ACS Synthetic Biology最新文献

The ATP-dependent ClpXP-SspB protease complex is responsible for the degradation of intracellular proteins and is maintained at low levels in Escherichia coli to avoid nonspecific degradation. The rate-limiting step in the protease complex leads to proteolytic queueing, where the proteins form waiting lines, and their overall degradation rate is slowed. Synthetic biologists have leveraged proteolytic queueing to design robust synthetic circuits by tagging proteins with the SsrA tag, an 11-amino acid sequence recognized by the complex. Previous work has demonstrated the binding site of each component of the ClpXP-SspB complex to the SsrA tag. However, the precise component responsible for queueing was unknown. To identify the bottleneck in the complex, we designed different SsrA tag variants depending on the chaperone binding sequences. We further overexpressed each protein in the ClpXP-SspB complex in vivo to determine how an increased amount of each component affects the tagged protein levels. Based on the degradation of the SsrA variants, upon overexpression of each component of the ClpXP-SspB system, evidence supports that ClpX (the ATP-dependent chaperone) is responsible for queueing but not ClpP (the protease) or SspB (the adapter, ATP-independent chaperone). In the process, we identified LAA-LAA, a 6-amino acid ClpX-dependent tag that degraded in vivo faster than the original SsrA tag, AANDENYALAA. We speculated that this high degradation tag could be useful in a dynamic-synthetic circuit, so we modified the well-characterized dual-feedback oscillator by replacing its original SsrA tag with the LAA-LAA tag to form the LAA-LAA-Osc oscillator. Both population and single-cell level experiments show that the new and old oscillators have distinct frequencies. Like the original oscillator, thousands of cells containing the new oscillator could be synchronized by entrainment using an external signal. Thus, the new LAA-LAA-Osc oscillator retains the original oscillator's best characteristics (robustness to fluctuations, a steady oscillation period, and entrainment across 1000s of cells to an external signal) but oscillates at a different frequency.

The supply of artemisinin, the primary antimalarial drug recommended by the World Health Organization (WHO), is limited due to synthesis cost and supply constraints. This study explores novel chemo-enzymatic pathways for the efficient synthesis of dihydroartemisinic acid (DHAA), the penultimate precursor to artemisinin. The key concept here is to leverage the seamless integration of chemical and enzymatic steps for more thoroughly exploring synthesis alternatives. Using novoStoic, a biosynthetic pathway design tool, we identified previously unexplored carbon- and energy-balanced pathways for converting amorpha-4,11-diene (AMPD) to DHAA. For some of the enzymatically catalyzed steps lacking efficient enzymes, chemical catalysis alternatives were proposed and implemented, leading to a hybrid chemo-enzymatic pathway design. The proposed pathway converts AMPD directly to DHAA without going through artemisinic acid (AA), making it a shorter pathway compared with the existing synthesis routes for artemisinin. This effort paves the way for the systematic design of chemo-enzymatic pathways and provides insight into decision strategies between chemical synthesis and enzymatic synthesis steps. It serves as an example of how synthesis pathway design tools can be integrated with human intuition for accelerating retrosynthesis and how AI-based tools can identify and replace human intuitions to automate the decision processes. This can help reduce human-machine interventions and improve the development of future tools for synthesis planning.

Icaritin (ICT) is a naturally occurring flavonoid compound with notable anticancer properties, recently recognized for its efficacy in treating advanced hepatic carcinoma. Traditional methods of ICT production, including plant extraction and chemical synthesis, face challenges such as low yield and environmental concerns. This study leverages synthetic biology to construct a microbial cell factory using Yarrowia lipolytica for de novo ICT synthesis. We engineered the yeast by integrating the ICT synthesis pathway involving EsPT from Epimedium sagittatum and OsOMTm from Oryza sativa. By optimizing the metabolic pathways, including enhancing the supply of DMAPP via mevalonate pathway modifications, and fine-tuning the expression and catalytic efficiency of EsPT through truncation strategies, we significantly improved ICT yield to 247.02 mg/L─the highest microbial ICT titer reported to date. These findings lay a solid foundation for the large-scale industrial production of ICT and offer valuable insights into the biosynthesis of other flavonoid plant natural products.

Icaritin (ICT) is a naturally occurring flavonoid compound with notable anticancer properties, recently recognized for its efficacy in treating advanced hepatic carcinoma. Traditional methods of ICT production, including plant extraction and chemical synthesis, face challenges such as low yield and environmental concerns. This study leverages synthetic biology to construct a microbial cell factory using Yarrowia lipolytica for de novo ICT synthesis. We engineered the yeast by integrating the ICT synthesis pathway involving EsPT from Epimedium sagittatum and OsOMTm from Oryza sativa. By optimizing the metabolic pathways, including enhancing the supply of DMAPP via mevalonate pathway modifications, and fine-tuning the expression and catalytic efficiency of EsPT through truncation strategies, we significantly improved ICT yield to 247.02 mg/L─the highest microbial ICT titer reported to date. These findings lay a solid foundation for the large-scale industrial production of ICT and offer valuable insights into the biosynthesis of other flavonoid plant natural products.

Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed by failed dimer resolution during replication. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains of Escherichia coli. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, transforming multimers into JM109 can yield strains that contain no singlet plasmids. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.

Plasmids are an essential tool for basic research and biotechnology applications. To optimize plasmid-based circuits, it is crucial to control plasmid integrity, including the formation of plasmid multimers. Multimers are tandem repeats of entire plasmids formed by failed dimer resolution during replication. Multimers can affect the behavior of synthetic circuits, especially ones that include DNA-editing enzymes. However, occurrence of multimers is not commonly assayed. Here we survey four commonly used plasmid backbones for occurrence of multimers in cloning (JM109) and wild-type (MG1655) strains of Escherichia coli. We find that multimers occur appreciably only in MG1655, with the fraction of plasmids existing as multimers increasing with both plasmid copy number and culture passaging. In contrast, transforming multimers into JM109 can yield strains that contain no singlet plasmids. We present an MG1655 ΔrecA single-locus knockout that avoids multimer production. These results can aid synthetic biologists in improving design and reliability of plasmid-based circuits.

Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. Komagataeibacter xylinus was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in K. xylinus has not been reported. To facilitate metabolic pathway engineering in K. xylinus, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in K. xylinus, the strain’s ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (bcsH, bcsX, bcsY, and bcsZ) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in K. xylinus is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library, called the “Clickable CDR-H3 Library”, that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photoreactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in “protein–small molecule hybrid” format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multimodal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.

Effective employment of renewable carbon sources is highly demanded to develop sustainable biobased manufacturing. Here, we developed Escherichia coli strains to produce 2,3-butanediol and acetoin (collectively referred to as diols) using acetate as the sole carbon source by stepwise metabolic engineering. When tested in fed-batch experiments, the strain overexpressing the entire acetate utilization pathway was found to consume acetate at a 15% faster rate (0.78 ± 0.05 g/g/h) and to produce a 35% higher diol titer (1.16 ± 0.01 g/L) than the baseline diols-producing strain. Moreover, singularly overexpressing the genes encoding alternative acetate uptake pathways as well as alternative isoforms of genes in the malate-to-pyruvate pathway unveiled that leveraging ackA-pta and maeA is more effective in enhancing acetate consumption and diols production, compared to acs and maeB. Finally, the increased substrate consumption rate and diol production obtained in flask-based experiments were confirmed in bench-scale bioreactors operated in fed-batch mode. Consequently, the highest titer of 1.56 g/L achieved in this configuration increased by over 30% compared to the only other similar effort carried out so far.

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library, called the "Clickable CDR-H3 Library", that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photoreactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multimodal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.