanufacturing, as well as biological research.


In combination with the rise of synthetic biology, cell-free systems today have not only taken on a new role as a promising technology for just-in-time manufacturing of therapeutically important biologics and high-value small molecules, but have also been utilized for applications such as biosensing, prototyping genetic parts, and metabolic engineering.


Cell-free synthetic biology is a broad term that encompasses many different in vitro biotechnologies. Broadly, the term cell-free synthetic biology refers to different methods and technologies for engineering or using biological processes outside of a cell. For example, cell-free protein synthesis reactions enable the production of proteins within biochemical reactions. Thus, cell-free reactions typically make use of isolated cellular components (e.g., recombinant proteins) and/or cell extracts, rather than live whole-cells.


Four commonly used cell-free reaction formats are  (i) recombinant enzyme-based, (ii) protein synthesis using recombinant elements (PURE)-based cell-free protein synthesis, (iii) wildtype and/or engineered cell extract biotransformation or (iv) cell extract-based cell-free protein synthesis.


Recombinant enzyme-based reaction formats utilize purified enzymes, along with any required co-factors and pathway substrates, to produce fine chemicals, polymer monomers or other molecules of interest.


The PURE-based cell-free protein synthesis format reconstitutes the transcription and translation machinery from Escherichia coli using purified histidine (His)-tagged proteins. In this reaction format, the exact components are known, including the co-factors, substrates and energy mixes. Since PURE reaction components are known they can be standardized and rationally optimized. However, PURE cell-free reactions typically produce lower protein yields than cell-free protein synthesis reactions that use E. coli extracts.


The third cell-free reaction format uses cell extracts from lysed wildtype and/or engineered cells, which can be mixed together along with relevant required enzyme co-factors and substrates to form multicomponent biosynthetic pathways. Finally, the last format, cell extract-based cell-free protein synthesis (CFPS), uses the transcription and translation machinery from lysed cells, along with added co-factors and energy mixes to produce proteins in vitro.


It is important to note that these different cell-free reaction formats are not mutually exclusive and can be combined together. Recombinant enzymes or small molecule substrates can also be added into cell-free protein synthesis reactions to complete biosynthetic pathways, or to use exogenous chemistries within the reaction.


Cell-free system Applications

Biosensing is an area where cell-free systems have recently proven useful. They possess a unique advantage over whole cells because of their ability to detect species that are cytotoxic or impermeable to the cell wall. These systems have been deployed to detect pathogens such as norovirus, Ebola virus, and Zika virus. In addition, initial studies have shown that paper-based cell-free sensors can detect the presence of heavy metals such as mercury and drugs such as γ-hydroxybutyrate, by utilizing the transcriptional regulators, MerR and BlcR, respectively.


Arguably, today, the most widely used cell-free technology is cell-free protein synthesis (CFPS), an in vitro platform for protein transcription (TX) and translation (TL). Cell-free protein synthesis (CFPS) has been utilized in a wide range of applications from the expression of pharmaceutical proteins  to the production of libraries for protein evolution and structural genomics


CFPS has also been used in the development of minimal cells, the simplest cellular mimics that consist of only the genes essential for survival. Minimal cells are often described as biological analogs to the hydrogen atom, which has served to uncover many fundamental phenomena in chemistry


Debut Biotech Partners with DSM to Biomanufacture High Value Natural Ingredients

Debut Biotech entered into an agreement with Royal DSM to evaluate the Debut Biotech scalable cell-free biomanufacturing platform. The agreement will focus on enabling a proof-of-concept opportunity for Debut Biotech’s method, which unlocks and overcomes the limitations of traditional cell-based fermentation, according to Joshua Britton, CEO of Debut Biotech. The company’s platform will be used to create natural ingredients together with DSM for use in personal health, food, and lifestyle products.


