Development and large-scale production of human milk fat analog by fermentation of microalgae.

Background: Human milk contains a complex mixture of triacylglycerols (TAG), making it challenging to recreate using common ingredients. Objective: The study aimed to develop an innovative fermentation technique to produce essential human milk TAG, effectively tackling a significant hurdle in infant nutrition. Method: An in-depth analysis of the literature has been conducted to identify the specific TAG to be targeted. We used a microalgal oil production platform and a two-step procedure to modify its fatty acid and TAG composition. The palmitic acid (16:0) content has been increased by classical strain improvement techniques, followed by a step involving the expression of a lysophosphatidic acid acyltransferase (LPAAT) sequence capable of esterifying 16:0 specifically at the internal position (sn-2 palmitate) of TAG. Once the strain was stabilized, the fermentation was scaled up in a 50-L reactor to yield several kilograms of biomass. Subsequently, the oil was extracted and refined using standard oil processing conditions. Liquid chromatography-mass spectrometry was employed to monitor the TAG profile and the region specificity of 16:0 at the internal position (sn-2 palmitate) of TAG. Results: The initial strain had a 16:0 level of 25% of total fatty acids, which was increased to 30% by classical strain improvement. Simultaneously, the oleic acid level decreased from 61% to 57% of total fatty acids. Upon expression of an exogenous LPAAT gene, the level of the 16:0 esterified in the internal position of the TAG (sn-2 palmitate) increased by a factor of 10, to reach 73% of total palmitic acid. Consequently, the concentration of oleic acid in the internal position decreased from 81% to 22% of total fatty acids, with TAG analysis confirming that the primary TAG species in the oil was 1,3-dioleoyl-2-palmitoyl-glycerol (OPO). The 50-L-scale fermentation trial confirmed the strain's ability to produce oil with a yield of >150 g of oil per liter of fermentation broth in a timeframe of 5 days, rendering the process scalable for larger-scale industrialization. Conclusion: We have demonstrated the feasibility of producing a suitable TAG composition that can be effectively integrated into the formulations of infant nutrition in combination with other fats and oils to meet the infant feeding requirements.

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.

Reprogramming sphingolipid glycosylation is required for endosymbiont persistence in Medicago truncatula.

Plant endosymbiosis relies on the development of specialized membranes that encapsulate the endosymbiont and facilitate nutrient exchange. However, the identity and function of lipids within these membrane interfaces is largely unknown. Here, we identify GLUCOSAMINE INOSITOL PHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) as a sphingolipid glycosyltransferase highly expressed in Medicago truncatula root nodules and roots colonized by arbuscular mycorrhizal (AM) fungi and further demonstrate that this enzyme functions in the synthesis of N-acetyl-glucosamine-decorated glycosyl inositol phosphoryl ceramides (GIPCs) in planta. MtGINT1 expression was developmentally regulated in symbiotic tissues associated with the development of symbiosome and periarbuscular membranes. RNAi silencing of MtGINT1 did not affect overall root growth but strongly impaired nodulation and AM symbiosis, resulting in the senescence of symbiosomes and arbuscules. Our results indicate that, although M. truncatula root sphingolipidome predominantly consists of hexose-decorated GIPCs, local reprogramming of GIPC glycosylation by MtGINT1 is required for the persistence of endosymbionts within the plant cell.

Further engineering of R. toruloides for the production of terpenes from lignocellulosic biomass.

BACKGROUND: Mitigation of climate change requires that new routes for the production of fuels and chemicals be as oil-independent as possible. The microbial conversion of lignocellulosic feedstocks into terpene-based biofuels and bioproducts represents one such route. This work builds upon previous demonstrations that the single-celled carotenogenic basidiomycete, Rhodosporidium toruloides, is a promising host for the production of terpenes from lignocellulosic hydrolysates. RESULTS: This study focuses on the optimization of production of the monoterpene 1,8-cineole and the sesquiterpene α-bisabolene in R. toruloides. The α-bisabolene titer attained in R. toruloides was found to be proportional to the copy number of the bisabolene synthase (BIS) expression cassette, which in turn influenced the expression level of several native mevalonate pathway genes. The addition of more copies of BIS under a stronger promoter resulted in production of α-bisabolene at 2.2 g/L from lignocellulosic hydrolysate in a 2-L fermenter. Production of 1,8-cineole was found to be limited by availability of the precursor geranylgeranyl pyrophosphate (GPP) and expression of an appropriate GPP synthase increased the monoterpene titer fourfold to 143 mg/L at bench scale. Targeted mevalonate pathway metabolite analysis suggested that 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR), mevalonate kinase (MK) and phosphomevalonate kinase (PMK) may be pathway bottlenecks are were therefore selected as targets for overexpression. Expression of HMGR, MK, and PMK orthologs and growth in an optimized lignocellulosic hydrolysate medium increased the 1,8-cineole titer an additional tenfold to 1.4 g/L. Expression of the same mevalonate pathway genes did not have as large an impact on α-bisabolene production, although the final titer was higher at 2.6 g/L. Furthermore, mevalonate pathway intermediates accumulated in the mevalonate-engineered strains, suggesting room for further improvement. CONCLUSIONS: This work brings R. toruloides closer to being able to make industrially relevant quantities of terpene from lignocellulosic biomass.

Expression of a bacterial 3-dehydroshikimate dehydratase (QsuB) reduces lignin and improves biomass saccharification efficiency in switchgrass (Panicum virgatum L.).

