Cell-Free Systems: Platform for Sustainable Commercial Biomanufacturing of Biochemicals

Humaira1, Afsana Huseynova Anvar2, Shaukat Ali3, Marcelo Franco4, Muhammad Irfan1*

1Department of Biotechnology, University of Sargodha, Sargodha Pakistan

2Department of Molecular Biology and Biotechnology, Baku State University, Baku, Azerbaijan

3Department of Zoology, Government College University Lahore Pakistan

4Department of Exact Sciences, State University of Santa Cruz (UESC), Ilhéus, 45662-900 Brazil

Abstract | Increasing material consumption and associated environmental concerns demand a shift from current fossils-based commercial manufacturing practices to sustainable, ecofriendly production systems. Extensive metabolic engineering practices incorporating living systems for commercial manufacturing of cost-effective value-added products and biochemical from sustainable feed-stock show limited success as desired higher production rates run counter to life-sustaining metabolic activities. To address these challenges, cell-free approaches developed as promising platform to proceed towards sustainable bio-manufacturing through the development of self-sustaining, constantly operating systems employing renewable biomass to yield higher concentrations and productivity of extended-range of products. To-date established cell-free systems including extract-based and purified enzyme-based cell-free technologies have been widely exploited for producing commercially valuable biopolymers and biochemicals such as building-block chemical, biofuels, bioplastic and value-added products. However, implementation of these cell-free systems at industrial production scale requires key considerations of sustainable carbon source, co-factors and energy regeneration pathways and biocatalyst engineering and recycling strategies to proceed towards sustainable production. At present, extensive research is required to address challenges of high cost associated co-factor supply and optimization of protocols to exploit full-potential of these cell-free technologies.

Novelty Statement | This review article describe the potential utilization of cell free systems for the produc-tion of valuable chemicals through technological interventions which is the current demand of the society.

Article History

Received: September 16, 2023

Revised: July 05, 2024

Accepted: July 18, 2024

Published: November 19, 2024

Authors’ Contributions

Humaira wrote the original draft. AHA reviewed the literature. SA formatted figures and editing. MF did final editing. MI supervised the study, did final editing in the manuscript.

Keywords

Cell free systems, Biochemicals, Commercial production, Value added products

Copyright 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Corresponding Author: Muhammad Irfan

[email protected], [email protected]

To cite this article: Humaira, Anvar, A.H., Ali, S., Franco, M. and Irfan, M., 2024. Cell-free systems: Platform for sustainable commercial biomanufacturing of biochemicals. Punjab Univ. J. Zool., 39(2): 213-230. https://dx.doi.org/10.17582/journal.pujz/2024/39.2.213.230

Introduction

The primary goal of metabolic engineering and industrial biotechnology is to fulfill ever increasing demand of sustainable and cost-effective chemicals and natural products (Dudley et al., 2015). At present, fossil fuels serve as almost sole source of materials and chemicals (Moulijn et al., 2014). Petrochemical industry has traditionally relied upon a few (xylene, benzene, methanol, propene, toluene, butadiene, ethylene) high-volume, low-cost commodity chemicals from which approximately all other products are derived. Some serious outcomes of increasing dependence on fossils include limited supply and price fluctuations due to finite availability (Höök and Tang, 2013), lack of novelty as materials are derived from limited number of building blocks (Bozell and Petersen 2010) and irreversible climatic changes (Karl et al., 2009). In current era, this petrochemical based material’s synthesis cannot be substituted completely with other production systems; however, advances in engineering biological systems offer access to wide range of building-blocks for manufacturing novel chemicals and value-added products (Karl et al., 2009).

Currently, industrial biotechnology research mainly focused on bio-based systems that utilize specific, robust enzymes as catalyst of complex cascade of reactions in living systems to synthesize wide range of bio-molecules, polymers, biofuels, therapeutics, platform-chemicals and value-added products etc. (Lee et al., 2012; Curran and Alper, 2012; Nielsen et al., 2013). Microbes based bioconversions proved to be efficient because of their potential to utilize heterogeneous substrates, e.g., waste streams (BETO, 2017), biomass hydrolysates (Kim et al., 2012), hydrolyzed lignin (Linger et al., 2014) and mild operating parameters. Synthetic biology has arisen as growing scientific field that combine biological systems with engineering technologies (Khambhati et al., 2019). Synthetic biology holds great potential to engineer biological systems for extending range of applications from biofuels to biomedical research. High-throughput DNA sequencing and advanced biotechnology techniques (gene-editing) offer unlimited opportunities to alter cellular functioning for user-defined purposes (Lee et al., 2012).

Currently, microbes based manufacturing is still limited as cells inherently posed several problems (Green, 2011). For example, Engineering at cellular level is problematic particularly because of membrane barriers (hinder optimization of synthetic process, cause incompatibility, variability issues) and complicated metabolic pathways (Jiang et al., 2018; Yue et al., 2019). Moreover, either Suitable vectors are required for carrying genetic instructions to cells that should be maintained through chromosomal integration or selectable marker expression to allow instructions to be evaluated (Tinafar et al., 2019). Other limitations of cell-based systems are limited product yield due to toxicity effects, synthesis of competing by-products, difficulty in optimization of lab-scale cultures to commercial production-scale because of variable fermentation conditions and constraints in down-stream processing in case of intra-cellular accumulation of product (Takors, 2012).

