The telomere-to-telomere (T2T) genome of Peucedanum praeruptorum Dunn provides insights into the genome evolution and coumarin biosynthesis.

BACKGROUND: Traditional Chinese medicine has used Peucedanum praeruptorum Dunn (Apiaceae) for a long time. Various coumarins, including the significant constituents praeruptorin (A-E), are the active constituents in the dried roots of P. praeruptorum. Previous transcriptomic and metabolomic studies have attempted to elucidate the distribution and biosynthetic network of these medicinal-valuable compounds. However, the lack of a high-quality reference genome impedes an in-depth understanding of genetic traits and thus the development of better breeding strategies. RESULTS: A telomere-to-telomere (T2T) genome was assembled for P. praeruptorum by combining PacBio HiFi, ONT ultra-long, and Hi-C data. The final genome assembly was approximately 1.798 Gb, assigned to 11 chromosomes with genome completeness >98%. Comparative genomic analysis suggested that P. praeruptorum experienced 2 whole-genome duplication events. By the transcriptomic and metabolomic analysis of the coumarin metabolic pathway, we presented coumarins' spatial and temporal distribution and the expression patterns of critical genes for its biosynthesis. Notably, the COSY and cytochrome P450 genes showed tandem duplications on several chromosomes, which may be responsible for the high accumulation of coumarins. CONCLUSIONS: A T2T genome for P. praeruptorum was obtained, providing molecular insights into the chromosomal distribution of the coumarin biosynthetic genes. This high-quality genome is an essential resource for designing engineering strategies for improving the production of these valuable compounds.

Chromosome-level Alstonia scholaris genome unveils evolutionary insights into biosynthesis of monoterpenoid indole alkaloids.

Alstonia scholaris of the Apocynaceae family is a medicinal plant with a rich source of bioactive monoterpenoid indole alkaloids (MIAs), which possess anti-cancer activity like vinca alkaloids. To gain genomic insights into MIA biosynthesis, we assembled a high-quality chromosome-level genome for A. scholaris using nanopore and Hi-C data. The 444.95 Mb genome contained 35,488 protein-coding genes. A total of 20 chromosomes were assembled with a scaffold N50 of 21.75 Mb. The genome contained a cluster of strictosidine synthases and tryptophan decarboxylases with synteny to other species and a saccharide-terpene cluster involved in the monoterpenoid biosynthesis pathway of the MIA upstream pathway. The multi-omics data of A. scholaris provide a valuable resource for understanding the evolutionary origins of MIAs and for discovering biosynthetic pathways and synthetic biology efforts for producing pharmaceutically useful alkaloids.

Application of artificial scaffold systems in microbial metabolic engineering.

In nature, metabolic pathways are often organized into complex structures such as multienzyme complexes, enzyme molecular scaffolds, or reaction microcompartments. These structures help facilitate multi-step metabolic reactions. However, engineered metabolic pathways in microbial cell factories do not possess inherent metabolic regulatory mechanisms, which can result in metabolic imbalance. Taking inspiration from nature, scientists have successfully developed synthetic scaffolds to enhance the performance of engineered metabolic pathways in microbial cell factories. By recruiting enzymes, synthetic scaffolds facilitate the formation of multi-enzyme complexes, leading to the modulation of enzyme spatial distribution, increased enzyme activity, and a reduction in the loss of intermediate products and the toxicity associated with harmful intermediates within cells. In recent years, scaffolds based on proteins, nucleic acids, and various organelles have been developed and employed to facilitate multiple metabolic pathways. Despite varying degrees of success, synthetic scaffolds still encounter numerous challenges. The objective of this review is to provide a comprehensive introduction to these synthetic scaffolds and discuss their latest research advancements and challenges.

The construction and optimization of engineered yeast chassis for efficient biosynthesis of 8-hydroxygeraniol.

Microbial production of monoterpenoid indole alkaloids (MIAs) provides a sustainable and eco-friendly means to obtain compounds with high pharmaceutical values. However, efficient biosynthesis of MIAs in heterologous microorganisms is hindered due to low supply of key precursors such as geraniol and its derivative 8-hydroxygeraniol catalyzed by geraniol 8-hydroxylase (G8H). In this study, we developed a facile evolution platform to screen strains with improved yield of geraniol by using the SCRaMbLE system embedded in the Sc2.0 synthetic yeast and confirmed the causal role of relevant genomic targets. Through genome mining, we identified several G8H enzymes that perform much better than the commonly used CrG8H for 8-hydroxygeraniol production in vivo. We further showed that the N-terminus of these G8H enzymes plays an important role in cellular activity by swapping experiments. Finally, the combination of the engineered chassis, optimized biosynthesis pathway, and utilization of G8H led to the final strain with more than 30-fold improvement in producing 8-hydroxygeraniol compared with the starting strain. Overall, this study will provide insights into the construction and optimization of yeast cells for efficient biosynthesis of 8-hydroxygeraniol and its derivatives.