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Pseudomonas putidahave emerged as promising biocatalysts for the conversion of sugars and aromatics 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 inP. putidaM2, a strain capable of consuming both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), 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 co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation ofcrcvia CRISPRi led to faster growth and uptake of glucose andp-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose +p-coumarate. The increased abundance of the Eda and amino acids 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 inP. putidastrains, were lower under strong CCR (glucose +p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolatedPseudomonas putidastrain,P. putidaM2, can utilize both hexose and pentose sugars as well as aromatics 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 aromatics 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 inP. putidaM2. 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 thecrcgene and small RNAs in carbon catabolite repression.

While many heterologous molecules can be produced at trace concentrations via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth couplingpara-coumarate to indigoidine production and prototype two cut sets inPseudomonas putidaKT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth onp-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY inpara-coumarate minimal media. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields.

Multiple heavy metal contamination is an increasingly common global problem. Heavy metals have the potential to disrupt microbially mediated biogeochemical cycling. However, systems-level studies on the effects of combinations of heavy metals on bacteria are lacking. For this study, we focused on the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface which is contaminated with several heavy metals and high concentrations of nitrate. Using a native Bacillus cereus isolate that represents a dominant species at this site, we assessed the combined impact of eight metal contaminants, all at site-relevant concentrations, on cell processes through an integrated multi-omics approach that included discovery proteomics, targeted metabolomics, and targeted gene-expression profiling. The combination of eight metals impacted cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Exposure to the metal mixture elicited a global iron starvation response not observed during individual metal exposures. This disruption of iron homeostasis resulted in decreased activity of the iron-cofactor-containing nitrate and nitrite reductases, both of which are important in biological nitrate removal at the site. We propose that the combinatorial effects of simultaneous exposure to multiple heavy metals is an underappreciated yet significant form of cell stress in the environment with the potential to disrupt global nutrient cycles and to impede bioremediation efforts at mixed waste sites. Our work underscores the need to shift from single- to multi-metal studies for assessing and predicting the impacts of complex contaminants on microbial systems.

Journal of the American Chemical Society : JACS 145(16), 8822-8832 (2023). doi:10.1021/jacs.2c11027, Published by American Chemical Society Publications, Washington, DC

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.

The method of X-ray footprinting mass spectrometry was used to investigate the effect of X-ray irradiation on various proteins in solution under both fully aerated and low dissolved oxygen conditions, and as a function of protein concentration., 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.

High-throughput screening technologies are widely used for elucidating biological activities. These typically require trade-offs in assay specificity and sensitivity to achieve higher throughput. Microfluidic approaches enable rapid manipulation of small volumes and have found a wide range of applications in biotechnology providing improved control of reaction conditions, faster assays, and reduced reagent consumption. The integration of mass spectrometry with microfluidics has the potential to create high-throughput, sensitivity, and specificity assays. This review introduces the widely-used mass spectrometry ionization techniques that have been successfully integrated with microfluidics approaches such as continuous-flow system, microchip electrophoresis, droplet microfluidics, digital microfluidics, centrifugal microfluidics, and paper microfluidics. In addition, we discuss recent applications of microfluidics integrated with mass spectrometry in single-cell analysis, compound screening, and the study of microorganisms. Lastly, we provide future outlooks towards online coupling, improving the sensitivity and integration of multi-omics into a single platform., The integration of mass spectrometry with microfluidics has the potential to create high-throughput, sensitivity, and specificity assays.

Globally, multiple heavy metal contamination is an increasingly common problem. As heavy metals have the potential to disrupt microbially-mediated biogeochemical cycling, it is critical to understand their impact on microbial physiology. However, systems-level studies on the effects of a combination of heavy metals on bacteria are lacking. Here, we use a native Bacillus cereus isolate from the subsurface of the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) — representing a highly abundant species at the site— to assess the combined impact of eight metal contaminants. Using this metal mixture and individual metals, all at concentrations based on the ORR site geochemistry, we performed growth experiments and proteomic analyses of the B. cereus strain, in combination with targeted MS-based metabolomics and gene expression profiling. The combination of eight metals impacts cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Specifically, exposure to the metal mixture elicited global iron starvation responses not observed in any of the individual metal treatments. As nitrate is also a significant contaminant at the ORR site and nitrate and nitrite reductases are iron-containing enzymes, we also examined the effects of the metal mixture on reduction of nitrogen oxides. We found that the metal mixture inhibits the activity of these enzymes through a combination of direct enzymatic damage and post-transcriptional and post-translational regulation. Altogether, these data suggest that metal mixture studies are critical for understanding how multiple rather than individual metals influence microbial processes in the environment.