Thus, to overcome these limitations synthetic biologists and biotechnologists have adopted an alternative approach cell-free synthesis. This strategy permits the activation of biological machinery through direct control of transcription, translation, and metabolism in cell-free environment. This in-vitro biomanufacturing offers several advantages as it separates target product’s yield from cell-growth. Absence of cell boundary permits easy manipulation, standardization, sampling, and monitoring. Cell-free bioconversions eliminate requirements of long-term strain improvement programs thus, greatly enhance design-build-test-learn cycle speed (Takahashi et al., 2015). Other advantages include high production without any need to maintain cell biomass, eliminate unnecessary metabolic pathways which in turn limit the synthesis of undesired by-products, enhanced tolerance against toxins and high reaction rates due to enhanced biomass transfer-rates (Carlson et al., 2012; Dudley, 2015; Rollin et al., 2013). Cell-free systems offer opportunity to manipulate biological parts at four levels; DNA, RNA, Proteins, and metabolism to develop complex systems for synthesis of sustainable biochemicals, functional biomolecules and bioenergy. Currently, purified-enzyme system and crude-extract based system represent two well-established cell-free technologies (Zhang, 2015). This review highlights the features of these currently established cell-free approaches, potential applications for sustainable biomanufacturing of commercially valuable products, considerations for scaleup efforts, challenges that should be addressed to develop these cell-free technologies as competitive production platform and future prospects.

Cell-free systems

Currently, two well-developed and commonly exploited cell-free systems are: Extract-based systems and purified enzyme-based systems (Rollin et al., 2018; Taniguchi et al., 2017; Dudley et al., 2015). Both of these systems possess several common features i.e. both have potential to deliver ~100 theoretical yield as these are entirely orthogonal to biocatalyst production and accomplish primary goal of metabolic engineering through system design. For example, in vitro conversion of glycerol into 1,3-propanediol was reported to be 0.95 mol/mol that is much higher than reported in traditional fermentation (0.6mol/mol) mainly due to elimination of competing byproduct formation (Rieckenberg et al., 2014). As in-vitro bioconversions do not show homeostasis; thus, these systems possess potential to be manipulated rapidly like chemical pathway engineering and easy to scale-up (Takahashi et al., 2015). Furthermore, these systems offer opportunity to circumvent obstacles in situations where membrane barrier limit product removal or substrate uptake and ultimately leads to higher productivity (Jewett et al., 2008).

Extract-based system

Extract-based cell-free approach utilize cell-lysate along with other supplements i.e. template DNA, NTPs, energy generating substrates, transcription and translational factors, tRNAs, salts, amino-acids, co-factors and RNA-polymerase for in-vitro transcription and protein expression. Crude-extracts can be derived both from prokaryotic and eukaryotic cells (Zemella et al., 2015). Some extract-based cell-free systems developed from prokaryotes include E. coli (Shrestha et al., 2012; Ninomiya et al., 2014), Sulfolobus solfataricus (Nishimuar et al., 2013), Bacillus subtilis (Kelwick et al., 2016) and Thermococcus kodakaraensis (Yamaji et al., 2009) extract-based systems. While reported eukaryotic extract-based systems include Streptomyces lividans (Li et al., 2017), Saccharomyces cerevisiae (Gan and Jewett, 2014; Schoborg, 2014), tobacco BY-2 (Buntru et al., 2015), wheat germ (Arumugam et al., 2014), Trichoplusia ni (Ezure et al., 2006), Spodoptera frugiperda (Ezure et al., 2014), rabbit reticulocytes (Douthwaite, 2012), human K562 (Brodel et al., 2015), Chinese Hamsters-Ovary (Brodal et al., 2014), HeLa cell-lines (Yadavalli and Sam-Yellowe, 2015), HEK293 (Bradrick et al., 2013), mouse embryonic fibroblast (Zeenko et al., 2008) and Leishmania tarentolae (Mureev et al., 2009). However, E. coli extract is the mostly widely exploited extract-based cell-free system (Lee et al., 2012).

 

These extract-based systems provide cost-effective routes for protein synthesis and can be easily scalable for commercial fermentation (Cai et al., 2015; Zawada et al., 2011). Several protocols have been established for crude-extract preparation that rely on isolating the cells at exponential growth-phase when intra-cellular translation is at the highest level. After harvesting cell are washed, lysed (Kwon and Jewett, 2015), followed by run-off treatment to activate crude-extract in cell-free environment as shown in Figure 1. This process probably degrades genomic DNA and endogenous mRNA transcripts to considerably reduce in-vitro translational efficacy (Moore et al., 2017). Further, small inhibitory molecules can be removed by dialysis, but it depends upon user preference (Kwon and Jewett, 2015). To-date several commercial extract-based batch systems have been established with improved energy-regeneration pathways that are designed by groups of jewett (Kwon and Jewett, 2015; Harris and Jewett, 2012), swartz (Yang et al., 2012) and Noireaux (Gramela et al., 2016; Caschera and Noireaux, 2104). Besides simplicity and ease to scale-up, key limitation of extract-based system cell-free system is their ability to sustain un-desired metabolic pathways; thus, additional engineering or purification may be required for their elimination (Moore et al., 2017).

Purified enzyme-based cell-free system

This system incorporates enzymes that are first over-expressed and purified individually followed by their reassembling to reconstruct biosynthetic-pathways in open-environment (Dudley et al., 2015). These purified enzyme-based cell-free systems proved to be extremely useful particularly if immobilization, encapsulation or engineered spatial-arrangement is required, also permit the easy optimization of each pathway enzyme and provide dynamic process control (Jin and Myung, 2019). Moreover, these enzymes-based approaches offer the opportunity to design novel synthetic pathways with aim of producing only the targeted product (Jinn and Myung, 2019). Researchers have also commenced commercial scale-up efforts and demonstrated the capacity of these systems up-to 20,000 L while maintaining high yield (~5g/L/h) (You et al., 2017). To date, several purified enzyme-based systems have been reported for manufacturing of commercially valuable products including mono-terpenes (Korman et al., 2017), iso-butanol (Sherkhanov et al., 2020), polyhydroxy-butyrate (PHB) (Opgenorth et al., 2016) styrene (Grubbe et al., 2020), biohydrogen (Rollin et al., 2015) and many others. However, absence of cellular biochemical regeneration system and high-cost associated with cofactors mainly limit scale-up of these enzyme-based cell-free technologies (Jinn and D-Myung, 2019; Dudley et al., 2015).

