生物设计研究(英文)最新文献

Vitamin A (retinoids) is essential for human metabolism and has extensive applications in medicine, health, and cosmetics. Microbial cell factories have been developed for retinoid biosynthesis, coupled with two-phase fermentation for in situ extraction. Given that notable portions of retinoids remained in the cells, promotion of retinoid secretion is expected for further production improvements. This study investigates the potential of yeast endogenous PDR family proteins to enhance retinoid efflux by overexpressing them in retinoid-producing Saccharomyces cerevisiae strains. Among the PDR proteins tested, the transcriptional factor Pdr3p and the transporters Pdr10p and Snq2p significantly enhanced retinol and retinal secretion and production, while the transcriptional factor Pdr8p and the transporters Pdr11p, Pdr12p, Pdr18p, and Aus1p markedly increased retinoic acid production. PDR3/PDR10 co-overexpression improved retinal production to a record 638.12 ​mg/L, while PDR8 overexpression led to 106.75 ​mg/L retinoic acid production in shake flasks. For retinol, synergistic overexpression of PDR3 and PDR10 elevated the extracellular proportion to 96.7 ​%. Given the ATP requirement of PDR protein-mediated transportation, ATP supply was strengthened by overexpressing the mitochondrial fusion-related gene MGM1 and introducing hemoglobin Vgb, both enhancing retinol secretion and production. Further precursor supply enhancement resulted in 727.30 ​mg/L retinol from 20 ​g/L glucose in shake flasks, with a carbon conversion rate of 7.62 ​%. These results confirm the combination of transport engineering, energy regulation and precursor supply enhancement as a pivotal strategy for augmenting vitamin A production.

Cannabis sativa has long been a cornerstone of both medicinal and cultural practices, with its therapeutic use spanning over 2700 years. Central to its therapeutic effects are cannabinoids, which interact with the endocannabinoid system to influence various physiological processes such as anxiety, pain, and inflammation. Despite its benefits, cannabinoid production faces challenges and scarcity from plant extraction. This work leverages Yarrowia lipolytica as a platform for cannabinoid biosynthesis. By optimizing the precursor supply, engineering biomolecular condensate-like dual prenyltransferase expression and expanding endogenous metabolism with a noncanonical polyketide synthase, we achieved the de novo biosynthesis of various cannabinoids and their analogs. Our engineered Y. lipolytica produced ∼3.5 ​mg/L cannabigerolic acid, 18.8 ​mg/L orsellinic acid, and 0.5 mg/L cannabigerorcinic acid. Additionally, the CBGA titer reached 15.7 ​mg/L with olivetolic acid supplementation. This work demonstrates the versatility of Y. lipolytica as a promising host for the production of cannabinoids and their analogs, which opens avenues for further research and medicinal applications.

Glycerol, a major byproduct of biodiesel production, serves as a promising and versatile feedstock for synthesizing high-value chemicals. In this study, the NZN111, a potential succinic acid (SA) producer, was subjected to adaptive laboratory evolution (ALE) under selective pressure imposed by sodium acetate (NaAC) pressure. By analyzing the differences in the transcript levels of genes related to substrate utilization and product generation in the evolved strains, it was found that NaAC metabolism in Escherichia coli involves a series of enzymatic reactions that are apparently benefit for glycerol metabolism and SA biosynthesis. To further enhance the strain's performance, a metabolic engineering strategy was implemented, including introduction of exogenous carboxykinases and HCO₃ ^- transporter proteins. The engineered strain produced 84.27 ​g/L SA in an anaerobic fermentation medium containing 100 ​g/L glycerol with a yield of 1.25 ​g/g glycerol, which was 1.38 and 1.16 times higher than those of the original strain, respectively. These findings highlight the potential of combining ALE with targeted metabolic engineering to develop robust microbial platforms for the efficient conversion of glycerol into high-value chemicals, contributing to the sustainable utilization of biodiesel byproducts.

3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a recreational drug under clinical investigation for the treatment of central nervous system disorders, including post-traumatic stress disorder. Chemical synthesis of MDMA mainly relies on the use of safrole and piperonal as starting materials. We report a novel strategy integrating bioconversion and biocatalysis in the bioproduction of MDMA and other methamphetamine derivatives. For the initial step, a yeast-based bioconversion system was used to produce phenylacetylcarbinol (PAC) derivatives from ring-substituted benzaldehyde precursors, including piperonal, using variants of pyruvate decarboxylase (PDC). Among seven wildtype enzymes tested, Candida tropicalis PDC (CtPDC) showed the highest yield from piperonal, and a CtPDC1-I479A mutant further improved the titer. Five of sixteen ring-substituted benzaldehyde analogs (i.e., piperonal, 6-chloropiperonal, 4-acetylbenzaldehyde, 2-fluoro-4-methoxybenzaldehyde, and 2-fluoro-4-propoxybenzaldehyde) yielded corresponding PAC derivatives with yields between 20 and 70 ​%, which allowed the purification of multiple milligram quantities of each product. Three stereoselective ω-transaminases were evaluated for their ability to catalyze the transamination of PAC derivatives, with the (R)-selective enzyme ATA-117-Rd11 able to convert all five isolated PAC derivatives. Isolated transamination products were subsequently N-methylated using human phenylethanolamine N-methyltransferase. Chemical reduction facilitated the final production of MDMA and its analog 6-chloro-MDMA. Our work represents the first reported bioproduction method leading to MDMA and other methamphetamine derivatives, suitable for future pathway and strain optimization.

