生物设计研究(英文)最新文献

Terpenes are natural secondary metabolites with isoprene as the basic structural unit; they are widely found in nature and have potential applications as advanced fuels, pharmaceutical ingredients, and agricultural chemicals. However, traditional methods are inefficient for obtaining terpenes because of complex processes, low yields, and environmental unfriendliness. The unconventional oleaginous yeast Yarrowia lipolytica, with a clear genetic background and complete gene editing tools, has attracted increasing attention for terpenoid synthesis. Here, we review the synthetic biology tools for Y. lipolytica, including promoters, terminators, selection markers, and autonomously replicating sequences. The progress and emerging trends in the metabolic engineering of Y. lipolytica for terpenoid synthesis are further summarized. Finally, potential future research directions are envisioned.

Dihydroxy acid dehydratase (DHAD) is the third enzyme in the plant branched-chain amino acid biosynthetic pathway and the target for commercial herbicide development. We have previously reported the discovery of fungal natural product aspterric acid (AA) as a submicromolar inhibitor of DHAD through self-resistance gene directed genome mining. Here, we reveal the mechanism of AA inhibition on DHAD and the self-resistance mechanism of AstD, which is encoded by the self-resistance gene astD. As a competitive inhibitor, the hydroxycarboxylic acid group of AA mimics the binding of the natural substrate of DHAD, while the hydrophobic moiety of AA occupies the substrate entrance cavity. Compared to DHAD, AstD has a relatively narrow substrate channel to prevent AA from binding. Several mutants of DHAD were generated and assayed to validate the self-resistance mechanism and to confer Arabidopsis thaliana DHAD with AA resistance. These results will lead to the engineering of new type of herbicides targeting DHAD and provide direction for the ecological construction of herbicide-resistant crops.

Microbial cell factories (MCFs) are extensively used to produce a wide array of bioproducts, such as bioenergy, biochemical, food, nutrients, and pharmaceuticals, and have been regarded as the "chips" of biomanufacturing that will fuel the emerging bioeconomy era. Biotechnology advances have led to the screening, investigation, and engineering of an increasing number of microorganisms as diverse MCFs, which are the workhorses of biomanufacturing and help develop the bioeconomy. This review briefly summarizes the progress and strategies in the development of robust and efficient MCFs for sustainable and economic biomanufacturing. First, a comprehensive understanding of microbial chassis cells, including accurate genome sequences and corresponding annotations; metabolic and regulatory networks governing substances, energy, physiology, and information; and their similarity and uniqueness compared with those of other microorganisms, is needed. Moreover, the development and application of effective and efficient tools is crucial for engineering both model and nonmodel microbial chassis cells into efficient MCFs, including the identification and characterization of biological parts, as well as the design, synthesis, assembly, editing, and regulation of genes, circuits, and pathways. This review also highlights the necessity of integrating automation and artificial intelligence (AI) with biotechnology to facilitate the development of future customized artificial synthetic MCFs to expedite the industrialization process of biomanufacturing and the bioeconomy.

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.