Potential commercial applications

Currently, we are living in a material world (Callén-Moreu and López-Gómez, 2019). Approximately 15billion trees are cut-down each year (Crowther et al., 2015) and worldwide fiber production is predicted to be more than 30million-tonns annually (Townsend and Sette, 2016). Our ecosystem is largely shaped by commercial manufacturing, processing, and disposal of extended range of synthetic materials. As we are proceeding towards irreparable climate change (Lenton et al., 2019), it becomes essential to balance advancements in material-sciences against adverse environmental outcomes associated with material consumption. So, to-date main challenge is to shift manufacturing practices from global mass-manufacturing to localized sustainable manufacturing approaches (Kleer and Piller, 2019). For example, in-contrast with current fossils-based chemical-industries next generation building-block chemicals or value-added products may derive from ecofriendly, renewable feedstock through advancements in sustainable biotechnology, synthetic biology and growing bio-based economy (Freemont, 2019; French, 2019). As biological systems are extremely complicated and initial efforts to engineer these systems for user defined-purposed show limited success (Kelwick et al., 2020). Thus, cell-free manufacturing systems offers highly promising strategy for commercial manufacturing of sustainable, cost-effective industrially valuable platform-chemicals and value-added products. Potential applications of currently established cell-free systems for sustainable manufacturing of some commercially important products are listed here.

Isoprenoids

Isoprenoids are important class of compounds that have wide-range of applications in flavoring, disinfectants, pesticides, fragrances, chemical feedstock and pharmaceutical industry (Leavell et al., 2016). Although, novel biosynthetic pathways for isoprenoid synthesis have been successfully expressed in microbes (George et al., 2015) but efforts to design and characterize metabolic pathways in living systems is sufficiently time-consuming process (Du et al., 2012; Biggs et al., 2014). Thus, cell-free systems offer unique alternative strategy for commercial production of isoprenoids. To date, several cell-free systems has been reported for isoprenoid production. For example, Enzyme enriched E. coli extracts have been successfully utilized for cell-free synthesis of limonene (Dudley et al., 2019). Study reported that cell-free biosynthetic pathway has been constructed for limonene synthesis by mixing several E. coli extracts each containing one overexpressed pathway enzyme. First, six-enriched lysates along with mevalonate (substrate), cofactors and energy-source were employed to synthesize limonene then system was extended further to utilize glucose as substrate. As extracts exhibit indigenous pathways to metabolize glucose to acetyl-CoA; hence, three additional enzymes were added to reaction mixture that catalyze the bioconversion of acetyl-CoA to mevalonate. Dudley et al. (2019) reported that by adjusting the concentration of co-factors, CoA and ATP this system has potential to produce 90.2mg/L limonene over 24 h. This enzyme-enriched extract-based system comprising of 20 metabolic-steps and incorporating 9 heterologous enzymes for conversion of glucose into limonene highlights the flexibility of extract-based system to sustain complex metabolic reactions in open environment (Dudley et al., 2019).

Korman et al. (2017) have designed 27 enzyme containing biosynthetic-pathway for cell-free conversion of glucose into monoterpenes along with generation of ATP and NAD(P)H both of which serve as co-factors for terpene synthesis. Study reported that by altering the concentration of terpene-synthase enzyme this purified enzyme-based cell-free system have successfully synthesized pinene, sabinene and limonene with single glucose addition and maintained metabolic activity for ~ 5 days (Korman et al., 2017). Moreover, Ward et al. (2019) reported cell-free synthesis of isoprenoids from isopentenol though construction of “isopentenol-utilization pathway” (IUP) in an open environment that appears to be promising substitute of native MVA (Mevalonate) and MEP (2C-methyl-D-erythritol-4-phosphate) pathways. This enzyme-based IUP utilize just four enzymes for bioconversion of isopentenol in presence of ATP into mono-, di-, and sesquiterpenoids; thus, offer highly flexible strategy for commercial production of isoprenoids. Metabolic-control analysis demonstrated that IUP is mainly regulated by Choline kinase (CK) and conversion efficacy is not influenced by any pathway intermediate. Study reported that this in-vitro established IUP can synthesize 220 mg/L of diterpene-taxadiene, over 9 h that is nearly 3-times greater than any other cell-free system reported for isoprenoid production (Ward et al., 2019).

Recently, Niu et al. (2020) have developed cell-free system for biosynthesis of α-Pinene from glucose. Besides its widespread applications in pharmaceuticals, α-Pinene also represents promising substitute of jet-fuel due to its high-energy content, low hygroscopicity and high-fluidity at low-temperature (Niu et al., 2017). Thus, biotechnological production of pinene has received much attention. Niu et al. (2020) reported that by using mixed-match approach concurrent addition of E. coli lysate containing overexpressed native 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, Pinus taeda pinene-synthase, SufBCD Fe−S cluster assembly-protein and isopentenyl-diphosphate isomerase significantly improved pinene production. NAD+, NADH and ammonium-acetate were found to be the key parameters for regulation of pinene synthesis (Niu et al., 2020). After optimizing the components of in-vitro reaction-mixture through Plackett−Burman experimental-design; pinene yield was further improved to 57 % by increasing enzyme-concentration in E. coli-extract to achieve overall productivity of 104.7 mg/ L/ h (Niu et al., 2020).

