cell-free-protein-synthesis-system-for-building-synthetic-cells

Cell-Free Protein Synthesis System for Building Synthetic Cells

The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for ...

Jiangsu Normal University

Aquaporins are involved in CO2 transport from the leaf intercellular air space to the chloroplast, which contributes to CO2 assimilation. However, the mechanism of CO2 transport by rice (Oryza sativa L.) aquaporins is unknown. Here, we investigated the function of the aquaporin OsPIP1;2 in CO2 diffusion-associated photosynthesis and phloem sucrose transport. Moreover, the grain yield of rice lines overexpressing OsPIP1;2 was determined. OsPIP1;2 was localized to the plasma membrane and the relative expression of OsPIP1;2 was approximately 5-fold higher in leaves in the presence of an elevated CO2 concentration. Overexpression of OsPIP1;2 increased mesophyll conductance by approximately 150% compared with wild-type (WT) rice. The OsPIP1;2-overexpressing lines had higher biomass than the WT, possibly due to increased phloem sucrose transport. In addition, the grain yield of OsPIP1;2-overexpressing lines was approximately 25% higher than that of the WT in three-season field experiments, due to the increased numbers of effective tillers and spikelets per panicle. Our results suggest that OsPIP1;2 modulates rice growth and grain yield by facilitating leaf CO2 diffusion, which increases both the net CO2 assimilation rate and sucrose transport.

Overexpression of rice aquaporin OsPIP1;2 improves yield by enhancing mesophyll CO2 conductance and phloem sucrose transport.

Erratum: Overexpression of rice aquaporin OsPIP1;2 improves yield by enhancing mesophyll CO2 conductance and phloem sucrose transport.

Aquaporins are key proteins in regulating water transport, plant growth and development. In this study, we investigated the function of plasma membrane intrinsic proteins (PIPs) in both yeast (Saccharomyces cerevisiae) and rice (Oryza sativa cv. Nipponbare). Three OsPIP1s (OsPIP1;1, OsPIP1;2 and OsPIP1;3) and four OsPIP2s (OsPIP2;1, OsPIP2;3, OsPIP2;4 and OsPIP2;5) were successfully amplified and expressed in yeast. Overexpression of OsPIP2s, especially OsPIP2;1, increased yeast membrane water permeability (P). Root hydraulic conductivity (Lpr) was decreased by approximately four-fold in OsPIP2; 1 RNAi knock-down plants, resulting in a decrease in OsPIP2;1 expression levels of 70% and 50% in line 3 and line 4, respectively, compared to the wild type (WT) plants. No significant differences in the photosynthetic rate, transpiration rate, mesophyll conductance and chloroplast CO concentration were observed between WT and OsPIP2; 1 RNAi plants. Higher stomatal conductance and intercellular CO concentrations were observed in line 3 plants than in WT plants. In addition, lower root total length, surface area, root volume and fewer root tips were found in the RNAi plants than in the WT plants. Finally, the RNAi plants were more sensitive to drought stress. The results indicate that PIP2; 1 plays an important role in the regulation of water transport and plant growth.

Aquaporin PIP2;1 affects water transport and root growth in rice (Oryza sativa L.).

The quest for a minimal cell not only sheds light on the fundamental principles of life but also brings great advances in related applied fields such as general biotechnology. Minimal cell projects came from the study of a plausible route to the origin of life. Later on, research extended and also referred to the construction of artificial cells, or even more broadly, as in vitro synthetic biology. The cell-free protein synthesis (CFPS) techniques harness the central cellular activity of transcription/translation in an open environment, providing the framework for multiple cellular processes assembling. Therefore, CFPS systems have become the first choice in the construction of the minimal cell. In this review, we focus on the recent advances in the quantitative analysis of CFPS and on its advantage for addressing the bottom-up assembly of a minimal cell and illustrate the importance of systemic chassis behavior, such as stochasticity under a compartmentalized micro-environment.

Cell-Free Protein Synthesis: Chassis toward the Minimal Cell.

In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.

Design of Sealable Custom-Shaped Cell Mimicries Based on Self-Assembled Monolayers on CYTOP Polymer.

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from  and  using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and PIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an  Based Cell-Free Protein Synthesis System.

