Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae.

Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30-40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation. ONE SENTENCE SUMMARY: An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

Stereotactic radiotherapy of intracranial tumor beds on a ring-mounted Halcyon LINAC.

PURPOSE: This study sought to evaluate the feasibility and efficacy of the Halcyon Ring Delivery System (RDS) for delivering stereotactic radiotherapy (SRT) treatments for intracranial tumors beds. METHODS: Ten previously treated brain SRT patients for 30 Gy in five fractions with non-coplanar HyperArc plans on TrueBeam (6MV-FFF) were replanned on Halcyon (6MV-FFF) using the same number of arcs and Eclipse's AcurosXB dose engine. Plan quality evaluation metrics per SRT protocol included: PTV coverage, GTV dose (minimum and mean), target conformity indices (CI), heterogeneity index (HI), gradient index (GI), maximum dose 2 cm away from the PTV (D2cm), and doses to organs-at-risk (OAR). Additionally, patient-specific quality assurance (QA) results and beam-on-time (BOT) were analyzed. RESULTS: The Halcyon RDS provided highly conformal SRT plans for intracranial tumor beds with similar dose to target. When benchmarked against clinically delivered HyperArc plans, target coverage, CI(s) and HI were statistically similar. The Halcyon plans saw no statistical difference in maximum OAR doses to the brainstem, spinal cord, and cochlea. Due to the machine's coplanar geometry, the Halcyon plans showed a decrease in optic pathway dose (0.75 Gy vs. 2.08 Gy, p = 0.029). Overall, Halcyon's coplanar geometry resulted in a larger GI (3.33 vs. 2.72, p = 0.008) and a larger D2cm (39.59% vs. 29.07%, p < 0.001). In this cohort, multiple cases had the PTV and the optic pathway in the same axial plane. In one such instance, the PTV was <2 cm away from the optic pathway but even at this close proximity OAR, Halcyon still adequately spared the optic pathway. Additionally, the Halcyon's geometry provided slightly larger amount of normal brain dose receiving 24.4 Gy (8.99 cc vs. 7.36 cc) and 28.8 Gy (2.9 cc vs. 2.5 cc), although statistically insignificant. The Halcyon plans achieved similar delivery accuracy, quantified by patient-specific QA results evaluated with a 2%/2 mm gamma criteria (99.42% vs. 99.70%). For both plans, independent Monte Carlo second checks calculation agreed within 1%. Average Halcyon BOT was slightly higher by 0.35 min (p = 0.045), however, due to the one-step patient set-up and verification overall estimated treatment times on Halcyon were lower compared to HyperArc treatments (7.61 min vs. 10.26 min, p < 0.001). CONCLUSIONS: When benchmarked against clinically delivered HyperArc treatments, the Halcyon brain SRT plans provided similar plan quality and delivery accuracy but achieved faster overall treatment times. We have started treating select brain SRT patients on the Halcyon RDS for patients having tumor beds greater than 1 cm in diameter with the closest OAR distance of greater than 2 cm away from the target. We recommend other clinics to consider commissioning SRT treatments on their Halcyon systems-allowing including remote Halcyon-only clinics to provide exceptionally high-quality therapeutic brain SRT treatments to an otherwise underserved patient cohort.

Biosynthesis and synthetic biology of psychoactive natural products.

Psychoactive natural products play an integral role in the modern world. The tremendous structural complexity displayed by such molecules confers diverse biological activities of significant medicinal value and sociocultural impact. Accordingly, in the last two centuries, immense effort has been devoted towards establishing how plants, animals, and fungi synthesize complex natural products from simple metabolic precursors. The recent explosion of genomics data and molecular biology tools has enabled the identification of genes encoding proteins that catalyze individual biosynthetic steps. Once fully elucidated, the "biosynthetic pathways" are often comparable to organic syntheses in elegance and yield. Additionally, the discovery of biosynthetic enzymes provides powerful catalysts which may be repurposed for synthetic biology applications, or implemented with chemoenzymatic synthetic approaches. In this review, we discuss the progress that has been made toward biosynthetic pathway elucidation amongst four classes of psychoactive natural products: hallucinogens, stimulants, cannabinoids, and opioids. Compounds of diverse biosynthetic origin - terpene, amino acid, polyketide - are identified, and notable mechanisms of key scaffold transforming steps are highlighted. We also provide a description of subsequent applications of the biosynthetic machinery, with an emphasis placed on the synthetic biology and metabolic engineering strategies enabling heterologous production.

