生物设计研究(英文)最新文献

Collagenases, a class of enzymes that are specifically responsible for collagen degradation, have garnered substantial attention because of their pivotal roles in tissue repair, remodeling, and medical interventions. This comprehensive review investigates the diversity, structures, and mechanisms of collagenases and highlights their therapeutic potential. First, it provides an overview of the biochemical properties of collagen and highlights its importance in extracellular matrix function. Subsequently, it meticulously analyzes the sources of collagenases and their applications in tissue engineering and food processing. Notably, this review emphasizes the predominant role played by microbial collagenases in commercial settings while discussing their production and screening methods. Furthermore, this study elucidates the methodology employed for determining collagenase activity and underscores the importance of an accurate evaluation for both research purposes and clinical applications. Finally, this review highlights the future research prospects for collagenases, with a particular focus on promoting wound healing and treating scar tissue formation and fibrotic diseases.

Engineered bacteria have the potential to deliver therapeutic payloads directly to tumors, with synthetic biology enabling precise control over therapeutic release in space and time. However, it remains unclear how to optimize therapeutic bacteria for durable colonization and sustained payload release. Here, we characterize nonpathogenic Escherichia coli expressing the bacterial toxin Perfringolysin O (PFO) and dynamic strategies that optimize therapeutic efficacy. While PFO is known for its potent cancer cell cytotoxicity, we present experimental evidence that expression of PFO causes lysis of bacteria in both batch culture and microfluidic systems, facilitating its efficient release. However, prolonged expression of PFO leads to the emergence of a mutant population that limits therapeutic-releasing bacteria in a PFO expression level-dependent manner. We present sequencing data revealing the mutant takeover and employ molecular dynamics to confirm that the observed mutations inhibit the lysis efficiency of PFO. To analyze this further, we developed a mathematical model describing the evolution of therapeutic-releasing and mutant bacteria populations revealing trade-offs between therapeutic load delivered and fraction of mutants that arise. We demonstrate that a dynamic strategy employing short and repeated inductions of the pfo gene better preserves the original population of therapeutic bacteria by mitigating the effects of mutational escape. Altogether, we demonstrate how dynamic modulation of gene expression can address mutant takeovers giving rise to limitations in engineered bacteria for therapeutic applications.

Ephedra-type alkaloids represent a large class of natural and synthetic phenylpropanolamine molecules with great pharmaceutical values. However, the existing methods typically rely on chemical approaches to diversify the N-group modification of Ephedra-type alkaloids. Herein, we report a 2-step enzymatic assembly line for creating structurally diverse Ephedra-type alkaloids to replace the conventional chemical modification steps. We first identified a new carboligase from Bacillus subtilis (BsAlsS, acetolactate synthase) as a robust catalyst to yield different phenylacetylcarbinol (PAC) analogs from diverse aromatic aldehydes with near 100% conversions. Subsequently, we screened imine reductases (IREDs) for the reductive amination of PAC analogs. It was found that IRG02 from Streptomyces albidoflavus had good activities with conversions ranging from 37% to 84% for the reductive alkylamination with diverse amine partners such as allylamine, propargylamine, and cyclopropylamine. Overall, 3 new bio-modifications at the N-group of Ephedra-type alkaloids were established. Taken together, our work lays a foundation for the future implementation of biocatalysis for synthesizing structurally diverse Ephedra-type alkaloids with potential new pharmaceutical applications.

Plants and their use as bioreactors for the generation of recombinant proteins have become one of the hottest topics in the field of Plant Biotechnology and Plant Synthetic Biology. Plant bioreactors offer superior engineering potential compared to other types, particularly in the realm of subcellular accumulation strategies for increasing production yield and quality. This review explores established and emerging strategies for subcellular accumulation of recombinant proteins in tobacco bioreactors, highlighting recent advancements in the field. Additionally, the review provides reference to the crucial initial step of selecting an optimal subcellular localization for the target protein, a design that substantially impacts production outcomes.

Terpenoids of substantial industrial interest are mainly obtained through direct extraction from plant sources. Recently, microbial cell factories or in vitro enzymatic biosystems have emerged as promising alternatives for terpenoid production. Here, we report a route for the synthesis of α-farnesene based on an in vitro enzyme cascade reaction using methanol as an inexpensive and renewable C1 substrate. Thirteen biocatalytic reactions divided into 2 modules were optimized and coupled to achieve methanol-to-α-farnesene conversion via integration with natural thylakoid membranes as a green energy engine. This in vitro enzymatic biosystem driven by light enabled the production of 1.43 and 2.40 mg liter^-1 α-farnesene using methanol and the intermediate glycolaldehyde as substrates, respectively. This work could provide a promising strategy for developing light-powered in vitro biosynthetic platforms to produce more natural compounds synthesized from C1 substrates.

