Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Complete integration of carbene-transfer chemistry into biosynthesis.

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Author Correction: Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid.

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Development of Corynebacterium glutamicum as a monoterpene production platform.

Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation. Corynebacterium glutamicum, a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes in C. glutamicum have remained relatively limited. In this study, we report a further expansion of the C. glutamicum biosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers in C. glutamicum to date at 1504.6 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined that C. glutamicum first oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.

Systematic engineering for production of anti-aging sunscreen compound in Pseudomonas putida.

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

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.

Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida.

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway.

Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase.

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.

Revealing oxidative pentose metabolism in new Pseudomonas putida isolates.

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well-characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth-promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion.

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Expression of blue pigment synthetase a from Streptomyces lavenduale reveals insights on the effects of refactoring biosynthetic megasynthases for heterologous expression in Escherichia coli.

High GC bacteria from the genus Streptomyces harbor expansive secondary metabolism. The expression of biosynthetic proteins and the characterization and identification of biological "parts" for synthetic biology purposes from such pathways are of interest. However, the high GC content of proteins from actinomycetes in addition to the large size and multi-domain architecture of many biosynthetic proteins (such as non-ribosomal peptide synthetases; NRPSs, and polyketide synthases; PKSs often called "megasynthases") often presents issues with full-length translation and folding. Here we evaluate a non-ribosomal peptide synthetase (NRPS) from Streptomyces lavenduale, a multidomain "megasynthase" gene that comes from a high GC (72.5%) genome. While a preliminary step in revealing differences, to our knowledge this presents the first head-to-head comparison of codon-optimized sequences versus a native sequence of proteins of streptomycete origin heterologously expressed in E. coli. We found that any disruption in co-translational folding from codon mismatch that reduces the titer of indigoidine is explainable via the formation of more inclusion bodies as opposed to compromising folding or posttranslational modification in the soluble fraction. This result supports that one could apply any refactoring strategies that improve soluble expression in E. coli without concern that the protein that reaches the soluble fraction is differentially folded.

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization.

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4.

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH4) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH4 by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH4 in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 µM of RLs, a 600-fold increase compared with the parent strain, solely from CH4. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH4.

Biosynthesis and secretion of the microbial sulfated peptide RaxX and binding to the rice XA21 immune receptor.

The rice immune receptor XA21 is activated by the sulfated microbial peptide required for activation of XA21-mediated immunity X (RaxX) produced by Xanthomonas oryzae pv. oryzae (Xoo). Mutational studies and targeted proteomics revealed that the RaxX precursor peptide (proRaxX) is processed and secreted by the protease/transporter RaxB, the function of which can be partially fulfilled by a noncognate peptidase-containing transporter component B (PctB). proRaxX is cleaved at a Gly-Gly motif, yielding a mature peptide that retains the necessary elements for RaxX function as an immunogen and host peptide hormone mimic. These results indicate that RaxX is a prokaryotic member of a previously unclassified and understudied group of eukaryotic tyrosine sulfated ribosomally synthesized, posttranslationally modified peptides (RiPPs). We further demonstrate that sulfated RaxX directly binds XA21 with high affinity. This work reveals a complete, previously uncharacterized biological process: bacterial RiPP biosynthesis, secretion, binding to a eukaryotic receptor, and triggering of a robust host immune response.

Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry.

In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments.

Machine learning for metabolic engineering: A review.

Machine learning provides researchers a unique opportunity to make metabolic engineering more predictable. In this review, we offer an introduction to this discipline in terms that are relatable to metabolic engineers, as well as providing in-depth illustrative examples leveraging omics data and improving production. We also include practical advice for the practitioner in terms of data management, algorithm libraries, computational resources, and important non-technical issues. A variety of applications ranging from pathway construction and optimization, to genetic editing optimization, cell factory testing, and production scale-up are discussed. Moreover, the promising relationship between machine learning and mechanistic models is thoroughly reviewed. Finally, the future perspectives and most promising directions for this combination of disciplines are examined.