Plant-microbe interactions are critical to ecosystem resilience and substantially influence crop production. From the perspective of plant science, two important focus areas concerning plant-microbe interactions include: 1) understanding plant molecular mechanisms involved in plant-microbe interfaces and 2) engineering plants for increasing plant disease resistance or enhancing beneficial interactions with microbes to increase their resilience to biotic and abiotic stress conditions. Molecular biology and genetics approaches have been used to investigate the molecular mechanisms underlying plant responses to various beneficial and pathogenic microbes. While these approaches are valuable for elucidating the functions of individual genes and pathways, they fall short of unraveling the complex cross-talk across pathways or systems that plants employ to respond and adapt to environmental stresses. Also, genetic engineering of plants to increase disease resistance or enhance symbiosis with microbes has mainly been attempted or conducted through targeted manipulation of single genes/pathways of plants. Recent advancements in synthetic biology tool development are paving the way for multi-gene characterization and engineering in plants in relation to plant-microbe interactions. Here, we briefly summarize the current understanding of plant molecular pathways involved in plant interactions with beneficial and pathogenic microorganisms. Then, we highlight the progress in applying plant synthetic biology to elucidate the molecular basis of plant responses to microbes, enhance plant disease resistance, engineer synthetic symbiosis, and conduct in situ microbiome engineering. Lastly, we discuss the challenges, opportunities, and future directions for advancing plant-microbe interactions research using the capabilities of plant synthetic biology.

Tropical crops play a pivotal role in ensuring global food security and driving sustainable agricultural development, particularly in regions with limited resources. However, the complexity of tropical crop genomes and the lack of integrated omics data pose significant challenges to genetic improvement and biodesign research. TropiCODB is a comprehensive multi-omics database designed to address these challenges by integrating high-quality genomic, variome, transcriptomic, metabolomic, and phenotypic datasets for eight economically significant tropical crops, including cassava, sugarcane, and oil palm. Advanced tools such as gene family analysis, miRNA profiling, and precision guideRNA design are seamlessly incorporated, offering unprecedented support for functional genomics and molecular breeding. TropiCODB's user-friendly interface and dynamic visualization capabilities enable researchers to efficiently explore key genetic mechanisms and design targeted breeding strategies. By leveraging innovative technologies and curated datasets, TropiCODB provides a robust foundation for advancing tropical agriculture through precision biodesign.

Agrobacterium mediated plant transformation largely depends on two distinct strain lineages - C58 and Ach5. To better serve the plant transformation community, we have created a suite of auxotrophic and auxotrophic recombinant deficient mutants of C58 derivatives EHA105, GV3101::pMP90, and Ach5 derivative LBA4404. While these derivatives are useful, having additional strain backgrounds available would help expand the repertoire for plant transformation even further. Toward that end, two underutilized hypervirulent strains are K599 (NCPPB 2659), and Chry5-but disarmed variants are not easily accessible. To improve availability, we produced disarmed versions of A. rhizogenes strain K599 and A. tumefaciens strain Chry5 and introduced the same desirable mutations as with the other lineages. Each thymidine auxotrophy and recombination deficiency were introduced to existing and newly disarmed Agrobacterium strains via loss of function mutations conferred to thyA and recA, respectively, through CRISPR-mediated base-editing of codons amenable to nonsense mutation. To streamline the editing process, we created a series of visually marked single component base-editor vectors and a corresponding guide-filtering Geneious Prime wrapper plugin for expedited guide filtering. These new strains, the simplified CRISPR-mediated base-editor plasmids, and streamlined workflow will improve the ease with which future Agrobacterium strain derivatives are created while also supporting plant transformation at large.

Approaches are needed to reduce the impact of climate change and provide freshwater. Synthetic biology, and plant synthetic biology specifically, is in an exceptional position to address these needs. Plants are powerful synthetic biology platforms as they derive their energy from the sun (photosynthesis) while capturing the potent greenhouse gas, carbon dioxide. Historically, plants were used in specialized circumstances to provide and store freshwater. Even today many mangrove species can filter seawater while some plants can store massive amounts of water. I propose that starting from nature's own design principles we could engineer plants to filter seawater and/or store freshwater. Moreover, synthetic biologists could derive components found in diverse life forms, modified by water's evolutionarily adaptive properties, to provide source material for engineering plants to filter and transport water. By combining components and inspiration from nature, with advanced synthetic biology tools such as computational protein design and directed evolution, we have the knowledge and technology to make an impact on climate change. Synthetic biologists could engineer plant platforms with the ability to filter saltwater while producing freshwater thus providing a powerful and sustainable means to address freshwater needs while reducing the impact of climate change. With creativity and time, plant synthetic biology could thus help provide sustainable solutions.

Throughout thousands of years, the yeast Saccharomyces cerevisiae has been acting as a cell factory in food production. Lately, it has further functioned as a platform cell factory for the production of a multitude of different compounds, spanning from high-volume fuels to high-value pharmaceuticals. The past decade has witnessed the fact that the innovative tools in synthetic biology have driven the rapid progress of the yeast cell factory, enabling us to edit the genetic systems of organisms efficiently and "reprogram" elements or systems such as genes, circuits, pathways, and networks of organisms. Here, we will offer a brief review that highlights the most recent significant advances and perspectives regarding the innovative tools in yeast synthetic biology. These tools encompass genome editing tools, computational tools, adaptive laboratory evolution, and the standardization of biological DNA parts, with the intention of providing a practical guide for the implementation of novel, effective, and efficient development of the customized yeast cell factory.

Stability is a crucial factor influencing applicability of industrial biocatalysts, and enzymatic operations in partial organic solvents and higher temperatures are preferable since organic solvents may improve the solubility of the substrates, and the reaction rates are often higher at elevated temperatures. This review aims to summarize recent advances for engineering enzymes to meet the industrial needs for solvent and thermostability and offer insights in the advances in methodologies utilizing B-factors, ancestral reconstructions, or machine learning approaches.