Platform chemicals

The European Commission’s Circular Economy Strategy and Strategic Plan 2014 and Action-Plan 2015 have raised considerable debate regarding the role and affiliation of circular-bioeconomy and agricultural industries including forestry (DeBoer et al., 2020), biofuel (Erickson, 2018) and plastic production (Blank et al., 2020); animals (Horton, 2019) and textile industry (Aznar, 2019) along-with several other examples. This Circular-Bioeconomy demands absolute carbon-efficacy and development of alternative sources to replace fossils-feedstock on which chemical industry has traditionally relied as energy and carbon source. Biotechnological methods offer opportunities to utilize non-traditional feedstock (CO2, agricultural waste, domestic waste, glycerol, methane etc.) along-with conventional substrates (starch, molasses) as carbon source (Bergquist et al., 2020). Extensive research has been conducted to engineer living systems to produce cost-effective commodity-chemicals from biomass ranging from low-value products e.g., building-block chemicals, plastic, fuels to valuable natural products e.g., cannabinoids (Chae et al., 2017; Chubukov et al., 2016). Although, these metabolically engineered microbes are advantageous as they utilize renewable-biomass as feedstock, mitigate release of green-house gases and produce biodegradable-products but mainly low volumetric production because of cellular toxicity make these bio-derived products economically less-viable (Bowie et al., 2020).

Thus, to address this challenge cell-free systems received attention for sustainable production of platform chemicals and value-added products. For example, Kay and Jewett (2019) have designed E. coli’s extract-based cell-free system to produce 2-3 Butane-diol (2,3-BDO) from glucose. 2,3-BDO is a promising aviation fuel and important platform-chemical that is widely used to produce industrially valuable chemicals e.g., acetoin, diacetyl, butadiene, methyl-ethyl-ketone. Single E. coli strain was metabolically engineered to overexpress three pathway enzymes required for conversion of pyruvate to 2,3- Butanediol that include 2,3-BD-dehydrogenase (BDH) from Klebsiella pneumoniae, acetolactate-decarboxylase (ALDC) and acetolactate-synthase (ALS) from Bacillus subtilis. Addition of cofactors (ATP, NAD+) and glucose to E. coli extract containing endogenous glycolytic enzymes first metabolize glucose to pyruvate which then is subsequently converted to 2,3-BDO by heterologous enzymatic pathway expressed in E. coli extract (Kay and Jewett, 2019). Yi et al. (2018) have developed first hybrid cell-free system for metabolic conversion of starch into 2,3-BDO. Recombinant E. coli strain was developed to express 2,3-BDO synthetic pathway enzymes including acetolactate-synthase (Mycobacterium tuberculosis), acetolactate decarboxylase and butanediol-dehydrogenase (Lactococcus lactis). As, E. coli extract naturally has limited potential to metabolize starch into pyruvate; thus, addition of cyanobacteria-lysate into cell-free mixture significantly improves pyruvate synthesis from starch which is then subsequently converted into 2,3-BDO by heterologous enzymatic pathway. This hybrid system yields 1.6-folds higher titer of 2,3-BDO than concentration obtained by employing only E. coli extract (Yi et al., 2018). Gao et al. (2019) have reported purified-enzyme based cell-free system for metabolic conversion of biomass-derived D-xylose into D-1,2,4-buatnetriol (BT). Under optimized conditions this in-vitro constructed biosynthetic pathway produce 6.1 g/L BT from 18 g/L D-xylose over 40 h with an overall productivity of 48 % (Gao et al., 2019).

Generally, multiple sugar monomers in biomass hydrolysate adversely affect fermentation, results in lower productivity and by-product formation that subsequently influence downstream processing (Sutiono et al., 2021). Recently Sutiono et al. (2021) have utilized sequence-based identification strategy to design promiscuous enzymes-based cell-free system that successfully metabolize biomass derived L-Arabinose and D-Xylose to 1,4-butanediol (BDO) at rate of 3 g/L/h with an overall productivity of >95 %. This in-vitro system was extended further to yield α -ketoglutarate (2-KG) from biomass-derived pentoses by employing O2 that serve as co-substrate for cofactor regeneration and synthesize 2-KG at rate of 4.2 g/L/h with an overall productivity of 99 % (Sutiono et al., 2021). Zhang et al. (2017), have designed cell-free biosynthetic pathway for conversion of ethanol into C4 platform-chemicals including 2-butanol, acetoin and 2,3-butanediol. This purified enzyme-based cell-free system incorporate five heterologous enzymes (formolase, diol-dehydratase, ethanol-dehydrogenase, NADH-oxidase and 2,3-butanediol-dehydrogenase) to convert bioethanol into industrially valuable C4 chemicals. Under optimized conditions, maximum of 88.28 %, 88.78 % and 27.25 % titer of 2,3-BDO, acetoin and 2-butanol were obtained respectively from 100 mM ethanol (Zhang et al., 2017). Moreover, glycerol that constitute about 10 % of biodiesel production as a byproduct; is a valuable, non-toxic and biodegradable green-solvent.

This biomass-derived glycerol is a promising substrate for biocatalysis and to produce industrially important products (Bergquist et al., 2020). Li et al. (2018) have designed cell-free biosynthetic pathway for metabolic conversion of glycerol into L-Lactate. This cell-free synthetic pathway have incorporated four thermostable-enzymes (dihydroxy-acid-dehydratase, L-Lactate dehydrogenase, alditol-oxidase, and catalase) along-with Formate-dehydrogenase that serve as NADH regeneration system to produce 34.4 mM L-Lactate from 50 g/L glycerol over 72-h (Li et al., 2018). Meng et al. (2018) have designed cell-free biosynthetic-pathway incorporating 3-enzymes for metabolic conversion of biomass-derived cello-dextrin into energy-rich phosphorylated-oligosaccharides e.g., glucose-6-P. Authors reported that this cell-free system successfully achieved much higher titers of inositol from cellodextrose without any toxicity effect that mainly limit its production in E. coli expression system (Meng et al., 2018). Some other cell-free systems reported for manufacturing of industrially valuable building-block chemicals and value-added products produced from biomass derived monomers are listed in Table 1.

