Verazine Biosynthesis from Simple Sugars in Engineered Saccharomyces cerevisiae.

Steroidal alkaloids are FDA-approved drugs (e.g., Zytiga) and promising drug candidates/leads (e.g., cyclopamine); yet many of the ≥ 697 known steroidal alkaloid natural products remain underutilized as drugs because it can be challenging to scale their biosynthesis in their producing organisms. Cyclopamine is a steroidal alkaloid produced by corn lily (Veratrum spp.) plants, and it is an inhibitor of the Hedgehog (Hh) signaling pathway. Therefore, cyclopamine is an important drug candidate/lead to treat human diseases that are associated with dysregulated Hh signaling, such as basal cell carcinoma and acute myeloid leukemia. Cyclopamine and its semi-synthetic derivatives have been studied in (pre)clinical trials as Hh inhibitor-based drugs. However, challenges in scaling the production of cyclopamine have slowed efforts to improve its efficacy and safety profile through (bio)synthetic derivatization, often limiting drug development to synthetic analogs of cyclopamine such as the FDA-approved drugs Odomzo, Daurismo, and Erivedge. If a platform for the scalable and sustainable production of cyclopamine were established, then its (bio)synthetic derivatization, clinical development, and, ultimately, widespread distribution could be accelerated. Ongoing efforts to achieve this goal include the biosynthesis of cyclopamine in Veratrum plant cell culture and the semi-/total chemical synthesis of cyclopamine. Herein, this work advances efforts towards a promising future approach: the biosynthesis of cyclopamine in engineered microorganisms. We completed the heterologous microbial production of verazine (biosynthetic precursor to cyclopamine) from simple sugars (i.e., glucose and galactose) in engineered Saccharomyces cerevisiae (S. cerevisiae) through the inducible upregulation of the native yeast mevalonate and lanosterol biosynthetic pathways, diversion of biosynthetic flux from ergosterol (i.e., native sterol in S. cerevisiae) to cholesterol (i.e., biosynthetic precursor to verazine), and expression of a refactored five-step verazine biosynthetic pathway. The engineered S. cerevisiae strain that produced verazine contains eight heterologous enzymes sourced from seven different species. Importantly, S. cerevisiae-produced verazine was indistinguishable via liquid chromatography-mass spectrometry from both a commercial standard (Veratrum spp. plant-produced) and Nicotiana benthamiana-produced verazine. To the best of our knowledge, this is the first report describing the heterologous production of a steroidal alkaloid in an engineered yeast. Verazine production was ultimately increased through design-build-test-learn cycles to a final titer of 83 ± 3 µg/L (4.1 ± 0.1 µg/g DCW). Together, this research lays the groundwork for future microbial biosynthesis of cyclopamine, (bio)synthetic derivatives of cyclopamine, and other steroidal alkaloid natural products.

Identification of ascorbate- and salicylate-responsive miRNAs and verification of the spectral control of miR395 in Arabidopsis.

We assumed that miRNAs might regulate the physiological and biochemical processes in plants through their effects on the redox system and phytohormones. To check this hypothesis, the transcriptome profile of wild-type Arabidopsis and lines with decreased ascorbate (Asc), glutathione (GSH), or salicylate (Sal) levels were compared. GSH deficiency did not influence the miRNA expression, whereas lower levels of Asc and Sal reduced the accumulation of 9 and 44 miRNAs, respectively, but only four miRNAs were upregulated. Bioinformatics analysis revealed that their over-represented target genes are associated with the synthesis of nitrogen-containing and aromatic compounds, nucleic acids, and sulphate assimilation. Among them, the sulphate reduction-related miR395 - ATP-sulfurylase couple was selected to check the assumed modulating role of the light spectrum. A greater induction of the Asc- and Sal-responsive miR395 was observed under sulphur starvation in far-red light compared to white and blue light in wild-type and GSH-deficient Arabidopsis lines. Sal deficiency inhibited the induction of miR395 by sulphur starvation in blue light, whereas Asc deficiency greatly reduced it independently of the spectrum. Interestingly, sulphur starvation decreased only the level of ATP sulfurylase 4 among the miR395 target genes in far-red light. The expression level of ATP sulfurylase 3 was higher in far-red light than in blue light in wild-type and Asc-deficient lines. The results indicate the coordinated control of miRNAs by the redox and hormonal system since 11 miRNAs were affected by both Asc and Sal deficiency. This process can be modulated by light spectrum, as shown for miR395.