The underlying idea of synthetic biology is that biological reactions/modules/systems can be precisely engineered and controlled toward desired products. Numerous efforts in the past decades in deciphering the complexity of biological systems in vivo have led to a variety of tools for synthetic biology, especially based on recombinant DNA. However, one generic limitation of all living systems is that the vast majority of energy input is dedicated to maintain the system as a whole, rather than the small part of interest. Cell-free synthetic biology is aiming at exactly this fundamental limitation, providing the next level of flexibility for engineering and designing biological systems in vitro. New technology has continuously inspired cell-free biology and extended its applications, including gene circuits, spatiotemporally controlled pathways, coactivated catalysts systems, and rationally designed multienzyme pathways, in particular, minimal cell construction. In the context of this special issue, discussing work being carried out in the "MaxSynBio" consortium, the advances in characterizing stochasticity and dynamics of cell-free protein synthesis within cell-sized compartments, as well as the molecular crowding effect, are discussed. The organization of spatial heterogeneity is the key prerequisite for achieving hierarchy and stepwise assembly of minimal cells from the bottom-up.

Cell-Free Protein Synthesis and Its Perspectives for Assembling Cells from the Bottom-Up.

Aquaporins are essential water channel proteins found in all kingdoms of life. Although the water permeability of aquaporins has been well characterized, sample preparation for aquaporin water permeability assays remains challenging and time-consuming. Besides the difficulty in overexpressing membrane proteins in a cell-based expression system, the unique requirement for homogeneity in aquaporin proteoliposome sample preparations for water transport assays further increases the complexity. In this study, a complementary Cell-free Protein Synthesis (CFPS) method is described in detail, providing three different strategies for the preparation of aquaporin proteoliposome samples. Aquaporin can be produced either as a pellet fraction and then resolubilized, or co-translationally as a detergent-soluble fraction. Furthermore, aquaporin can be directly incorporated into liposomes, which was included in the CFPS reactions. Although proteoliposomes tend to fuse during the incubation of the CFPS reactions, an additional treatment of the fused samples with detergent, followed by a detergent removal step, can re-form homogenously sized proteoliposomes suitable for functional analysis. Using this method, we successfully characterized aquaporins from both prokaryotic and eukaryotic organisms. In particular, in the presence of liposomes, the developed CFPS expression system is a fast and convenient method for sample preparation for the functional analysis of aquaporins.

Functional Analysis of Aquaporin Water Permeability Using an -Based Cell-Free Protein Synthesis System.

The polyethylene terephthalate (PET) is one of the major plastics with a huge annual production. Alongside with its mass production and wide applications, PET pollution is threatening and damaging the environment and human health. Although mechanical or chemical methods can deal with PET, the process suffers from high cost and the hydrolyzed monomers will cause secondary pollution. Discovery of plastic-degrading microbes and the corresponding enzymes emerges new hope to cope with this issue. Combined with synthetic biology and metabolic engineering, microbial cell factories not only provide a promising approach to degrade PET, but also enable the conversion of its monomers, ethylene glycol (EG) and terephthalic acid (TPA), into value-added compounds. In this way, PET wastes can be handled in environment-friendly and more potentially cost-effective processes. While PET hydrolases have been extensively reviewed, this review focuses on the microbes and metabolic pathways for the degradation of PET monomers. In addition, recent advances in the biotransformation of TPA and EG into value-added compounds are discussed in detail.

Microbial degradation and valorization of poly(ethylene terephthalate) (PET) monomers.

Aquaporins are transmembrane channels that allow for the passive permeation of water and other small molecules across biological membranes. Their channel activities are sensitive to mercury ions. Intriguingly, while most aquaporins are inhibited by mercury ions, several aquaporins are activated by mercury ions. The molecular basis of the opposing aquaporin regulation by mercury remains poorly understood. Herein, we investigated AqpZ inhibition and AQP6 activation upon binding of mercury ions using solid-state NMR (ssNMR) and molecular dynamics (MD) simulations. Based on the structure of the Hg-AqpZ complex constructed by MD simulations and ssNMR, we identified that the pore closure was caused by mercury-induced conformational changes of the key residue R189 in the selectivity filter region, while pore opening was caused by conformational changes of residues H181 and R196 in the selectivity filter region in AQP6. Both conformational changes were caused by the disruption of the H-bond network of R189/R196 by mercury. The molecular details provided a structural basis for mercury-mediated functional changes in aquaporins.

Molecular Mechanisms of Mercury-Sensitive Aquaporins.