Recently, Grubbe et al. (2020) reported cell-free synthesis of styrene. Styrene is a valuable petroleum-derived product that serve as building-block for manufacturing of different plastics e.g., foamed-packaging material, disposable silverware. This petroleum-derived styrene significantly contributes to global warming as emit over 100-million tons of green-house gases annually (Zheng and Shu, 2019). Thus, engineered E. coli strains was demonstrated as a potential substitute for bioconversion

 

Table 1: Industrially important plat-form chemicals and value-added products from biomass derived monomers.

Substrate	Product	Yield	Biosynthetic pathway enzymes	Industrial applications	References
Glycerol	D-fagomine	70 g/L/h	3	Anti-diabatic activity	Hartley et al. (2019)
Sucrose	Glucaric acid	35 mM (70 %)	7	Therapeutic agent, intermediate in slow release of fertilizers, detergent, degradable polymer	Su et al. (2019)
Glucose	Lactate	48.2 mM (>90 %)	5	Pathway intermediate	Xie et al. (2018)
Chitin	Pyruvate	0.62 mM	12	Precursor for alcohols, amino acid and other building-blocks.	Honda et al. (2017)
mannose	Lactic acid	4.4 mM	4	Used in medical, textile food industry, monomer for biodegradable poly-lactic-acid (PLA) manufacturing.	Kopp et al. (2019)
Glycerol	Di-hydroxy-acetone phosphate	88 %, 4.1 µM/s	4	Intermediate or precursor for di-hydroxy acetone synthesis	Hartley et al. (2017)
Tyrosine	Raspberry ketone	55 mg/L (33.7 %)	5	Natural additive in food and sport industry	Moore et al. (2017)
Glucose	n-butanol	82 %	16	Promising next-generation biofuel	Krutsakorn et al. (2013)
Starch	Myo-inositol	2.9 g/L/h (>90 %)	4	Used in the pharmaceutical, cosmetic and food and feed industries	You et al. (2017)
Pyruvate	2,3-BDO	11.3 g /L/h	3	Used in inks, perfumes, explosives, polymers, paint, flavorings & pharmaceuticals manufacturing	Kay and Jewett (2015)
D-Xylose	Myoinositol	96.6 %	11	Used in the pharmaceutical, cosmetic and food and feed industries	Cheng et al. (2019)

 

of glucose into-styrene through shikimate-pathway. However, cellular toxicity and prerequisite for product removal limit in-vivo synthesis of styrene (Liu et al., 2018; Lee et al., 2019). Grubbe et al. (2020) have established cell-free biosynthetic pathway for styrene biosynthesis from L- phenylalanine. Recombinant E. coli strain was developed to express heterologous enzymatic pathway incorporating “ferulic-acid decarboxylase 1 (FDC1)” (saccharomyces cerevisiae) and phenylalanine-ammonia lyase 2 (PAL2) (Arabidopsis thaliana) responsible for bioconversion of L-phenylalanine to styrene then simply heat-treatment release lysate of engineered E. coli strain which is then employed for in-vitro styrene synthesis. This cell-free system was optimized to produce maximum of 40mM styrene, nearly more than 2-fold higher productivity than reported in engineered in-vivo system (Grubbe et al., 2020).

Biofuels

Ever-increasing energy-consumption and environmental concerns associated with the exploitation of fossil-fuels leads to rising demand for sustainable and cost-effective production of biofuels. Global biofuel market is estimated to comprised of about 85 % bioethanol and 15 % biodiesel (Gao et al., 2015). Although, several cellular systems have been metabolically engineered to synthesize extended range of biofuels, but development of living cells-based competitive platform for commercial production of biofuels is still challenging (Chubukov et al., 2016; Chen and Liao, 2016; Chae et al., 2017) particularly due to low product-yield (0.2 % v/v), feedback inhibition of substrate, toxicity effects that disrupt life-sustaining processes and lack of cost-effective downstream processing. To overcome these limitations biotechnology research, move towards the development of cell-free systems for biofuel production (Zhang et al., 2011; Zhang, 2011). For example, yeast’s extract-based cell-free system was reported for ethanol production from glucose without any prerequisite for pathway prototyping and strain engineering (Khattak et al., 2014). Yeast cells cultivated in waste beer fermentation-broth (WBFB) were acquired, in-vitro grown, lysed, buffered, and blended with glucose to produce ethanol at rate of approximately 4 g/L/h. Strikingly, authors reported that concentrations of NAD+ (0.11 mMol) and ATP (1.8 mMol) in yeast’s-cell extract were sufficiently high to maintain metabolic reactions even without adding supplements (Khattak et al., 2014).

Recently, Cui et al. (2020) have established extract-based cell-free system to identify factors limiting ethanol yield in Clostridium thermocellum. Study reported 25 mM ethanol yield from 15 mM biomass-derived cellobiose at rate of approximately 0.5 mM/h in cell-free environment. Two different approaches (exogenous enzyme addition to cell-free extract and feeding various substrates) are used to determine enzymes responsible for limiting metabolic-flux. NADH recycling was found to be rate limiting factor and major improvement was reported by addition of yeast “Alcohol-dehydrogenase” (ADH) to extract (Cui et al., 2020).

 

Table 2: Recent studies of using Biomass waste as a potential feedstock to produce biofuels (Bio-diesel, Bio-oil) (Modified from Quevedo-Amador et al., 2023).