CRISPRi repression of nonhomologous end-joining for enhanced genome engineering via homologous recombination in Yarrowia lipolytica.

In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies.

Feasibility of a Single-Fraction Stereotactic Dose of 30 Gy to Solitary Lung Lesions on Halcyon.

Purpose We sought to explore the feasibility of using the current co-planar Halcyon ring delivery system (RDS) with a novel multileaf collimator (MLC) aperture shape controller in delivering a single high dose of 30 Gy to solitary lung lesions via stereotactic body radiotherapy (SBRT). Materials and methods Thirteen non-small-cell lung cancer (NSCLC) patients previously treated with a single dose of 30 Gy to lung lesions via SBRT on the TrueBeam (6MV-FFF) using non-coplanar volumetric modulated arc therapy (VMAT) arcs were anonymized and replanned onto the Halcyon RDS (6MV-FFF) following RTOG-0915 single-fraction criteria. The Halcyon plans utilized a novel dynamic conformal arc (DCA)-based MLC-fitting approach before VMAT optimization with a user-defined aperture shape controller option. The clinical TrueBeam and Halcyon plans were compared via their protocol compliance, target conformity, gradient index, and dose to organs-at-risk (OAR). Treatment delivery efficacy and accuracy were assessed through end-to-end quality assurance (QA) tests on Halcyon and independent dose verification via in-house Monte Carlo (MC) second-check validation. Results All Halcyon lung SBRT plans met RTOG-0915 protocol's requirements for target coverage, conformity, and gradient indices, and maximum dose 2 cm away from the target (D2cm) while being statistically insignificant (p > 0.05) when compared to clinical TrueBeam plans. Additionally, Halcyon provided a similar dose to OAR except for the ribs, where Halcyon demonstrated a lower maximum dose (15.22 Gy vs 17.01 Gy, p < 0.001). However, Halcyon plans required a higher total monitor unit (8892 MU vs 7413 MU, p < 0.001), resulting in a higher beam modulation factor (2.96 MU/cGy vs 2.47 MU/cGy, p < 0.001) and an increase in beam-on time by a factor of 2.1 (11.11 min vs 5.3 min, p < 0.005). End-to-end QA measurements demonstrate that Halcyon plans were clinically acceptable with an average gamma passing rate of 99.8% for 2%/2mm criteria and independent MC 2nd checks within ±2.86%. Conclusion Our end-to-end testing and validation study demonstrates that by utilizing a DCA-based MLC aperture shape controller before VMAT optimization, Halcyon can be used for delivering a single dose of lung SBRT treatment. However, future improvements of Halcyon RDS are recommended to allow higher output rates, rotational couch corrections, and an integrated intrafraction motion management system that will further enhance Halcyon's capability for site-specific single dosage of SBRT.

Development and Quality Assurance of Multileaf Collimator (MLC) Auto-Feathering Junctions for Multi-Isocenter Supine Volumetric Modulated Arc Therapy (VMAT) Craniospinal Axis Irradiation on Halcyon.

Currently, there is a lack of methods and tools that efficiently evaluate the auto-feathering junctions created by multileaf collimator (MLCs) for supine volumetric modulated arc therapy (VMAT) craniospinal irradiation (CSI) plans. We have investigated the feasibility of stitching together multi-isocenter fluence maps to then analyze the feathered junctions for patient-specific quality assurance (QA). Furthermore, we investigated the capability of Halcyon for the treatment of CSI patients. Three patients, who previously underwent VMAT CSI treatment on TrueBeam (6-MV flattening filter-free (FFF)) for 36 Gy in 20 fractions were replanned for Halcyon. A multi-isocenter approach with only translational superior-inferior shifts was used for both platforms. Each isocenter consists of two full arcs with anterior avoidance sectors, ±5° collimator rotations between arcs, and 5-8 cm of overlapping MLC auto-feathering junctions. All plans were QA'd via electronic portal imaging device (EPID) portal dosimetry and analyzed with a gamma criteria of 3%/3 mm. A variety of plan quality metrics were analyzed to evaluate dose distributions to the target, doses to organs at risk (OARs), and integral dose to the patient. A MATLAB script was developed to stitch the calculated and measured fluence maps in order to perform patient-specific QA for the composite fluence. The Halcyon plans provided highly conformal and homogenous dose distributions to the entire CSI target, superior to the clinical TrueBeam plans, while sparing critical organs with significantly lower values of V10Gy and V18Gy by up to 2% and 2.5%, respectively. Qualitative depictions of vertical dose profiles from the stitched DICOM of the entire CSI target for both planned and delivered fluence maps demonstrated equivalency, with slightly lower average pass rates with Halcyon (97%) compared to TrueBeam (99.9%). This approach to stitch multiple measured versus calculated EPID fluence maps has shown to be a feasible and accurate method and will be helpful for comprehensive VMAT CSI QA on both platforms. Further implementation of this script will be used in examining dosimetric impacts of daily patient positioning errors at MLC auto-feathering junctions.