Multiple heavy metal contamination is an increasingly common global problem. Heavy metals have the potential to disrupt microbially mediated biogeochemical cycling. However, systems-level studies on the effects of combinations of heavy metals on bacteria are lacking. For this study, we focused on the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface which is contaminated with several heavy metals and high concentrations of nitrate. Using a native Bacillus cereus isolate that represents a dominant species at this site, we assessed the combined impact of eight metal contaminants, all at site-relevant concentrations, on cell processes through an integrated multi-omics approach that included discovery proteomics, targeted metabolomics, and targeted gene-expression profiling. The combination of eight metals impacted cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Exposure to the metal mixture elicited a global iron starvation response not observed during individual metal exposures. This disruption of iron homeostasis resulted in decreased activity of the iron-cofactor-containing nitrate and nitrite reductases, both of which are important in biological nitrate removal at the site. We propose that the combinatorial effects of simultaneous exposure to multiple heavy metals is an underappreciated yet significant form of cell stress in the environment with the potential to disrupt global nutrient cycles and to impede bioremediation efforts at mixed waste sites. Our work underscores the need to shift from single- to multi-metal studies for assessing and predicting the impacts of complex contaminants on microbial systems.

Phosphorylation ofEscherichia coliCheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY.FliMNwith and without D13K-Y106W have shown FliMNbound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMNand D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D13K at the D57 phosphorylation site and Y/W106 at the FliMNinterface; namely the closure of the α4-β4 hinge and inward rotation of Y/W106 with W58. We used hydroxy-radical foot-printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other sidechains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay sidechains reported by XFMS confirmed the intermediate conformation of the native CheY.FliMNcomplex, the inactive state of free D13K-Y106W CheY and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch with Y/W106 sidechain burial correlated with increased FliMNaffinity. Activation entrained CheY fold stabilization to FliMNaffinity. The CheY network could be partitioned into four dynamically coordinated community sectors. Residue substitutions mapped to sectors around D57 or the FliMNinterface according to phenotype. FliMNincreased sector size and interactions. These sectors fused between the substituted K13K-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far.Statement of SignificanceCheY affinity for FliMN, its binding target at the flagellar motor, is increased by phosphorylation to switch rotation sense. Atomistic simulations based on CheY and CheY.FliMNcrystal structures with and without the phospho-mimetic double substitution (D13K-Y106W) showed CheY compaction is entrained to increased FliMNaffinity. Burial of exposed aromatic sidechains drove compaction, as validated by tracking sidechain solvent accessibility with hydroxyl-radical foot-printing. The substitutions were localized at the phosphorylation pocket (D13K) and FliMNinterface (Y106W). Mutual information measures revealed these locations were allosterically coupled by a specialized conduit when the conformational landscape of FliMN-tethered CheY was modified by the substitutions. Novel surfaces stabilized by the conduit may bind additional motor sites, essential for the high cooperativity of the flagellar switch.

Nonribosomal peptide synthetase (NRPS) oxidase (Ox) domains oxidize protein-bound intermediates to install crucial structural motifs in bioactive natural products. The mechanism of this domain remains elusive. Here, by studying indigoidine synthetase, a single-module NRPS involved in the biosynthesis of indigoidine and several other bacterial secondary metabolites, we demonstrate that its Ox domain utilizes an active-site base residue, tyrosine 665, to deprotonate a protein-bound l-glutaminyl residue. We further validate the generality of this active-site residue among NRPS Ox domains. These findings not only resolve the biosynthetic pathway mediated by indigoidine synthetase but enable mechanistic insight into NRPS Ox domains.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge in PKS design is engineering a partially reductive module to produce a saturated β-carbon through a reductive loop exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, could provide a viable strategy to reductive loop exchanges. We first introduced a set of donor reductive loops of diverse genetic origin and chemical substrate structures into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with atom pair chemical similarity between the substrate of the donor reductive loops and recipient LipPKS1, reaching a titer of 165 mg/L of short chain fatty acids produced by Streptomyces albus J1074 harboring these engineered PKSs. Expanding this method to larger intermediates requiring bimodular communication, we introduced reductive loops of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host. Keywords: Natural products, Synthetic biology, Aromatic polyketides, CRISPR/Cas9, Polyketide, Production platform in eukaryotes, Metabolic engineering, Biotechnology