Recently, there has been increasing interest in the use of bacteria for cancer therapy due to their ability to selectively target tumor sites and inhibit tumor growth. However, the complexity of the interaction between bacteria and tumor cells evokes unpredictable therapeutic risk, which induces inflammation, stimulates the up-regulation of cyclooxygenase II (COX-2) protein, and stimulates downstream antiapoptotic gene expression in the tumor microenvironment to reduce the antitumor efficacy of chemotherapy and immunotherapy. In this study, we encapsulated celecoxib (CXB), a specific COX-2 inhibitor, in liposomes anchored to the surface of Escherichia coli Nissle 1917 (ECN) through electrostatic absorption (C@ECN) to suppress ECN-induced COX-2 up-regulation and enhance the synergistic antitumor effect of doxorubicin (DOX). C@ECN improved the antitumor effect of DOX by restraining COX-2 expression. In addition, local T lymphocyte infiltration was induced by the ECN to enhance immunotherapy efficacy in the tumor microenvironment. Considering the biosafety of C@ECN, a hypoxia-induced lysis circuit, pGEX-Pvhb-Lysis, was introduced into the ECN to limit the number of ECNs in vivo. Our results indicate that this system has the potential to enhance the synergistic effect of ECN with chemical drugs to inhibit tumor progression in medical oncology.

In the biotechnological industry, multicopy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins and to assemble multigene biochemical pathways. In this study, we developed copper-induced in vivo gene amplification in budding yeast for multicopy gene expressions. To make copper as an effective selection pressure, we first constructed a copper-sensitive yeast strain by deleting the CUP1 gene encoding a small metallothionein-like protein for copper resistance. Subsequently, the reporter gene fused with a proline-glutamate-serine-threonine-destabilized CUP1 was integrated at the δ sites of retrotransposon (Ty) elements to counter the copper toxicity at 100 μM Cu²⁺. We further demonstrated the feasibility of modulating chromosomal rearrangements for increased protein expression under higher copper concentrations. In addition, we also demonstrated a simplified design of integrating the expression cassette at the CUP1 locus to achieve tandem duplication under high concentrations of copper. Taken together, we envision that this method of copper-induced in vivo gene amplification would serve as a robust and useful method for protein overproduction and metabolic engineering applications in budding yeast.

Plants are complex systems hierarchically organized and composed of various cell types. To understand the molecular underpinnings of complex plant systems, single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for revealing high resolution of gene expression patterns at the cellular level and investigating the cell-type heterogeneity. Furthermore, scRNA-seq analysis of plant biosystems has great potential for generating new knowledge to inform plant biosystems design and synthetic biology, which aims to modify plants genetically/epigenetically through genome editing, engineering, or re-writing based on rational design for increasing crop yield and quality, promoting the bioeconomy and enhancing environmental sustainability. In particular, data from scRNA-seq studies can be utilized to facilitate the development of high-precision Build-Design-Test-Learn capabilities for maximizing the targeted performance of engineered plant biosystems while minimizing unintended side effects. To date, scRNA-seq has been demonstrated in a limited number of plant species, including model plants (e.g., Arabidopsis thaliana), agricultural crops (e.g., Oryza sativa), and bioenergy crops (e.g., Populus spp.). It is expected that future technical advancements will reduce the cost of scRNA-seq and consequently accelerate the application of this emerging technology in plants. In this review, we summarize current technical advancements in plant scRNA-seq, including sample preparation, sequencing, and data analysis, to provide guidance on how to choose the appropriate scRNA-seq methods for different types of plant samples. We then highlight various applications of scRNA-seq in both plant systems biology and plant synthetic biology research. Finally, we discuss the challenges and opportunities for the application of scRNA-seq in plants.

The golden age has passed for antibiotic discovery, and while some antibiotics are currently in various phases of clinical trials in the United States, many pharmaceutical companies have abandoned antibiotic research. With the need for antibiotics, we should expand our horizon for therapeutic mining and can look toward understudied sources such as ice cores. Ice cores contain microorganisms and genetic material that have been frozen in time for thousands of years. The antibiotics used by these organisms are encoded in their genomes, which can be unlocked, identified, and characterized with modern advances in molecular biology, genetic sequencing, various computational approaches, and established natural product discovery pipelines. While synthetic biology can be used in natural product discovery approaches, synthetic biology and bioengineering efforts can also be leveraged in the selection and biodesign of increased compound yields, potency, and stability. Here, we provide the perspective that ice cores can be a source of novel antibiotic compounds and that the tools of synthetic biology can be used to design better antimicrobials.

The third-generation (3G) biorefinery aims to use microbial cell factories or enzymatic systems to synthesize value-added chemicals from one-carbon (C1) sources, such as CO₂, formate, and methanol, fueled by renewable energies like light and electricity. This promising technology represents an important step toward sustainable development, which can help address some of the most pressing environmental challenges faced by modern society. However, to establish processes competitive with the petroleum industry, it is crucial to determine the most viable pathways for C1 utilization and productivity and yield of the target products. In this review, we discuss the progresses that have been made in constructing artificial biological systems for 3G biorefineries in the last 10 years. Specifically, we highlight the representative works on the engineering of artificial autotrophic microorganisms, tandem enzymatic systems, and chemo-bio hybrid systems for C1 utilization. We also prospect the revolutionary impact of these developments on biotechnology. By harnessing the power of 3G biorefinery, scientists are establishing a new frontier that could potentially revolutionize our approach to industrial production and pave the way for a more sustainable future.