生物设计研究(英文)最新文献

Violacein is a natural purple secondary metabolite with a wide range of biological activities including antibacterial, anticancer, antioxidant, and antiparasitic properties, rendering it a highly promising candidate for applications in medicine, agriculture, and food industries. Despite its availability from natural sources, a profound understanding of its production mechanisms has long been lacking. High-level production of violacein has been achieved through integrated strategies, including heterologous expression of its biosynthetic pathway in recombinant strains, enhancement of tryptophan precursor supply, and optimization of fermentation conditions. These approaches offer a flexible and scalable platform for violacein biosynthesis. Furthermore, recent efforts have focused on utilizing agro-industrial waste as a cost-effective and sustainable feedstock to further improve production efficiency and environmental compatibility. This review provides a comprehensive overview of the latest advancements in violacein production, examines the challenges associated with its application, and proposes strategies for optimizing gene expression, refining fermentation protocols, and utilizing low-cost raw materials to facilitate the efficient and sustainable violacein production.

With the rapid advancements in sustainable development and green chemistry, biotransformation has become increasingly pivotal in the synthesis of bulk chemicals and high-value products. Because natural evolution predominantly favors cellular survival, many valuable compounds, such as 2,4-dihydroxybutanoic acid and 1,2-butanediol, lack corresponding biosynthetic pathways in nature. This limitation calls for the development of fully nonnatural metabolic pathways. By enabling modular design and incorporating novel reactions, such pathways allow efficient de novo synthesis of compounds without known natural biosynthetic pathways. Nonetheless, their implementation may introduce new challenges, such as increased metabolic burden and the accumulation of toxic intermediates. Expanding the scope and efficiency of biotransformation through rational nonnatural pathways has become a key challenge. To address this, researchers have developed various computational methods for nonnatural pathway design, and two major types of methods, template-based and template-free methods, are reviewed here. We evaluate their practical applications in guiding the construction of microbial cell factories and analyze their effectiveness. Additionally, we compiled 55 experimentally validated nonnatural pathways from recent literature to establish a dataset for evaluating the strengths and limitations of these pathway design methods. By simulating a wide range of experimentally verified pathways, we highlight the gaps between computational predictions and empirical feasibility. Finally, we propose potential strategies to bridge these gaps, offering theoretical insights and practical guidance for integrating computational tools with experimental synthetic biology.

Real-time sensing of viral infection in live cells is crucial for virology research and antiviral development. However, existing methods face challenges of low signal sensitivity and the necessity for viral manipulation and cell fixation. Here, we develop a Viral-Engineered RNA-based Activation System (VERAS) that harnesses the viral replicase to induce transgene expression upon viral infection. VERAS is designed to detect real-time viral transcription and replication in live cells, which can trigger the translation of reporter and therapeutic genes. By integrating a viral packaging sequence, VERAS can also be transmitted to neighboring cells through progeny virions, effectively acting as a 'Trojan Horse'. The negative-stranded VERAS elements demonstrated effective detection of several coronaviruses, including 229E and OC43, due to the conservation of cis-acting RNA structures across coronaviruses. Notably, VERAS functions as a dual-purpose system, acting both as an infection detector and inducible antiviral system. VERAS has the potential for basic virology research applications and can be adopted in improving the inducible expression of mRNA medicines for future coronaviruses.

A great number of multifactorial diseases, including neoplastic, metabolic, and autoimmune diseases, have been associated with microbiota dysbiosis. Recently, there has been an increasing understanding of the importance of microbiome and their impact on human health. Advances in synthetic biology have led to the development of probiotics as diagnostic tools and disease treatment approaches. In this review, we briefly summarize recent examples of engineered probiotic-based therapeutics in human diseases, including cancers, gastrointestinal disorders, infectious diseases, and metabolic disorders. Finally, we discuss the challenges and opportunities in developing engineered probiotics for disease treatments.

In this study, a platform was constructed for the efficient biosynthesis of a lactate-based copolymer using a phosphite dehydrogenase (PtxD)-based NADH regeneration strategy. PtxD catalyzes the conversion of phosphite to phosphate while reducing NAD ​⁺ ​to NADH. The latter is an essential cofactor for lactate synthesis in Escherichia coli. This strategy allows the decoupling of NADH regeneration from carbon metabolism flow, providing sufficient NADH for lactate synthesis. Different concentrations of isopropyl β-d-1-thiogalactopyranoside (IPTG) were used to control the intensity of PtxD expression, and the lactate fraction in the copolymer synthesized by the engineered strain ranged from 6.2 to 16.7 ​mol%. The ptxD gene was integrated into the genome of strain WJPCTP-01, which successfully synthesized 3.24 ​g/L P(3HB-co-23.0 ​mol% LA) and 2.23 ​g/L P(3HB-co-39.0 ​mol% LA) using glucose and xylose as substrates, respectively, in shake flask cultures. In 5 ​L bioreactor fermentations, the titer of P(3HB-co-41.3 ​mol% LA) reached 8.57 ​g/L, with a synthesis rate of 0.12 ​g/L/h when xylose was used as a substrate. These findings indicate that the PtxD-based NADH regeneration strategy enhances lactate synthesis without any significant negative impact on bacterial growth or the synthesis of P(3HB-co-LA).