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 (

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

This protocol details steps to normalize the amount of protein for tryptic digestion in quantitative proteomic workflows by using a Biomek NX liquid handler system. It is optimized to normalize protein concentrations in a 96-well plate format and add TCEP, IAA, and trypsin. This protocol works best as part of a semi-automated proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Quantitation with the Biomek-FX liquid handler system

Cyclopropane-functionalized hydrocarbons are excellent fuels due their high energy density. However, the organic synthesis of these molecules is challenging. In this work, we produced polycyclopropanated fatty acids in bacteria. These molecules can be converted into renewable fuels for energy-demanding applications such as shipping, long-haul transport, aviation, and rocketry. We explored the chemical diversity encoded in thousands of bacterial genomes to identify and repurpose naturally occurring cyclopropanated molecules. We identified a set of candidate iterative polyketide synthases (iPKSs) predicted to produce polycyclopropanated fatty acids (POP-FAs), expressed them in Streptomyces coelicolor, and produced POP-FAs. We determined the structure of the molecules and increased their production 22-fold. Finally, we produced polycyclopropanated fatty acid methyl esters (POP-FAMEs). Our POP fuel candidates can have net heating values of more than 50 MJ/L. Our research shows that the POP-FAMEs and other POPs have the energetic properties for energy-demanding applications for which sustainable alternatives are scarce.

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

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.

Plants are an attractive sourceof renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components, such as aromatics and amino acids, may improve the efficiency of biomass conversion. Pseudomonas putida is a promising host for its ability to metabolize a wide variety of organic compounds. P. putida was engineered to produce methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics. Unexpectedly, P. putida methyl ketone production using Arabidopsis thaliana hydrolysates was enhanced 2-5-fold compared with sugar controls derived from engineered plants that overproduce lignin-related aromatics. This enhancement was more pronounced (~seven-fold increase) with hydrolysates from nonengineered switchgrass. Proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a nine-fold increase in methyl ketone titer (1.1 g/L).

How membrane viscosity affects respiration In bacteria, energy production by the electron transport chain occurs at cell membranes and can be influenced by the lipid composition of the membrane. Budin et al. used genetic engineering to influence the concentration of unsaturated branched-chain fatty acids and thus control membrane viscosity (see the Perspective by Schon). Experimental measurements and mathematical modeling indicated that rates of respiratory metabolism and rates of cell growth were dependent on membrane viscosity and its effects on diffusion. Experiments on yeast mitochondria also showed similar effects. Maintaining efficient respiration may thus place evolutionary constraints on cellular lipid composition. Science , this issue p. 1186 ; see also p. 1114

This protocol details steps in discovery proteomic data-dependent acquisition with a standard-flow UHPLC-Obitrap system and a subsequent Mascot database search. It was adapted from González Fernández-Niño, S. M., et al. "Standard flow liquid chromatography for shotgun proteomics in bioenergy research."Frontiers in bioengineering and biotechnology, 3 (2015): 44.

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.

We adapted a high-throughput sample preparation workflow for Gram-negative bacteria to work with yeast. It consists of a bead-beating step, cell lysis, protein precipitation, protein resuspension, protein quantification, and normalization of protein concentration followed by standard bottom-up proteomic procedures of reducing and blocking cysteine residues and tryptic digestion. This protocol was adapted from the manual sample preparation method found in Chen, Y., et al. "Automated “Cells-To-Peptides” Sample Preparation Workflow for High-Throughput, Quantitative Proteomic Assays of Microbes."Journal of proteome research 18.10 (2019): 3752-3761.