Unlike chemical manufacturing, biomanufacturing uses cells and proteins, the natural building blocks of plants, to produce biological molecules and materials on a commercial scale. We truly believe that biomanufacturing will be how we create ingredients and materials in the future. However, standard fermentation is limiting this transition and this needs to be overcome. Debut is tired of continually overcoming cell-based problems such as long test-build cycles, low titers, cell-wall problems and product toxicity – this has to change.


Debut Biotech uses cell-free biomanufacturing to unlock and overcome the problems of using cells. To overcome the limits of the cell, we get rid of the cell. We use a platform technology we have pioneered called cell-free biomanufacturing. Here the good parts of the cell are kept such as enzymes and the bad parts such as the cell wall are removed to remove the limitations of biomanufacturing. Debut is unlocking latent markets and providing access to sustainably produced ingredients that no others can.


The company’s cell-free platform has the ability to transform low-value bio-renewable materials into high-value specialty chemicals, unlocking latent markets and providing unprecedented access to sustainably produced ingredients. Debut Biotech’s method provides a sustainable approach to biomanufacturing well into the future. By going cell-free, Debut Biotech is able to manufacture up to 1000% more than with previous methods. What’s more, the approach is far more sustainable than existing processes. Debut’s manufacturing platform uses exponentially less space and water, avoiding many of the chemicals used in traditional chemical synthesis and dramatically reduces a reliance on traditional petroleum-based products. Debut Biotech has advanced biomanufacturing to be cell-free, leveraging natural enzymes to make valuable materials across all major industries, including food, agricultural chemicals, cosmetics, beverages, health and wellness, additives, and dyes.


“Biomanufacturing holds promise for the sustainable production of materials and ingredients, but the traditional cell-based fermentation approach has limitations— especially for high-value specialty ingredients,” said Britton. “To truly innovate and move forward as an industry, we need to create tools that allow us to move away from the cell. This has allowed our team to break into wholly new product categories using biomanufacturing processes to produce rare, expensive, and new compounds. We are excited about our partnership with DSM because it will allow us to demonstrate our platform’s capabilities in the specialty ingredients market with a recognized global leader.”


Debut Biotech has advanced biomanufacturing to be cell-free, leveraging natural enzymes to make valuable materials across all major industries, including food, agricultural chemicals, cosmetics, beverages, health and wellness, additives, and dyes, added Britton. “The company’s cell-free platform has the ability to transform low-value bio-renewable materials into high-value specialty chemicals, unlocking latent markets and providing unprecedented access to sustainably produced ingredients,” he continued, noting that Debut Biotech’s method provides a sustainable approach to biomanufacturing.


Debut’s manufacturing platform uses exponentially less space and water, avoiding many of the chemicals used in traditional chemical synthesis and dramatically reduces a reliance on traditional petroleum-based products, Britton pointed out. “We’re excited to partner with Debut Biotech, and are thrilled to see how their cell-free method can support DSM’s role as a leader and innovator in the specialty food and nutritional ingredients market,” said Ross Zirkle of DSM. “The biomanufacturing of natural products has the potential to produce high value ingredients that customers are increasingly demanding.”


Cell-Free Technology, Growing Trend In The Synthetic Biology Market

The global synthetic biology market size is expected to grow from $10.07 billion in 2021 to $13.11 billion in 2022 at a compound annual growth rate (CAGR) of 30.2%. The Russia-Ukraine war disrupted the chances of global economic recovery from the COVID-19 pandemic, at least in the short term. The war between these two countries has led to economic sanctions on multiple
countries, surge in commodity prices, and supply chain disruptions, effecting many markets across the globe. The synthetic biology market is expected to reach $33.63 billion in 2026 at a CAGR of 26.6%.

Cell-Free Technology is gaining popularity in the synthetic biology market. In this cell-free technology, cell transcription and translation machinery are exploited to express only one or
several genes of interest in vitro. The global cell-free protein expression market size was valued at USD 229.7 million in 2021 and is expected to expand at a compound annual growth rate (CAGR) of 8.48 % from 2022 to 2030.

