Fernando Guzmán-Chávez

Recombinant proteins have transformative potential in biomedicine, but their production is often costly and carries contamination risks. Plants offer an attractive alternative, with low growth costs and reduced pathogen risk, yet their slow growth cycles limit their use for rapid protein engineering. Here, we establish Marchantia polymorpha , a genetically tractable liverwort with a short life cycle, as a new platform for recombinant protein production. Using stable Agrobacterium-mediated transformation, we expressed an anti-mCherry nanobody fused to the fluorescent protein mTurquoise2 with different purification tags. Expression levels reached up to ∼120 µg/g fresh weight, and nanobody functionality was validated through a microscopy-based bead-binding assay. This yield rivals that of established systems such as Nicotiana benthamiana . Our results position M. polymorpha as a scalable, safe, and efficient chassis for protein engineering, with broad potential for applications in synthetic biology. This work opens the door to exploiting liverwort biotechnology for fast, cost-effective, and biosafe production of valuable recombinant proteins. Graphical Table of Contents:

Eukaryotic cell division is controlled by cyclins and cyclin-dependent kinases (CDKs). The high number of cyclin-CDK pairs in flowering plants hinders functional analysis due to redundancy, and how this system might have worked in early land plant ancestors remains unresolved. Our phylogenetic analysis showed that non-seed plants have a simple system of cell cycle genes, suggesting that the complexity in seed plants is a derived feature. To explore simpler systems, we studied the liverwort Marchantia polymorpha, which possesses a reduced, nonredundant set of core cell cycle genes. Single-cell RNA-seq and live imaging of fluorescent reporters revealed phase-specific expression of cell cycle genes during cell division, characterized by 1 predominant cyclin per phase in the vegetative gametophyte, with limited overlap at transitions. Live imaging of tagged cyclins indicated that protein turnover and localization contribute to phase specificity. Functional studies revealed that MpCYCD;1 is sufficient to promote cell cycle re-entry, while overexpression of MpCYCA and MpCYCB;1 causes growth arrest, consistent with their roles in the G1, S, and G2/M transitions. Our findings reveal conserved features of cell cycle control across eukaryotes and the ancestral state of land plants. Marchantia thus provides a powerful framework for understanding multicellular proliferation and its evolution, with the potential for engineering plant growth and development.

The regulation of cell division is broadly conserved across eukaryotes, governed by cyclins and cyclin-dependent kinases (CDKs) to coordinate progression through the cell cycle. Plants have evolved a complex set of cell-cycle genes with unique features. The high number of cyclin-CDK pairs in flowering plants complicates functional studies due to redundancy and diversification. It is critical to study simple systems in other plant lineages to better understand the functional integration of the cell-cycle control machinery and its evolution across land plants. Through a comprehensive phylogenetic analysis, we show that non-seed plants possess a simple repertoire of cyclin and CDK proteins, suggesting that the observed complexity in seed plants is a derived trait. The liverwort Marchantia polymorpha possesses a streamlined set of core cell cycle genes with minimal redundancy during vegetative development. Using single-cell RNA-seq and fluorescent reporters, we found a precise, phase-specific expression pattern for cell cycle genes. We demonstrated in vivo that only three cyclins are active, one at a given phase, without redundancy. Functional analyses revealed that Mp CYCD;1 promotes cell cycle re-entry and disrupts differentiation, while overexpression of Mp CYCA or Mp CYCB;1 arrests the cell cycle, consistent with their respective roles at G1, S, and G2/M progression. Our findings highlight the functional conservation of mechanisms for cell-cycle control across eukaryotes and provide insights into its ancestral state, revealing a minimal set of functional components required for multicellular development. This study advances our understanding of fundamental aspects of cell-cycle regulation and opens new possibilities for engineering plant growth.

Genome mining has emerged as a revolutionary tool for discovering new ribosomally synthesized and post-translationally modified peptides (RiPPs) in various genomes. Recently, these approaches have been used to detect and explore unique environments as sources of RiPP-producing microorganisms, particularly focusing on endophytic microorganisms found in medicinal plants. Some endophytic actinobacteria, especially strains of Streptomyces, are notable examples of peptide producers, as specific biosynthetic clusters encode them. To uncover the genetic potential of these organisms, we analyzed the genome of the endophytic actinobacterium Embleya sp. NF3 using genome mining and bioinformatics tools. Our analysis led to the identification of a putative class I lanthipeptide. We cloned the core biosynthetic genes of this putative lanthipeptide, named embleporicin, and expressed them in vitro using a cell-free protein system (CFPS). The resulting product demonstrated antimicrobial activity against Micrococcus luteus ATCC 9341. This represents the first RiPP reported in the genus Embleya and the first actinobacterial lanthipeptide produced through cell-free technology.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterization of genetic elements would make heterologous gene expression more predictable in this test bed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (_pro35S × 2) provided the highest yield of proteins, although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia genes for ETHYLENE RESPONSE FACTOR 1 and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER protein drove expression to higher levels across all tissues without a growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed RUBY, a polycistronic betalain synthesis cassette linked by P2A sequences, to demonstrate coordinated expression of metabolic enzymes. A heat-shock-inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing tool kit for gene expression in Marchantia and provided new resources for the Marchantia research community.

