A de novo originated gene depresses budding yeast mating pathway and is repressed by the protein encoded by its antisense strand

Recent transcription profiling studies have revealed an unexpectedly large proportion of antisense transcripts in eukaryotic genomes. These antisense genes seem to regulate gene expression by interacting with sense genes. Previ- ous studies have focused on the non-coding antisense genes, but the possible regulatory role of the antisense protein is poorly understood. In this study, we found that a protein encoded by the antisense gene ADF1 acts as a transcription suppressor, regulating the expression of sense gene MDF1 in Saccharomyces cerevisiae. Based on the evolutionary, ge- netic, cytological and biochemical evidence, we show that the protein-coding sense gene MDF1 most likely originated de novo from a previously non-coding sequence and can significantly suppress the mating efficiency of baker＇s yeast in rich medium by binding MATa2 and thus promote vegetative growth. These results shed new light on several im- portant issues, including a new sense-antisense interaction mechanism, the de novo origination of a functional gene, and the regulation of yeast mating pathway.

Zanthoxylum-specific whole genome duplication and recent activity of transposable elements in the highly repetitive paleotetraploid Z.bungeanum genome

Zanthoxylum bungeanum is an important spice and medicinal plant that is unique for its accumulation of abundant secondary metabolites,which create a characteristic aroma and tingling sensation in the mouth.Owing to the high proportion of repetitive sequences,high heterozygosity,and increased chromosome number of Z.bungeanum,the assembly of its chromosomal pseudomolecules is extremely challenging.Here,we present a genome sequence for Z.bungeanum,with a dramatically expanded size of 4.23 Gb,assembled into 68 chromosomes.This genome is approximately tenfold larger than that of its close relative Citrus sinensis.After the divergence of Zanthoxylum and Citrus,the lineage-specific whole-genome duplication event q-WGD approximately 26.8 million years ago(MYA)and the recent transposable element(TE)burst~6.41 MYA account for the substantial genome expansion in Z.bungeanum.The independent Zanthoxylum-specific WGD event was followed by numerous fusion/fission events that shaped the genomic architecture.Integrative genomic and transcriptomic analyses suggested that prominent speciesspecific gene family expansions and changes in gene expression have shaped the biosynthesis of sanshools,terpenoids,and anthocyanins,which contribute to the special flavor and appearance of Z.bungeanum.In summary,the reference genome provides a valuable model for studying the impact of WGDs with recent TE activity on gene gain and loss and genome reconstruction and provides resources to accelerate Zanthoxylum improvement.

A new era for paclitaxel biosynthesis is coming

Paclitaxel(Taxol)stands out as a tetracyclic diterpenoid natural product derived from the endangered plant Taxus.Recognized as a pivotal broad-spectrum anticancer drug,it has garnered widespread attention due to its low yield,intricate structure,unique anticancer mechanism,and remarkable efficacy(Tong et al.,2021).Although chemists achieved the total synthesis of paclitaxel 30 years ago,after decades of research,the natural biosynthetic pathway for its production remains an enigma(Ajikumar et al.,2010;Malci et al.,2023).A recent breakthrough,published in Molecular Plant by Alisdair R.Fernie's team,unveils a minimal gene set of 18 genes required for paclitaxel biosynthesis(Zhang et al.,2023),representing a significant mark toward unraveling the entire biosynthetic pathway and enhancing paclitaxel production efficiency.

Cyclization mechanism of monoterpenes catalyzed by monoterpene synthases in dipterocarpaceae

Monoterpenoids are typically present in the secretory tissues of higher plants,and their biosynthesis is catalyzed by the action of monoterpene synthases(MTSs).However,the knowledge about these enzymes is restricted in a few plant species.MTSs are responsible for the complex cyclization of monoterpene precursors,resulting in the production of diverse monoterpene products.These enzymatic reactions are considered exceptionally complex in nature.Therefore,it is crucial to understand the catalytic mechanism of MTSs to elucidate their ability to produce diverse or specific monoterpenoid products.In our study,we analyzed thirteen genomes of Dipterocarpaceae and identified 38 MTSs that generate a variety of monoterpene products.By focusing on four MTSs with different product spectra and analyzing the formation mechanism of acyclic,monocyclic and bicyclic products in MTSs,we observed that even a single amino acid mutation can change the specificity and diversity of MTS products,which is due to the synergistic effect between the shape of the active cavity and the stabilization of carbon-positive intermediates that the mutation changing.Notably,residues N340,I448,and phosphoric acid groups were found to be significant contributors to the stabilization of intermediate terpinyl and pinene cations.Alterations in these residues,either directly or indirectly,can impact the synthesis of single monoterpenes or their mixtures.By revealing the role of key residues in the catalytic process and establishing the interaction model between specific residues and complex monoterpenes in MTSs,it will be possible to reasonably design and engineer different catalytic activities into existing MTSs,laying a foundation for the artificial design and industrial application of MTSs.

pUGTdb:A comprehensive database of plant UDP-dependent glycosyltransferases

Dear Editor,Plant UDP-dependent glycosyltransferases(UGTs),belonging to the carbohydrate-active enzyme glycosyltransferase 1 family(Louveau and Osbourn,2019),not only play important roles in adaptation to various environments(Cai et al.,2020;Pastorczyk-Szlenkier and Bednarek,2021)but also endow plant natural products with great pharmaceutical and ecological significance(Margolin et al.,2020).In recent years,an increasing number of plant UGTs have been characterized to function in the biosynthesis of many bioactive compounds such as ginsenosides(Wei et al.,2015),breviscapine(Liu et al.,2018),and rubusoside(Xu et al.,2022).