Engineered Production of Strictosidine and Analogues in Yeast.

Monoterpene indole alkaloids (MIAs) are an expansive class of plant natural products, many of which have been named on the World Health Organization's List of Essential Medicines. Low production from native plant hosts necessitates a more reliable source of these drugs to meet global demand. Here, we report the development of a yeast-based platform for high-titer production of the universal MIA precursor, strictosidine. Our fed-batch platform produces ∼50 mg/L strictosidine, starting from the commodity chemicals geraniol and tryptamine. The microbially produced strictosidine was purified to homogeneity and characterized by NMR. Additionally, our approach enables the production of halogenated strictosidine analogues through the feeding of modified tryptamines. The MIA platform strain enables rapid access to strictosidine for reconstitution and production of downstream MIA natural products.

CRISPR Interference and Activation to Modulate Transcription in Yarrowia lipolytica.

Recent developments in RNA-guided nuclease technologies have advanced the engineering of a wide range of organisms, including the nonconventional yeast Yarrowia lipolytica. Y. lipolytica has been the focus of a range of synthetic biology and metabolic engineering studies due to its high capacity to synthesize and accumulate intracellular lipids. The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully adapted and used for genome editing in Y. lipolytica. However, as engineered strains are moved closer to industrialization, the need for finer control of transcription is still present. To overcome this challenge, we have developed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems to allow modulating the transcription of endogenous genes. We begin this protocol chapter by describing how to use the CRISPRi system to repress expression of any gene in Y. lipolytica. A second method describes how to use the CRISPRa system to increase expression of native Y. lipolytica genes. Finally, we describe how CRISPRi or CRISPRa vectors can be combined to enable multiplexed activation or repression of more than one gene. The implementation of CRISPRi and CRISPRa systems improves our ability to control gene expression in Y. lipolytica and promises to enable more advanced synthetic biology and metabolic engineering studies in this host.

Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function.

The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that facilitate high expression of sgRNA and improved CRISPR-Cas9 function. We provide selection criteria of Pol III promoters, efficient promoter construction methods, and a sample screening technique to test the efficiency of the hybrid promoters. A hybrid promoter system developed for Yarrowia lipolytica will serve as a model.

Host and Pathway Engineering for Enhanced Lycopene Biosynthesis in Yarrowia lipolytica.

Carotenoids are a class of molecules with commercial value as food and feed additives with nutraceutical properties. Shifting carotenoid synthesis from petrochemical-based precursors to bioproduction from sugars and other biorenewable carbon sources promises to improve process sustainability and economics. In this work, we engineered the oleaginous yeast Yarrowia lipolytica to produce the carotenoid lycopene. To enhance lycopene production, we tested a series of strategies to modify host cell physiology and metabolism, the most successful of which were mevalonate pathway overexpression and alleviating auxotrophies previously engineered into the PO1f strain of Y. lipolytica. The beneficial engineering strategies were combined into a single strain, which was then cultured in a 1-L bioreactor to produce 21.1 mg/g DCW. The optimized strain overexpressed a total of eight genes including two copies of HMG1, two copies of CrtI, and single copies of MVD1, EGR8, CrtB, and CrtE. Recovering leucine and uracil biosynthetic capacity also produced significant enhancement in lycopene titer. The successful engineering strategies characterized in this work represent a significant increase in understanding carotenoid biosynthesis in Y. lipolytica, not only increasing lycopene titer but also informing future studies on carotenoid biosynthesis.