Miltiradiene, the major biosynthetic precursor of abietane diterpenoid natural products, has downstream metabolic derivatives with a significant potential for pharmacologically development. The formation of the characteristic abietane skeleton is attained through the rearrangement of the C13 methyl group in the pimarane intermediate, but the key enzyme mechanism responsible for this rearrangement remains elusive. Previous studies have shown that IrKSL3a in Isodon rubescens generates the pimarane diterpene isopimaradiene, and the insertion of two amino acids in the flexible region distal to the enzyme active center can change the enzyme's function to produce the abietane diterpene miltiradiene. In this paper, mutation studies were conducted on another isopimaradiene synthase IrKSL6 in Isodon rubescens and the miltiradiene synthase SmKSL1 in Salvia miltiorrhiza at these two sites. It was found that the insertion at these two sites is conservative in changing the enzyme function, and it can also affect the solubility of SmKSL1 and its affinity for the substrate. The titer of miltiradiene in the mutant SmKSL1: E550 + KR engineered bacteria increased by approximately 44 ​% compared to the wild type, the solubility of the protein increased by 24 ​%, and the catalytic efficiency (Kcat/Km) increased by 26 ​%. This paper conducted a preliminary study on the influence of the distal flexible region of the protein on the physicochemical properties of the enzyme, establishing critical molecular targets for the rational design of abietane diterpene synthases, while the high-performance mutants obtained provide superior enzymatic components for constructing biosynthesis platforms of pharmacologically relevant metabolites.

White biotechnology stands as a major sustainable alternative to address pressing environmental issues arising from our heavy dependence on petrochemical synthesis. However, reaching this goal, both technologically and economically, will take time, resources and money. A major reason is within the biological system itself, as it has evolved into a bow-tie structure in which carbon and energy are converted, via highly regulated, complex and interconnected metabolic networks, into cellular components for growth and homeostasis. This objective is fundamentally at odds with that of biotechnology, which aims to convert carbon and energy into bioproducts. Engineering of microorganism using systems and synthetic biological systems tools has been developed to provide a compromise between these two objectives. However, these genetic and metabolic interventions have revealed often unexpected physiological behaviors, in part due to the fact that a large proportion of metabolic enzymes are catalyzing other reactions than those for which they were evolved. While this promiscuity is the source of an underground metabolism that can prove very advantageous in building high-performance production routes, it is also responsible for loss of yield and production due to metabolic disturbances, negative cross-talks between natural and heterologous pathways as well as it is at the onset of metabolic damages. Identifying these promiscuous enzymes and thus anticipating their opportunities or weaknesses in engineering microbial cell factories for bioproduction is a major challenge in order to improve their performance. It is foreseen that machine learning tools operating on databases continuously fed by genetic, metabolic, enzymatic and fermentation processes data can help to overcome these challenges and provide a better understanding of the physiological functioning of the microbial system.

Clascoterone (cortexolone 17α-propionate), a novel therapeutic agent for acne, provides enhanced safety and efficacy over conventional therapies. However, the traditional synthesis of clascoterone, especially the final step, suffers from the use of hazardous and excessive organic solvents, along with suboptimal yields due to side reactions and incomplete conversion, which poses environmental and economic challenges. In this study, we developed a regioselective solvent-free alcoholysis method with the commercially available immobilized lipase Novozyme 435 as a catalyst and isopropanol (IPA) as an alcohol donor, achieving a conversion rate of over 98.0 ​% with 20 ​g/L substrate. Furthermore, we implemented a continuous flow microreactor process that increased the conversion rate to 100 ​% with 4 ​g/L substrate, demonstrating scalability for industrial manufacturing.

Lignocellulose-derived fuels and chemicals are vital to breaking the world's dependence on fossil fuels. Though plant biomass is notoriously resistant to deconstruction, lignocellulolytic thermophiles are especially adept at degrading its constituent polysaccharides into mono- and oligo-saccharides for catabolism. And many thermophiles, whether lignocellulolytic or not, can be engineered to ferment lignocellulose-derived sugars into valuable fuels and chemicals as part of consolidated bioprocesses. Although the past twenty years have seen major advances in the genetic and metabolic engineering of individual thermophiles, the strategy of co-culturing thermophilic strains as part of synthetic communities has not been well established. Synthetic communities unlock synergistic interactions that outperform monocultures, thereby enhancing product titers, rates, and yields. While limited genetic tools once hindered the development of synthetic thermophilic communities, recent advances now offer robust systems for engineering these industrially relevant organisms. Here, we propose that this expanded genetic malleability enables engineering of 1) transport specialization to reduce substrate competition between strains and 2) division of labor strategies whereby one strain focuses on lignocellulose deconstruction while another strain dedicates metabolic burden for product synthesis. We draw on examples of engineered thermophiles like Clostridium thermocellum, Thermoanaerobacter saccharolyticum, and Anaerocellum bescii to illustrate how these mechanisms have been applied in thermophilic co-cultures. In brief, this perspective outlines design principles to construct effective thermophilic communities for lignocellulose bioprocessing.