Origin and evolution of the main starch biosynthetic enzymes

Starch,a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications.Despite the starch biosynthetic pathway’s main enzymes have been characterized,their origin and evolution remained a subject of debate.In this study,we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes:starch synthase(SS),starch branching enzyme(SBE)and isoamylase-type debranching enzyme(ISA)from 51,151 annotated genomes.Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway.Initially,the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor(LECA)via horizontal gene transfer(HGT).This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen.Furthermore,during the emergence of Archaeplastida,one clade of SS was transferred from Deltaproteobacteria by HGT,while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer(EGT).Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway.Subsequently,after the divergence of Viridiplantae from Rhodophyta,all three enzymes underwent multiple duplications and N-terminus extension domain modifications,resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway.By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway,this study provides important insights into the evolutionary events of plants.

Multilayered regulation of secondary metabolism in medicinal plants

Medicinal plants represent a huge reservoir of secondary metabolites(SMs),substances with significant pharmaceutical and industrial potential.However,obtaining secondary metabolites remains a challenge due to their low-yield accumulation in medicinal plants;moreover,these secondary metabolites are produced through tightly coordinated pathways involving many spatiotemporally and environmentally regulated steps.The first regulatory layer involves a complex network of transcription factors;a second,more recently discovered layer of complexity in the regulation of SMs is epigenetic modification,such as DNA methylation,histone modification and small RNA-based mechanisms,which can jointly or separately influence secondary metabolites by regulating gene expression.Here,we summarize the findings in the fields of genetic and epigenetic regulation with a special emphasis on SMs in medicinal plants,providing a new perspective on the multiple layers of regulation of gene expression.

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway

Taxus,commonly known as yew,is a well-known gymnosperm with great ornamental and medicinal value.In this study,by assembling a chromosome-level genome of the Himalayan yew(Taxus wallichiana)with 10.9 Gb in 12 chromosomes,we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome,resulting in the main genes for paclitaxel biosynthesis,i.e.those encoding the taxadiene synthase,P450s,and transferases,being clustered on the same chromosome.The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization.Furthermore,we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway.The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodi-versity of taxoids in gymnosperms.

Raising the production of phloretin by alleviation of by-product of chalcone synthase in the engineered yeast

Phloretin is an important skin-lightening and depigmenting agent from the peel of apples. Although de novo production of phloretin has been realized in microbes using the natural pathway from plants, the efficiency of phloretin production is still not enough for industrial application. Here, we established an artificial pathway in the yeast to produce phloretin via assembling two genes of p-coumaroyl-CoA ligase(4CL) and chalcone synthase(CHS). CHS is a key enzyme which conventionally condenses a CoA-tethered starter with three molecules of malonyl-CoA to form the backbone of flavonoids. However, there was 33% of byproduct generated via CHS by condensing two molecules of malonyl-CoA during the fermentation process. Hence, we introduced a more efficient CHS and improved the supply of malonyl-CoA through two pathways;the by-product ratio was decreased from 33% to 17% and the production of phloretin was improved from 48 to 83.2 mg L^(-1). Finally, a fed-batch fermentation process was optimized and the production of phloretin reached 619.5 mg L^(-1), which was 14-fold higher than that of the previous studies. Our work established a platform for the biosynthesis of phloretin from the low-cost raw material 3-(4-hydroxyphenyl) propanoic acid and also illustrated the potential for industrial scale bio-manufacturing of phloretin.

Synthetic biology of plant natural products: From pathway elucidation to engineered biosynthesis in plant cells

Plant natural products(PNPs)are the main sources of drugs,food additives,and new biofuels and have become a hotspot in synthetic biology.In the past two decades,the engineered biosynthesis of many PNPs has been achieved through the construction of microbial cell factories.Alongside the rapid development of plant physiology,genetics,and plant genetic modification techniques,hosts have now expanded from single-celled microbes to complex plant systems.Plant synthetic biology is an emerging field that combines engineering principles with plant biology.In this review,we introduce recent advances in the biosynthetic pathway elucidation of PNPs and summarize the progress of engineered PNP biosynthesis in plant cells.Furthermore,a future vision of plant synthetic biology is proposed.Although we are still a long way from overcoming all the bottlenecks in plant synthetic biology,the ascent of this field is expected to provide a huge opportunity for future agriculture and industry.