Waste	Treatment/ Process	Operating conditions	Yield or conversion, %	References
Goat bone	Thermal	Methanol/oil ratio: 11/1, 2% catalyst, 3h, 60 °C	92 (bio-diesel)	Mamo and Mekonnen (2019)
Garlic peel	Thermochemical	Methanol/oil ratio: 10/1, 8% catalyst, 3.5 h, 60 °C	96 (bio-diesel)	Wei et al. (2022)
Banana pseudostem (Dwarf Cavendish)	Thermal	60 °C, 2 h, methanol/oil ratio: 9.35/1, 4.7% of catalyst	98 (bio-diesel)	Daimary et al. (2023)
Banana peel	Thermal	Methanol/oi ratio: 6/1, 5 °C, 1.5 h, 2% of catalyst	98 (bio-diesel)	Husin et al. (2023)
Karanja seed shell	Thermal	Methanol/oil ratio: 10/1, 2% catalyst, 1 h, 65 °C	96 (bio-diesel)	Prajapati et al. (2023)
Rice husk	Thermochemica	Methanol/oil ratio: 16/1, 9.9% catalyst, 4.8 h, 74.8 °C	98 (bio-diesel)	Saidi et al. (2023)
Corncob	Catalytic pyrolysis	3 g biomass, 500 °C, 5 mL/min, 40 min, HZSM-5/activated carbon ratio: 2/1, biomass /catalyst ratio: 1/1	44 (bio-oil)	Duan et al. (2022)
Pistacia lentiscus L seeds	Pyrolysis	20 g biomass, 475 °C, heating rate of 25 °C/min, particle size: 0.3–0.6 mm	64 (bio-oil)	Farissi et al. (2022)
Date seed and plastic waste	Co-pyrolysis	100 g date seed, 70 g plastic, 500 °C, heating rate of 50 °C/min, 100 mL/min N2, 12.5 MPa, 1200 rpm	59 (bio-oil)	Inayat et al. (2022)
Food and plastics waste	Co-pyrolysis	Batch Reactor, 200 g biomass, 400 °C, 1 h, food waste/plastic constant ratio: 2/1	Fish bone+plastic: 16 Chicken bone +plastic: 20 Rice +plastic: 29 (bio-oil)	Lim et al. (2022)
Eucalyptus wood	Catalytic fast pyrolysis	500 °C, 1.5 h, 11 L/min, fuidized bed reactor: 160 g/h, biomass/catalyst mass ratio: 0.4	11 (bio-oil)	Promsampao et al. (2022)
Linseed residue	Pyrolysis	50 g biomass, 500 °C, 1.5 h, 200 cm3/min, heating rate: 20 °C/min	79 (bio-oil)	Bahadorian et al. (2023)
Reutealis trisperma oil	Pyrolysis	Non-catalytic, reactor semibatch, 450 °C, 190 min Catalytic, dolomite/ Reutealis trisperma oil mass ratio: 1/10	68 (bio-oil) 77	Buyang et al. (2023)
Sawdust and rice husk	Co-pyrolysis	Fluidized bed reactor, 500 °C, 20 L/min, blending ratio of sawdust/rice husk: 50/50	52 (bio-oil)	Fadhilah et al. (2023)
Biomass of water hyacinth	Hydrothermal liquefaction	Impregnation of biomass of water hyacinth with CuCl2 0.2 MHydrothermal liquefaction: Cu-impregnated biomass of water hyacinth/water ratio of 1/9, 270 °C, 0.5 h	83 (bio-oil)	Gao et al. (2023)
Municipal (household) and horticultural wastes	Co-pyrolysis	50 g biomass, waste blends (0–100 wt%), 550 °C, 1 min, 200 mL/min N2, 0–15% of catalyst	58 (bio-oil)	Ghorbannezhad et al. (2023)
Corncob	Hydrothermal liquefaction	0.25 h, 300 °C, 10 mL/min	10 (bio-oil)	Martins-Vieira et al. (2023)
Hydnocarpus de-oiled seed cake, waste electrical and electronic plastic	Microwave co-pyrolysis	Hydnocarpus de-oiled seed cake /Waste electrical and electronic plastic weight ratio: 50/50 (g/g), 500 °C, 10 g catalyst, heating rate of 30 °C/ min, microwave power of 1100 W	26 (bio-oil)	Muniyappan et al. (2023)
Cassava residue	Pyrolysis	500 °C, 1–2 h, 0.5–3.5 L/min N2, fuidized bed reactor: 100 g/h	54 (bio-oil)	Rueangsan et al. (2023)
Lychee	Pyrolysis	350 °C, 125 min, heating rate of 120 °C/min, 110 mL/min Ar	38 (bio-oil)	Singh et al. (2023)
Corn straw and hair waste	Pyrolysis/Co-pyrolysis	5 g biomass, corn straw/hair waste blend ratio: 25/75% (w/w), 450 °C, 20 min, 80 mL/min	Corn straw: 10 Hair waste: 48 Corn straw/hair waste: 46 (bio-oil)	Xiong et al. (2023)

 

Besides ethanol, isobuatnol is considered be a promising next-generation biofuel. Isobuatnol display superior features as compared to ethanol as a fuel e.g., high energy-content, less hygroscopic, less-volatile, comparable to gasoline, supported by current infrastructure. Recently, Sherkhanov et al. (2020) have established purified enzyme-based cell-free system that employed 16-enzymes-incorporated biosynthetic-pathway for conversion of glucose into isobutanol. Through continuous product removal from bioreactor this system provides maximum yield of 4 g/L/h and 95 % productivity over approximately 5 days. Authors reported that this production rate is higher even from the highly developed ethanol-fermentation (Sherkhanov et al., 2020). Wong et al. (2019) reported conversion of ketoisovaleric acid into isobutanol through immobilized-enzymes based cell-free system. This system utilized ketoacid-decarboxylase immobilized on maltose-binding protein along with Formate-dehydrogenase and alcohol-dehydrogenase to metabolically convert ketoisovaleric-acid to isobutanol at concentration of 2 g/L with overall productivity of 43 % (Wong et al., 2019). Grimaldi et al. (2016). have also established immobilized enzyme-based cell-free system for isobutanol production from ketoisovaleric acid. Two pathway enzymes “Alcohol dehydrogenase” (ADH) and “ketoacid-decarboxylase” (KdcA) covalently immobilized on methacrylate matrix and one enzyme “Formate-dehydrogenase” (FDH) in reaction mixture effectively convert ketoisovaleric-acid into isobutanol (55 % conversion efficiency) at concentration of 0.135 mol/mol. Study reported that this cell-free system achieved 8-20 times higher isobutanol yield than highest reported isobutanol titer in S. cerevisiae (Grimaldi et al., 2016).