Reversible membrane targeting of proteins is one of the key regulators of cellular interaction networks, for example, for signaling and polarization. So-called "membrane switches" are thus highly attractive targets for the design of minimal cells but have so far been tricky to reconstitute in vitro. Here, we introduce cell-free prenylated protein synthesis (CFpPS), which enables the synthesis and membrane targeting of proteins in a single reaction mix including the prenylation machinery. CFpPS can confer membrane affinity to any protein via addition of a 4-peptide motif to its C-terminus and offers robust production of prenylated proteins not only in their soluble forms but also in the direct vicinity of biomimetic membranes. Thus, CFpPS enabled us to reconstitute the prenylated polarity hub Cdc42 and its regulatory protein in vitro, implementing a key membrane switch. We propose CFpPS to be a versatile and effective platform for engineering complex features, such as polarity induction, in synthetic cells.

Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression.

Roots are the main organs through which plants absorb water and nutrients. As the key phytohormone involved in root growth, auxin functions in plant environmental responses by modulating auxin synthesis, distribution and polar transport. The  gene  can improve root architecture, and  mutants have significantly shortened primary roots. However, the mechanism underlying the short roots of the  mutant and the upstream signaling pathway and downstream genes regulated by  are unclear. Here, we demonstrated that the  gene could promote auxin accumulation in -overexpressing plants. By comparing the transcriptomic data of  and wild-type roots, we found several upregulations of auxin-related genes, including ,  and , may play an important role in the  gene-mediated auxin transport signaling pathway, ultimately leading to changes in auxin content and primary root length. Moreover, increased  expression can regulate primary root growth and lateral root elongation under different concentration of nitrate conditions. Overall, constitutive expression of  increased auxin contents and improved lateral root elongation, constituting a new method for improving the nitrogen utilization efficiency of plants.

The  trehalose-6-phosphate phosphatase gene  regulates primary root growth and lateral root elongation.

The rapid development of synthetic biology has enabled the production of compounds with revolutionary improvements in biotechnology. DNA manipulation tools have expedited the engineering of cellular systems for this purpose. Nonetheless, the inherent constraints of cellular systems persist, imposing an upper limit on mass and energy conversion efficiencies. Cell-free protein synthesis (CFPS) has demonstrated its potential to overcome these inherent constraints and has been instrumental in the further advancement of synthetic biology. Via the removal of the cell membranes and redundant parts of cells, CFPS has provided flexibility in directly dissecting and manipulating the Central Dogma with rapid feedback. This mini-review summarizes recent achievements of the CFPS technique and its application to a wide range of synthetic biology projects, such as minimal cell assembly, metabolic engineering, and recombinant protein production for therapeutics, as well as biosensor development for in vitro diagnostics. In addition, current challenges and future perspectives in developing a generalized cell-free synthetic biology are outlined.

Advancing synthetic biology through cell-free protein synthesis.

Despite the physiological significance of effective CO diffusion across biological membranes, the underlying mechanism behind this process is not yet resolved. Particularly debatable is the existence of CO-permeable aquaporins. The lipophilic characteristic of CO should, according to Overton's rule, result in a rapid flux across lipid bilayers. However, experimental evidence of limited membrane permeability poses a challenge to this idea of free diffusion. In this review, we summarized recent progress with regard to CO diffusion, and discussed the physiological effects of altered aquaporin expression, the molecular mechanisms of CO transport via aquaporins, and the function of sterols and other membrane proteins in CO permeability. In addition, we highlight the existing limits in measuring CO permeability and end up with perspectives on resolving such argument either by determining the atomic resolution structure of CO permeable aquaporins or by developing new methods for measuring permeability.

Aquaporins and CO diffusion across biological membrane.

Technical advances in biotechnology have greatly accelerated the development of bottom-up synthetic biology. Unlike top-down approaches, bottom-up synthetic biology focuses on the construction of a minimal cell from scratch and the application of these principles to solve challenges. Cell-free protein synthesis (CFPS) systems provide minimal machinery for transcription and translation, from either a fractionated cell lysate or individual purified protein elements, thus speeding up the development of synthetic cell projects. In this review, we trace the history of the cell-free technique back to the first in vitro fermentation experiment using yeast cell lysate. Furthermore, we summarized progresses of individual cell mimicry modules, such as compartmentalization, gene expression regulation, energy regeneration and metabolism, growth and division, communication, and motility. Finally, current challenges and future perspectives on the field are outlined.