Discovery and modification of cytochrome P450 for plant natural products biosynthesis

Cytochrome P450s are widespread in nature and play key roles in the diversification and functional modification of plant natural products.Over the last few years,there has been remarkable progress in plant P450s identification with the rapid development of sequencing technology,“omics”analysis and synthetic biology.However,challenges still persist in respect of crystal structure,heterologous expression and enzyme engineering.Here,we reviewed several research hotspots of P450 enzymes involved in the biosynthesis of plant natural products,including P450 databases,gene mining,heterologous expression and protein engineering.

The origin and evolution of the diosgenin biosynthetic pathway in yam

Diosgenin,mainly produced by Dioscorea species,is a traditional precursor of most hormonal drugs in the pharmaceutical industry.The mechanisms that underlie the origin and evolution of diosgenin biosynthesis in plants remain unclear.After sequencing the whole genome of Dioscorea zingiberensis,we revealed the evolutionary trajectory of the diosgenin biosynthetic pathway in Dioscorea and demonstrated the de novo biosynthesis of diosgenin in a yeast cell factory.First,we found that P450 gene duplication and neofunctionalization,driven by positive selection,played important roles in the origin of the diosgenin biosynthetic pathway.Subsequently,we found that the enrichment of diosgenin in the yam lineage was regulated by CpG islands,which evolved to regulate gene expression in the diosgenin pathway and balance the carbon flux between the biosynthesis of diosgenin and starch.Finally,by integrating genes fromplants,animals,and yeast,weheterologously synthesized diosgenin to 10mg/l in genetically-engineered yeast.Our study not only reveals the origin and evolutionary mechanisms of the diosgenin biosynthetic pathway in Dioscorea,but also introduces an alternative approach for the production of diosgenin through synthetic biology.

PCPD:Plant cytochrome P450 database and web-based tools for structural construction and ligand docking

Plant cytochrome P450s play key roles in the diversification and functional modification of plant natural products.Although over 200,000 plant P450 gene sequences have been recorded,only seven crystalized P450 genes severely hampered the functional characterization,gene mining and engineering of important P450s.Here,we combined Rosetta homologous modeling and MD-based refinement to construct a high-resolution P450 structure prediction process(PCPCM),which was applied to 181 plant P450s with identified functions.Furthermore,we constructed a ligand docking process(PCPLD)that can be applied for plant P450s virtual screening.10 examples of virtual screening indicated the process can reduce about 80%screening space for next experimental verification.Finally,we constructed a plant P450 database(PCPD:http://p450.biodesign.ac.cn/),which includes the sequences,structures and functions of the 181 plant P450s,and a web service based on PCPCM and PCPLD.Our study not only developed methods for the P450-specific structure analysis,but also introduced a universal approach that can assist the mining and functional analysis of P450 enzymes.

Thirteen Dipterocarpoideae genomes provide insights into their evolution and borneol biosynthesis

Dipterocarpoideae,the largest subfamily of the Dipterocarpaceae,is a dominant component of Southeast Asian rainforests and is widely used as a source of wood,damar resin,medicine,and essential oil.However,many Dipterocarpoideae species are currently on the IUCN Red List owing to severe degradation of their habitats under global climate change and human disturbance.Genetic information regarding these taxa has only recently been reported with the sequencing of four Dipterocarp genomes,providing clues to the function and evolution of these species.Here,we report on 13 high-quality Dipterocarpoideae genome assemblies,ranging in size from 302.6 to 494.8 Mb and representing the five most species-rich genera in Dipterocarpoideae.Molecular dating analyses support the Western Gondwanaland origin of Dipterocarpaceae.Based on evolutionary analysis,we propose a three-step chromosome evolution scenario to describe the karyotypic evolution from an ancestor with six chromosomes to present-day species with 11 and 7 chromosomes.We discovered an expansion of genes encoding cellulose synthase(CesA),which is essential for cellulose biosynthesis and secondary cell-wall formation.We functionally identified five bornyl diphosphate synthase(BPPS)genes,which specifically catalyze the biosynthesis of borneol,a natural medicinal compound extracted from damar resin and oils,thus providing a basis for large-scale production of natural borneol in vitro.

Metabolic engineering of Yarrowia lipolytica for scutellarin production

Scutellarin related drugs have superior therapeutic effects on cerebrovascular and cardiovascular diseases.Here,an optimal biosynthetic pathway for scutellarin was constructed in Yarrowia lipolytica platform due to its excellent metabolic potential.By integrating multi-copies of core genes from different species,the production of scutellarin was increased from 15.11 mg/L to 94.79 mg/L and the ratio of scutellarin to the main by-product was improved about 110-fold in flask condition.Finally,the production of scutellarin was improved 23-fold and reached to 346 mg/L in fed-batch bioreactor,which was the highest reported titer for de novo production of scutellarin in microbes.Our results represent a solid basis for further production of natural products on unconventional yeasts and have a potential of industrial implementation.