Cell-free protein expression i.e. cell-free protein synthesis is a technique that allows scientists to rapidly manufacture and express small quantities of functional proteins. Compared to in-vivo techniques based on tissue culture or bacterial cells, CFPE is significantly faster as it does not require extensive protein purification, cell culture, or gene transfection. Increasing research and development in genomics and proteomics, high prevalence of infectious diseases and cancer, and structural modification and shorter-expression time required in CFPE are major factors driving the growth of the market.

As the world continues to struggle with the current effects of COVID-19, scientists are using synthetic biology, and precise, cell-free systems to advance novel vaccines, diagnostic tools, and treatments. For instance, biologists at the University of Toronto extracted enzymes required for translation and transcription from Escherichia coli to develop a cell-free expression system that identifies the parts of the SARS-CoV-2 genome and activates a molecular shift to generate a reporter protein. This technique also eliminated the requirement to purify the RNA from the sample and replaced RT-PCR for additional rapid diagnostic testing. Such novel developments increased the adoption of CFPS in the diagnosis of COVID-19 and contributed to the CFPS market growth.

Cell-free protein expression (CFPE) is one of the widely used techniques in molecular biology. Proteins can be produced in a few hours using CFPE whereas, protein production using cells usually takes about a few days to weeks. So, in comparison with the traditional approaches, CPSE-based protein production can be time-saving from the initial synthesis to downstream applications. For instance, Promega’s, transcription and translation systems offer a time-saving substitute for eukaryotic in vitro transcription by coupling the procedure in a single tube format. Similarly, The human cell-free protein expression system aids in easy and simple in vitro translation and transcription. The single tube reaction of protein expression can be completed in one hour. This expression system allows high-yield expression and purification of functional protein.

Moreover, cell-free expression kits are helping in the faster expression of desired proteins in a short duration. For instance, the AccuRapid CFPE kit helps in the expression of a required protein in a reaction tube within 3 hours using the template DNA. Therefore, a reduction in the expression time is increasing the adoption of CFPE in the production of proteins by various industries can drive the CFPE market in the future.

Cell-free protein expression systems are evolving into a key platform for synthetic biology applications. Many synthetic biology tools are depended on cell-based systems, however, their constraints have led to the progress of cell-free protein expression and their applications in synthetic biology.

Furthermore, cell-free methods are being developed for biomanufacturing and prototyping. In prototyping applications, cell-free systems are utilized to optimize and test biosynthetic pathways before administration in the live cells and scale-up. For instance, the enzymatic pathways are constructed by a modular assembly of lysates containing enzymes produced by CFPS rather than living cells which reduces the time taken to build the pathways from months to a few days. This approach coupled with machine learning and automation can quicken the tests for combination metabolic pathways. Some of the commercially available strains and plasmids used for cell-free prototyping pathways include NEB Turbo and BL21 manufactured by New England Biolabs.

Product Insights

The expression systems segment held the highest share of the market in 2021. E. coli lysates is one of the most preferred lysates commercially due to their cost-effectiveness and higher protein yield. Moreover, E. coli-based systems used in recombinant technology are used for a wide range of biological therapeutics, involving insulin.

Wheat Germ Cell-free Protein Expression System is expected to expand at the fastest CAGR. The wheat germ system offers the maximum translation efficiency among the eukaryotic CFPE approaches and has an extreme success rate for the expression of soluble proteins of exceptional quality. In addition, this system is a choice of preference for numerous applications in protein research comprising options for protein labeling and the expression of proteins such as multiple protein complexes and membrane proteins.

Application Insights

The enzyme engineering segment dominated the market with a share of 34.97% in 2021. With the continuous growth of enzyme engineering, it has played an important role in biomanufacturing synthetic biology, and medicine. Lately, cell-free synthetic biology approaches for enzyme engineering have been used as an alternative strategy. This method has no constraint on the cell membrane and does not need to maintain cell viability, and each of the biosynthetic pathways is highly flexible.