Bottom-Up Synthetic Biology Using Cell-Free Protein Synthesis.

Sweetpotato (Ipomoea batatas (L.) Lam.) holds a crucial position as one of the staple foods globally, however, its yields are frequently impacted by environmental stresses. In the realm of plant evolution and the response to abiotic stress, the RNA helicase family assumes a significant role. Despite this importance, a comprehensive understanding of the RNA helicase gene family in sweetpotato has been lacking. Therefore, we conducted a comprehensive genome-wide analysis of the sweetpotato RNA helicase family, encompassing aspects such as chromosome distribution, promoter elements, and motif compositions. This study aims to shed light on the intricate mechanisms underlying the stress responses and evolutionary adaptations in sweetpotato, thereby facilitating the development of strategies for enhancing its resilience and productivity. 300 RNA helicase genes were identified in sweetpotato and categorized into three subfamilies, namely IbDEAD, IbDEAH and IbDExDH. The collinearity relationship between the sweetpotato RNA helicase gene and 8 related homologous genes from other species was explored, providing a reliable foundation for further study of the sweetpotato RNA helicase gene family's evolution. Furthermore, through RNA-Seq analysis and qRT-PCR verification, it was observed that the expression of eight RNA helicase genes exhibited significant responsiveness to four abiotic stresses (cold, drought, heat, and salt) across various tissues of ten different sweetpotato varieties. Sweetpotato transgenic lines overexpressing the RNA helicase gene IbDExDH96 were generated using A.rhizogenes-mediated technology. This approach allowed for the preliminary investigation of the role of sweetpotato RNA helicase genes in the response to cold stress. Notably, the promoters of RNA helicase genes contained numerous cis-acting elements associated with temperature, hormone, and light response, highlighting their crucial role in sweetpotato abiotic stress response.

Article	Year
Overexpression of rice aquaporin OsPIP1;2 improves yield by enhancing mesophyll CO2 conductance and phloem sucrose transport. Journal of experimental botany\| PubMed ID: 30535321	2019
Erratum: Overexpression of rice aquaporin OsPIP1;2 improves yield by enhancing mesophyll CO2 conductance and phloem sucrose transport. Journal of experimental botany\| PubMed ID: 30869151	2019
Aquaporin PIP2;1 affects water transport and root growth in rice (Oryza sativa L.). Plant physiology and biochemistry : PPB\| PubMed ID: 30889480	2019
Cell-Free Protein Synthesis: Chassis toward the Minimal Cell. Cells\| PubMed ID: 30959805	2019
Design of Sealable Custom-Shaped Cell Mimicries Based on Self-Assembled Monolayers on CYTOP Polymer. ACS applied materials & interfaces\| PubMed ID: 31136146	2019
Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an Based Cell-Free Protein Synthesis System. Cells\| PubMed ID: 31717877	2019
Cell-Free Protein Synthesis and Its Perspectives for Assembling Cells from the Bottom-Up. Advanced biosystems\| PubMed ID: 32648712	2019
Functional Analysis of Aquaporin Water Permeability Using an -Based Cell-Free Protein Synthesis System. Frontiers in bioengineering and biotechnology\| PubMed ID: 32974321	2020
Microbial degradation and valorization of poly(ethylene terephthalate) (PET) monomers. World journal of microbiology & biotechnology\| PubMed ID: 35426614	2022
Molecular Mechanisms of Mercury-Sensitive Aquaporins. Journal of the American Chemical Society\| PubMed ID: 36413513	2022
Reconstitution of a Reversible Membrane Switch via Prenylation by One-Pot Cell-Free Expression. ACS synthetic biology\| PubMed ID: 36445320	2023
The trehalose-6-phosphate phosphatase gene regulates primary root growth and lateral root elongation. Frontiers in plant science\| PubMed ID: 36714693	2022
Advancing synthetic biology through cell-free protein synthesis. Computational and structural biotechnology journal\| PubMed ID: 37216017	2023
Aquaporins and CO diffusion across biological membrane. Frontiers in physiology\| PubMed ID: 37383148	2023
Bottom-Up Synthetic Biology Using Cell-Free Protein Synthesis. Advances in biochemical engineering/biotechnology\| PubMed ID: 37526707	2023
Genome-wide systematic survey and analysis of the RNA helicase gene family and their response to abiotic stress in sweetpotato. BMC plant biology\| PubMed ID: 38493089	2024

Lei Kai

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