Moreover, as compared to capacity of living systems cell-free approaches hold much-more potential for biohydrogen production. In natural system, during metabolism each glucose molecule generate just 4 H2 molecules (Chou et al., 2008). Although theoretically each glucose molecule should yield 12 H2 molecules. Rollin et al. (2015) reported purified enzyme-based cell-free system for biohydrogen production. First, corn-stover was pretreated (acid/ enzymatic hydrolysis) to generate xylose and glucose which were subsequently converted to hydrogen through cell-free biosynthetic pathway incorporating more than10 enzymes. Study reported that improved enzyme-availability, substrate concentration and kinetic modeling leads to 67-times increase in yield with overall productivity of 54 mmol H2 L/h (Rollin et al., 2015). Some recent studies, reporting the use of bio-waste from various sources as a potential feedstock for biofuel production are enlisted in Table 2.

Biosurfactants

Biosurfactants (BS) are amphoteric molecules synthesized extracellularly by wide range of bacteria, yeast and fungi (Santos et al., 2016). Members of Bacillus genus are recognized as best producers of lipopeptide-biosurfactants (Jacques, 2011). In contrast with synthetic surfactants, BS offer various advantages such as high-biodegradability, biocompatibility, low-toxicity, low-irritancy, digestibility along with diverse chemical structures and functions. Despite these benefits, commercial production of BS is low because of high production-cost (Banat et al., 2014). To address this challenge cell-free system might prove be an effective alternative for cost-effective large-scale production of BS. Satpute et al. (2018) have reported cell-free synthesis of BS from “Lactobacillus acidophilus NCIM 2903” and examined its antiadhesive, antibiofilm properties by employing Microfluidic approach. This in-vitro produced biosurfactant displayed antibacterial activity at titer of 625 µg/ml against P. aeruginosa, E. coli, S. aureus and B. subtilis. Moreover, antiadhesive and antibiofilm action was also demonstrated on Polydimethylsiloxane (PDMS) surface and catheter as intro-synthesized BS strongly inhibited biofilm formation by S. aureus and E. coli (Satpute et al., 2018).

Bioplastic

The large-scale manufacturing and use of oil-derived plastic has resulted in irreversible ecological damage (Suaria et al, 2016). Despite, serious environmental concerns petrochemical-derived plastic is yet in high-demand mainly due to versatility and cost-effective production. Even current recycling practices seems to be ineffective to affects future de-novo plastic manufacturing (Geyer et al., 2017). Furthermore, introduction of sustainable substitutes to petroleum-derived plastic faced considerable technological and societal challenges. However, advances in synthetic-biology and metabolic engineering offers a way for sustainable commercial production of biopolymer “polyhydroxyalkanoates” (PHA), that is a promising biodegradable alternative to oil-derived plastic (Chen et al., 2017; Dietrich et al., 2017). Extensive research has been conducted to construct effective-microbial PHA’s production system through metabolite-recycling (Beckers et al., 2016), pathway engineering (Tao et al., 2017) and the use of industrially viable cost-effective feedstock (Nielsen et al., 2017). However, microbial production of PHAs is currently more costly than petroleum-derived plastic. Thus, highly competent production-systems are required for sustainable commercial production of PHAs (Kelwick et al., 2018).

Cell-free biosynthesis serve as a highly promising platform for designing biosynthetic pathways for cost-effective and sustainable PHAs production. For example, Kelwick et al. (2018) have established numerous E. coli extract-based cell-free systems for prototyping PHAs synthesis operons and for identifying enzymes responsible for metabolite recycling. In-vitro transcription-translation prototyping and gas-chromatography, mass-spectrometry quantification of in-vitro synthesized 3-hydroxybutyrate (3HB) demonstrated variations in activities of native ΔPhaC_C319A (1.18 6 0.39 mM), C101 Δ PhaC_C319A (2.65 6 1.27 mM) and C104 ΔPhaC_C319A (4.62 6 1.31 mM) PhaCAB operon. C104 was reported to be the most active operon as synthesize higher concentration of PHAs as compared to native PhaCAB operon in both in-vivo as well as in-vitro analyses (Kelwick et al., 2018). Study reported that addition of optimal quantity of whey-permeate this cell-free reaction increase in-vitro GFP mut3b synthesis by approximately 50 %. Thus, data recommended that cell-free strategies help-out in-vivo workflows to demonstrate additional promising approaches for optimizing PHAs production (Kelwick et al., 2018).

Scale-up

Despite the obvious advantages of cell-free technologies practical implementation of these systems from lab-scale to industrial manufacturing scale still require considerable efforts to develop cost-effective strategies for synthesis and purification of components essential for bottom-up reconstitution of cell-free system (Claassens et al., 2019). Some promising considerations necessary in the designing of these system at commercial level to proceed towards sustainable production of biomolecules and value-added products are listed here.