CIPK-B is essential for salt stress signalling in Marchantia polymorpha.

Calcium signalling is central to many plant processes, with families of calcium decoder proteins having expanded across the green lineage and redundancy existing between decoders. The liverwort Marchantia polymorpha has fast become a new model plant, but the calcium decoders that exist in this species remain unclear. We performed phylogenetic analyses to identify the calcineurin B-like (CBL) and CBL-interacting protein kinase (CIPK) network of M. polymorpha. We analysed CBL-CIPK expression during salt stress, and determined protein-protein interactions using yeast two-hybrid and bimolecular fluorescence complementation. We also created genetic knockouts using CRISPR/Cas9. We confirm that M. polymorpha has two CIPKs and three CBLs. Both CIPKs and one CBL show pronounced salt-responsive transcriptional changes. All M. polymorpha CBL-CIPKs interact with each other in planta. Knocking out CIPK-B causes increased sensitivity to salt, suggesting that this CIPK is involved in salt signalling. We have identified CBL-CIPKs that form part of a salt tolerance pathway in M. polymorpha. Phylogeny and interaction studies imply that these CBL-CIPKs form an evolutionarily conserved salt overly sensitive pathway. Hence, salt responses may be some of the early functions of CBL-CIPK networks and increased abiotic stress tolerance required for land plant emergence.

3' Untranslated Region Structural Elements in CYP24A1 Are Associated With Infantile Hypercalcemia Type 1.

Loss-of-function mutations in the CYP24A1 protein-coding region causing reduced 25 hydroxyvitamin D (25OHD) and 1,25 dihydroxyvitamin D (1,25(OH)2 D) catabolism have been observed in some cases of infantile hypercalcemia type 1 (HCINF1), which can manifest as nephrocalcinosis, hypercalcemia and adult-onset hypercalciuria, and renal stone formation. Some cases present with apparent CYP24A1 phenotypes but do not exhibit pathogenic mutations. Here, we assessed the molecular mechanisms driving apparent HCINF1 where there was a lack of CYP24A1 mutation. We obtained blood samples from 47 patients with either a single abnormality of no obvious cause or a combination of hypercalcemia, hypercalciuria, and nephrolithiasis as part of our metabolic and stone clinics. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine serum vitamin D metabolites and direct sequencing to confirm CYP24A1 genotype. Six patients presented with profiles characteristic of altered CYP24A1 function but lacked protein-coding mutations in CYP24A1. Analysis upstream and downstream of the coding sequence showed single nucleotide variants (SNVs) in the CYP24A1 3' untranslated region (UTR). Bioinformatics approaches revealed that these 3' UTR abnormalities did not result in microRNA silencing but altered the CYP24A1 messenger RNA (mRNA) secondary structure, which negatively impacted translation. Our experiments showed that mRNA misfolding driven by these 3' UTR sequence-dependent structural elements was associated with normal 25OHD but abnormal 1,25(OH)2 D catabolism. Using CRISPR-Cas9 gene editing, we developed an in vitro mutant model for future CYP24A1 studies. Our results form a basis for future studies investigating structure-function relationships and novel CYP24A1 mutations producing a semifunctional protein. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters.

Next-generation sequencing (NGS) of small RNA (sRNA) cDNA libraries permits the identification and characterization of sRNA species de novo. However, the method through which these libraries are constructed can often introduce artifacts such as over- or underrepresentation of specific sequences or adapter oligonucleotides due to sequence biases held by the enzymes used. In this chapter we describe a protocol for sRNA library construction making use of high-definition (HD) adapters for the Illumina sequencing platform, which reduce ligation bias. This protocol leads to drastically reduced direct 5'/3' adapter ligation products and can be used for the synthesis of sRNA libraries from total RNA or sRNA of various plant, animal, and fungal samples. This protocol also includes a method for total RNA extraction from plant leaf and cultured cells or body fluids.