Cell-free systems for gene expression have gained attention as platforms for the facile study of genetic circuits and as highly effective tools for teaching. Despite recent progress, the technology remains inaccessible for many in low- and middle-income countries due to the expensive reagents required for its manufacturing, as well as specialized equipment required for distribution and storage. To address these challenges, we deconstructed processes required for cell-free mixture preparation and developed a set of alternative low-cost strategies for easy production and sharing of extracts. First, we explored the stability of cell-free reactions dried through a low-cost device based on silica beads, as an alternative to commercial automated freeze dryers. Second, we report the positive effect of lactose as an additive for increasing protein synthesis in maltodextrin-based cell-free reactions using either circular or linear DNA templates. The modifications were used to produce active amounts of two high-value reagents: the isothermal polymerase Bst and the restriction enzyme BsaI. Third, we demonstrated the endogenous regeneration of nucleoside triphosphates and synthesis of pyruvate in cell-free systems (CFSs) based on phosphoenol pyruvate (PEP) and maltodextrin (MDX). We exploited this novel finding to demonstrate the use of a cell-free mixture completely free of any exogenous nucleotide triphosphates (NTPs) to generate high yields of sfGFP expression. Together, these modifications can produce desiccated extracts that are 203–424-fold cheaper than commercial versions. These improvements will facilitate wider use of CFS for research and education purposes.

Following this recipe, you will cell-free extracts to perform Tx-TL reactions using plasmid or linear DNA based on P70a and T7 promoters. Likewise, this protocol describes how to prepare 4x wizard mix and setting up a CFPS reaction. NOTE : The protocol described here is an adaptation from these papers: Adam D. Silverman, Nancy Kelley-Loughnane, Julius B. Lucks, and Michael C. Jewett (2019).Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry. ACS Synthetic Biology, 403-414. DOI: 10.1021/acssynbio.8b00430. Andriy Didovyk, Taishi Tonooka, Lev Tsimring, and Jeff Hasty. (2017).Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synthetic Biology, 2198-2208. DOI: 10.1021/acssynbio.7b00253. Yang WC, Patel KG, Wong HE, Swartz JR.(2012). Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509. Please note that some steps were taken literally as they appear in these sources, and it is highly recommended to read these papers before starting. Our adaptations includes: Changes in buffers, volumes to perform the cell-free reactions using 12 µL as final volume.

This protocol details the procedures for the preparation of individual cell-free protein synthesis (CFPS) precursor solutions according to Yang et al., 2012, Caschera et al., 2014 and Arce et al, 2021 with some modifications. If volumes smaller or larger volumes than 2000-4000L are desired, the number of reagents and chemicals must be scaled down or up accordingly.The precursor solutions have typically been prepared using the following protocols in the following order: 1)Preparation of 10x Energy Mix solution 2)Preparation of 50 mM 19 Amino Acid Mix stock. 3)Preparation of a 2.5x Reaction Buffer 4)Perform cell-free reactions based on Maltodextrin as energy source # Equipment Note:Using a heated stir plate with temperature control along with a pH strips and/or pH meter will be helpful in preparing the CFPS precursor solutions.

This protocol details the procedures for the preparation of individual cell-free protein synthesis (CFPS) precursor solutions according to Yang et al., 2012.500 µL of the 4x premix is sufficient to perform 2mL of CFPS.If volumes smaller or larger volumes than 500 µL are desired, the number of reagents and chemicals must be scaled down or up accordingly.The precursor solutions have typically been prepared using the following protocols in the following order: 1)Preparation of a Magnesium Glutamate Solution 2)Preparation of a 10x Salt Solution 3)Preparation of a 25x Nucleotide Mix 4)Preparation of a 25x 19 amino acid mix 5)Preparation of a 25x PEP solution # Equipment Note: Using a heated stir plate with temperature control along with a pH strips and/or pH meter will be helpful in preparing the CFPS precursor solutions. NOTE : The protocol described here is an adaptation from these papers: Adam D. Silverman, Nancy Kelley-Loughnane, Julius B. Lucks, and Michael C. Jewett (2019).Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry. ACS Synthetic Biology, 403-414. DOI: 10.1021/acssynbio.8b00430. Andriy Didovyk, Taishi Tonooka, Lev Tsimring, and Jeff Hasty. (2017).Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synthetic Biology, 2198-2208. DOI: 10.1021/acssynbio.7b00253. Yang WC, Patel KG, Wong HE, Swartz JR.(2012). Simplifying and streamlining Escherichia coli-based cell-free protein synthesis.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509. Please note that some steps were taken literally as they appear in these sources, and it is highly recommended to read these papers before starting. Our adaptations includes: changes in reagents and volumes.

Work instruction for 2xYT and 2xYTG medium preparation Following this recipe, you will obtain 1L of 2XYT medium 2X YTis a standard growth medium used to cultivateE. coli. In cell-free system, this medium is commonly used to growth the cells. NOTE : The protocol described here is an adaptation from these papers: Adam D. Silverman, Nancy Kelley-Loughnane, Julius B. Lucks, and Michael C. Jewett (2019).Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry. ACS Synthetic Biology, 403-414. DOI: 10.1021/acssynbio.8b00430. Andriy Didovyk, Taishi Tonooka, Lev Tsimring, and Jeff Hasty. (2017).Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synthetic Biology, 2198-2208. DOI: 10.1021/acssynbio.7b00253.