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[学术文献 ] Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing 进入全文
nature genetics
Somatic embryogenesis is crucial for plant genetic engineering, yet the underlying mechanisms in cotton remain poorly understood. Here we present a telomere-to-telomere assembly of Jin668 and a high-quality assembly of YZ1, two highly regenerative allotetraploid cotton germplasms. The completion of the Jin668 genome enables characterization of ~30.1 Mb of centromeric regions invaded by centromeric retrotransposon of maize and Tekay retrotransposons, an ~8.1 Mb 5S rDNA array containing 25,190 copies and a ~75.1 Mb major 45S rDNA array with 8,131 copies. Comparative analyses of regenerative and recalcitrant genotypes reveal dynamic transcriptional patterns and chromatin accessibility during the initial regeneration process. A hierarchical gene regulatory network identifies AGL15 as a contributor to regeneration. Additionally, we demonstrate that genetic variation affects sgRNA target sites, while the Jin668 genome assembly reduces the risk of off-target effects in CRISPR-based genome editing. Together, the complete Jin668 genome reveals the complexity of genomic regions and cotton regeneration, and improves the precision of genome editing.
[学术文献 ] Natural variation of an E3 ubiquitin ligase encoding gene Chalk9 regulates grain chalkiness in rice 进入全文
nature communications
Grain chalkiness is an undesirable trait affecting rice quality, concerning both consumers and breeders. However, the genetic mechanisms underlying rice chalkiness remain largely elusive. Here, we identify Chalk9 as a major gene associated with grain chalkiness in a natural population. Chalk9 encodes an E3 ubiquitin ligase that targets OsEBP89 for its ubiquitination and degradation. Low expression of Chalk9 results in excessive accumulation of OsEBP89, disrupting the homeostasis of storage components and leading to the chalkiness phenotype. A 64-bp insertion/deletion in the Chalk9 promoter contributes to its differential transcriptional levels, thus causing chalkiness variation among rice varieties. Moreover, the elite allele Chalk9-L reduces grain chalkiness, without compromising yield. Chalk9-L is strongly selected in japonica but exhibits a complex evolutionary trajectory in indica. Our findings reveal the molecular and genetic mechanisms underlying chalkiness and provide a potential strategy for breeding rice varieties with improved quality.
[学术文献 ] A generalized platform for artificial intelligence-powered autonomous enzyme engineering 进入全文
nature communications
Proteins are the molecular machines of life with numerous applications in energy, health, and sustainability. However, engineering proteins with desired functions for practical applications remains slow, expensive, and specialist-dependent. Here we report a generally applicable platform for autonomous enzyme engineering that integrates machine learning and large language models with biofoundry automation to eliminate the need for human intervention, judgement, and domain expertise. Requiring only an input protein sequence and a quantifiable way to measure fitness, this automated platform can be applied to engineer a wide array of proteins. As a proof of concept, we engineer Arabidopsis thaliana halide methyltransferase (AtHMT) for a 90-fold improvement in substrate preference and 16-fold improvement in ethyltransferase activity, along with developing a Yersinia mollaretii phytase (YmPhytase) variant with 26-fold improvement in activity at neutral pH. This is accomplished in four rounds over 4 weeks, while requiring construction and characterization of fewer than 500 variants for each enzyme. This platform for autonomous experimentation paves the way for rapid advancements across diverse industries, from medicine and biotechnology to renewable energy and sustainable chemistry.
[学术文献 ] Targeting lignocellulolytic gene clusters in novel Trichoderma atroviride and Trichoderma harzianum strains through bacterial artificial chromosome–guided analysis 进入全文
Mycologia
Lignocellulosic biomass is a complex carbon source with recalcitrant properties whose degradation via industrial enzymatic hydrolysis is challenging, directly affecting the cost of reliable energy production. In nature, filamentous fungi, including Trichoderma species, degrade lignocellulose via an arsenal of hydrolytic and oxidative enzymes that act synergistically to process it into soluble sugar monomers. This work explored the genomic content of Trichoderma atroviride and Trichoderma harzianum strains with hydrolytic abilities by identifying regions possessing degradative enzyme–encoding genes, namely, hydrolytic clusters. We employed bacterial artificial chromosome (BAC) methodology to target specific genomic regions and explore their genetic organization, proximal gene context, and gene expression under degradative conditions. With this tool, it was possible to inspect the linear structure and expression profile of target hydrolytic-rich genomic regions. The present work offers a perspective on the organization of genome regions related to carbohydrate metabolism. This study revealed novel genes and genome regions that are positively regulated during cellulose degradation, contributing to elucidating differences in gene organization that potentially impact hydrolysis among Trichoderma species.
[学术文献 ] Machine learning-guided engineering of T7 RNA polymerase and mRNA capping enzymes for enhanced gene expression in eukaryotic systems 进入全文
Chemical Engineering Journal
The integration of synthetic biology tools into eukaryotic systems offers both significant opportunities and challenges, particularly in optimizing transcriptional and post-transcriptional processes. T7 RNA polymerase (T7 RNAP) and mRNA capping enzymes (CEs) have been fused to enable eukaryotic mRNA production within a single construct. However, the activity of the fusion construct between the African Swine Fever Virus capping enzyme (ASFVCE) and T7 RNAP was relatively low. To address this, we fused the Brazilian Marseillevirus capping enzyme (BMCE) to T7 RNAP and developed a machine learning (ML) pipeline to engineer greatly improved fusion variants. This approach enabled the additive integration of nine predicted single substitutions that improved gene expression in yeast, thereby generating fusion polymerases that exhibited over 10-fold improvements in gene expression efficiency relative to the original fusion enzyme. Not only were ML substitutions additive for gene expression, they could be further combined with variants identified via directed evolution for even higher activities. By allowing ML predictions to guide validations we could rapidly explore the sequence landscape for enzyme optimization, achieving superior results even when compared to directed evolution. The improved enzymes have potential impact for numerous synthetic biology applications, including metabolic engineering, mRNA therapeutics, and cell free systems.
[学术文献 ] Reprogramming yeast metabolism to Alter fatty acid profiles from even-chain to odd-chain Configuration 进入全文
Bioresource Technology
Odd-chain fatty acids have significant applications in biofuels and pharmaceutical industries. In this study, a yeast cell factory was engineered to produce odd-chain fatty acids and their derivatives. The threonine biosynthesis pathway was initially engineered to enable the de novo synthesis of odd-chain fatty acids, resulting in odd-chain fatty acids accounting for 24.7 % of the total fatty acids. Subsequently, silencing the native fatty acid synthase and introducing a fatty acid synthase from Rhodotorula toruloides, which exhibits higher affinity for propionyl-CoA than the native enzyme, increased the proportion of odd-chain fatty acids to 51.9 %. Further modifications to the lipid metabolism enabled the production of odd-chain free fatty acids (184.1 mg/L) and odd-chain triglycerides (75.2 mg/g). This study successfully shifted the metabolism of Saccharomyces cerevisiae from traditional even-chain fatty acids to a strain dominant in odd-chain fatty acids, demonstrating the potential to develop a novel platform strain for producing specific odd-chain fatty acids derivatives.