Moreover, the high throughput production system is expected to register the fastest CAGR. The usage of CFPS for high-throughput enzyme screening has been proven to be a competent technique to discover improved enzyme variants and novel biocatalysts. The incorporation of cutting-edge high-throughput technologies allows the application of a Cell-free protein expression system for speedy prototyping to fast-track the screening procedure of enzymes with enhanced characteristics.

Method Insights

The transcription & translation systems segment captured the highest revenue share of the market in 2021. Coupled translation and transcription systems provide investigators with time-saving options for eukaryotic in vitro translation and transcription, by connecting these procedures in single tube format. Transcription and translation systems are used for numerous applications in low-to-high throughput functional proteome and genome analyses.

End-use Insights

The pharmaceutical & biotechnological companies segment captured the highest revenue share of the market in 2021. The expansion of the current manufacturing capacities for biopharmaceuticals drives the demand for cell-free protein expression products. For instance, in February 2022 Takara Bio Inc. announced the opening of an expansion of its manufacturing facility, the Center for Gene and Cell Processing in Shiga, Japan. The GMP facility development will address the increasing demand for contract development and manufacturing companies It will also meet the necessity for a broader variety of raw materials and manufacturing modalities. Manufactured products will contain regenerative medicines, RNA and DNA vaccines, cancer immunotherapy drugs, and gene therapy

Other end-user segment is expected to expand at the fastest CAGR in the review period. Growing investment in pharmaceutical R&D, and a rising number of Antibody-Drug Conjugates clinical trials are some of the factors driving the market growth. For instance, globally more than 50 antibody-drug conjugates are in clinical trials and 250 are under development, antibody-drug conjugates’ product quality features are often extra complex than those for the therapeutic proteins alone. Cell-free synthesis provides the ability to include nonstandard amino acids, which allows rapid prototyping of proteins, the number of conjugation sites and control of the location, and consequent incorporation and scaling of potent toxins as loads.

Regional Insights

North America dominated the regional market with a share of 36.29% in 2021. This major share can be attributed to the growth in the biotechnology and pharmaceutical industries, technologically advanced products, and the rising incidence of diseases such as cancer coupled with investments and funding in drug discovery research. For instance, In April 2021 Promega announced the opening of the newly constructed R&D facility (Kornberg Center) on its Fitchburg, WI campus. The building will support essential technology and product development in genetic identity, cellular and molecular biology, life science research, scientific applications, clinical diagnostic, and training.

Many companies in the synthetic biology market are developing this method to gain large outputs in a small-time frame. For instance, in September 2020, OriCiro Genomics, a Japan-based genome technology company offering innovative products and services for synthetic biology, announced the launch of the OriCiro Cell-Free Cloning System, the world’s first technology enabling cell-free amplification of large circular DNA, which eliminates the reliance on E. coli cloning.

The Asia Pacific is estimated to be the fastest-growing region owing to the strategic activities of key market players. For instance, In July 2021, CELLFREE SCIENCES CO., LTD, a Japanese company, received funding from Ehime Prefecture under the Ehime Prefecture New Growth Manufacturing Company Comprehensive Support Program. The company will use its own technologies comprising a protein bead array platform to discover new therapeutic concepts and drug targets.

Key Companies and Market Share Insights

Key players in this market are adopting various organic and inorganic strategies such as partnerships, mergers and acquisitions, geographical expansion, and strategic collaborations to expand their market presence. For instance, In September 2021, Sutro Biopharma extended the cytokine derivative research program in partnership with Merck. Under the terms of this agreement, Merck and Sutro are performing preclinical R&D of cytokine derivatives using Sutro’s exclusive site-specific conjugation and cell-free protein synthesis platforms, Xpress CF+ and XpressCF.

Key companies include Thermo Fisher Scientific, Inc., Takara Bio Company, New England Biolabs, Promega Corporation, Jena Bioscience GmbH, GeneCopoeia, Inc., Biotechrabbit GmbH, Cube Biotech GmbH, CellFree Sciences Co., Ltd. Merck KGaA


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