Effective energy-regeneration system

A prerequisite to improve efficacy of cell-free biomanufacturing is the development of efficient energy-regeneration modules because ATP and NAD(P)H are required to derive biosynthetic reaction under in-vitro conditions (Silverman et al., 2020; Moon et al., 2019). Typically, cell-free systems rely on natural catabolic-pathways for energy regeneration (Bowie et al., 2020; Lin et al., 2020). Furthermore, addition of artificial vesicles encompassing ATP-synthase and cytochromes or purified E. coli membrane-vesicles that are metabolically active in cell-free environment can improve ATP supply for biosynthesis (Lin et al., 2019; Otrin et al., 2017). However, use of simple carbon substrate mainly limits ATP regeneration in cell-free system through oxidative-phosphorylation. To overcome this limitation, electrical energy can be employed along-with these sources to power biosynthetic pathways in open environment after being converted to chemical energy. For example, electrically active microbes such as Geobacter sulfurreducens, Shewanella oneidensis can be utilized as external energy-source to supply energy directly to biological energy-carriers (Fang et al., 2020). Such electrically derived in-vitro systems could comprised of hybrid reaction mixture incorporating defined content of lysate from electrically active microbes. Moreover, other promising sustainable energy source include solar-energy harnessed by employing plant-derived thylakoids or artificial vesicles comprising ATP-synthase and bacteriorhodopsin (Berhanu et al., 2019; Lee et al., 2018). Implementation of such energy-regeneration systems could greatly enhance economic viability and efficacy of cell-free biomanufacturing.

Sustainable carbon source

Currently, majority of cell-free approaches utilize glucose as carbon-source. However, there exist great potential to utilize cheaper, renewable biomass as carbon sources to proceed towards cost-effective in-vitro biochemical transformations (Rasor et al., 2021). Biopolymers such-as whey permeate, starch, cellulose after hydrolysis to monomers could be employed as feedstock as reported in both in-vitro and in-vivo systems (Mano et al., 2020; Tian et al., 2019; Yi et al., 2018). For example, study reported that addition of whey-permeate to E. coli MG1655 extract increase nearly 50 % protein synthesis in cell-free system without supplementing additional enzyme and has also been successfully utilized to enhance in-vitro manufacturing of 3-hydroxybutyrate (Kelwich et al., 2018). Moreover, single-carbon feedstock (CO2, methanol, CH4, formate) represents another potential substrate-group that has not been widely investigated as carbon source for biomanufacturing in an open-system (Cotton et al., 2020). CO2-fixation system is particularly promising as it exhibits potential to manufacture sustainable products from readily available, cheap carbon sources (Liew et al., 2016).

 

Recently established purified enzyme-based cell-free system “The CETCH cycle” utilize first synthetic CO2 fixation-pathway (Miller et al., 2020). These economically viable, renewable-feedstock could significantly reduce feedstock-cost; thus, possess potential to enhance efficiency of commercial biomanufacturing in cell-free systems in future (Rasor et al., 2021).

Biocatalyst engineering and recycling

One principal factor required to be considered for scale-up of enzyme-based systems is enzyme production and purification cost. In case of extract-based systems scale-up efforts are much easier and cost-effective as simply involve cells harvesting and lysing and use cell-lysate for in-vitro biomanufacturing (Rollin et al., 2018). While for purified enzyme-based approach economic viability of system largely depends upon host employed for enzyme production, expression level, enzymes purification method and stability. Higher enzyme-stability is vital parameter for commercial implementation of these enzyme-based biosynthetic pathways. As enzyme stability significantly reduce purification cost and allow enzyme recovery simply by heating the extract (Ninh et al., 2015). Furthermore, semi-rational and rational biocatalyst engineering strategies are particularly important as greatly increase biocatalyst’s robustness (Longwell et al., 2017; Sheldon and Pereira, 2017). Two promising approaches for biocatalyst recycling include in-situ product recovery and immobilization method (Rollin et al., 2018).

In-situ recovery approach permit separation of limited number of products as precipitate or in gas-phase. By employing concentrated substrate as feedstock, it is possible to operate cell-free biosynthesis at steady-state without losing components of aqueous-phase (Opgenorth et al., 2017). While second in-situ product recovery strategy use membranes for enzymes retention while permit product to be removed from reactor (Rollin et al., 2018). However, enzymes immobilization strategy involves enzymes impregnation on surfaces like hydrogels, membranes or appropriate matrixes (Jochems et al., 2011; Blanchette et al., 2016) or polymer capsules. Besides, these advancements still extensive efforts are required for scale-up of purified enzyme-based cell-free technology for sustainable biomanufacturing of extended range of products. In addition to these key considerations for scale-up of cell-free technologies some important considerations are shown in Figure 2, like increasing scale, high-value product manufacturing.

Challenges and future prospects

Although cell-free systems appear to be exciting technology but also pose several challenges that should be addressed to establish these systems as competitive production platform. Main challenges include high-cost associated with co-factors, energy supplements that are essential for in-vitro activation of biological machinery after extracting it from cells. Besides cost-factor, standardization of these in-vitro biosynthetic pathways is particularly problematic as different research-labs have developed complementary and competing systems by employing different extract preparation protocol, variable genetic strains of different organisms and acquired different result (Burgenson et al., 2018). Bio-machinery content can also exhibit variations even within same strain and its activity can also influenced by employing different fermentation media and fermenters. Moreover, in-vitro manufacturing can be operated in batch (Zawada, 2011), microfluidics (Timmy et al., 2016; Georgi et al., 2016), fed-batch (Salehi et al., 2016) and continuous mode (Li et al., 2017; Stech et al., 2014); although, these varied operational modes offer design-flexibility, but extensive research is required to optimize each product. However, current scenario suggests that development of innovative extract preparation protocols and utilization of renewable energy and carbon source can greatly help in advancement of these cell-free technologies as a competitive platform for commercial manufacturing that will probably replace currently well-established industrial fermentation in future.

Declarations

Funding

The study received no external funding.

Ethical statement

All applicable institutional, national and international guidelines for the care and use of animals were followed.

Statement of conflict of interest

The authors have declared no conflict of interest.

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