BACKGROUND: Lignin deposited in plant cell walls negatively affects biomass conversion into advanced bioproducts. There is therefore a strong interest in developing bioenergy crops with reduced lignin content or altered lignin structures. Another desired trait for bioenergy crops is the ability to accumulate novel bioproducts, which would enhance the development of economically sustainable biorefineries. As previously demonstrated in the model plant Arabidopsis, expression of a 3-dehydroshikimate dehydratase in plants offers the potential for decreasing lignin content and overproducing a value-added metabolic coproduct (i.e., protocatechuate) suitable for biological upgrading. RESULTS: The 3-dehydroshikimate dehydratase QsuB from Corynebacterium glutamicum was expressed in the bioenergy crop switchgrass (Panicum virgatum L.) using the stem-specific promoter of an O-methyltransferase gene (pShOMT) from sugarcane. The activity of pShOMT was validated in switchgrass after observation in-situ of beta-glucuronidase (GUS) activity in stem nodes of plants carrying a pShOMT::GUS fusion construct. Under controlled growth conditions, engineered switchgrass lines containing a pShOMT::QsuB construct showed reductions of lignin content, improvements of biomass saccharification efficiency, and accumulated higher amount of protocatechuate compared to control plants. Attempts to generate transgenic switchgrass lines carrying the QsuB gene under the control of the constitutive promoter pZmUbi-1 were unsuccessful, suggesting possible toxicity issues associated with ectopic QsuB expression during the plant regeneration process. CONCLUSION: This study validates the transfer of the QsuB engineering approach from a model plant to switchgrass. We have demonstrated altered expression of two important traits: lignin content and accumulation of a co-product. We found that the choice of promoter to drive QsuB expression should be carefully considered when deploying this strategy to other bioenergy crops. Field-testing of engineered QsuB switchgrass are in progress to assess the performance of the introduced traits and agronomic performances of the transgenic plants.

A bimodular PKS platform that expands the biological design space.

Traditionally engineered to produce novel bioactive molecules, Type I modular polyketide synthases (PKSs) could be engineered as a new biosynthetic platform for the production of de novo fuels, commodity chemicals, and specialty chemicals. Previously, our investigations manipulated the first module of the lipomycin PKS to produce short chain ketones, 3-hydroxy acids, and saturated, branched carboxylic acids. Building upon this work, we have expanded to multi-modular systems by engineering the first two modules of lipomycin to generate unnatural polyketides as potential biofuels and specialty chemicals in Streptomyces albus. First, we produce 20.6 mg/L of the ethyl ketone, 4,6 dimethylheptanone through a reductive loop exchange in LipPKS1 and a ketoreductase knockouts in LipPKS2. We then show that an AT swap in LipPKS1 and a reductive loop exchange in LipPKS2 can produce the potential fragrance 3-isopropyl-6-methyltetrahydropyranone. Highlighting the challenge of maintaining product fidelity, in both bimodular systems we observed side products from premature hydrolysis in the engineered first module and stalled dehydration in reductive loop exchanges. Collectively, our work expands the biological design space and moves the field closer to the production of "designer" biomolecules.

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism.

Despite intensive study, plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remains unvalidated. Recently we described a missing step in the D-lysine catabolism of Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (2HG) via hydroxyglutarate synthase (HglS), a DUF1338 family protein. Here we solve the structure of HglS to 1.1 Å resolution in substrate-free form and in complex with 2OA. We propose a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O2-dependent manner. Specificity is mediated by a single arginine, highly conserved across most DUF1338 proteins. An Arabidopsis thaliana HglS homolog coexpresses with known lysine catabolism enzymes, and mutants show phenotypes consistent with disrupted lysine catabolism. Structural and biochemical analysis of Oryza sativa homolog FLO7 reveals identical activity to HglS despite low sequence identity. Our results suggest DUF1338-containing enzymes catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes.

Chemoinformatic-Guided Engineering of Polyketide Synthases.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated ß-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs 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.

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.

Response of Pseudomonas putida to Complex, Aromatic-Rich Fractions from Biomass.

There is strong interest in the valorization of lignin to produce valuable products; however, its structural complexity has been a conversion bottleneck. Chemical pretreatment liberates lignin-derived soluble fractions that may be upgraded by bioconversion. Cholinium ionic liquid pretreatment of sorghum produced soluble, aromatic-rich fractions that were converted by Pseudomonas putida (P. putida), a promising host for aromatic bioconversion. Growth studies and mutational analysis demonstrated that P. putida growth on these fractions was dependent on aromatic monomers but unknown factors also contributed. Proteomic and metabolomic analyses indicated that these unknown factors were amino acids and residual ionic liquid; the oligomeric aromatic fraction derived from lignin was not converted. A cholinium catabolic pathway was identified, and the deletion of the pathway stopped the ability of P. putida to grow on cholinium ionic liquid. This work demonstrates that aromatic-rich fractions obtained through pretreatment contain multiple substrates; conversion strategies should account for this complexity.

Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer.

Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year (Zhang et al., 2017). To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 h of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts.

Optimization of the IPP-bypass mevalonate pathway and fed-batch fermentation for the production of isoprenol in Escherichia coli.

Isoprenol (3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals. Biological production of isoprenol via the mevalonate pathway has been developed and optimized extensively in Escherichia coli, but high ATP requirements and isopentenyl diphosphate (IPP) toxicity have made it difficult to achieve high titer, yield, and large-scale production. To overcome these limitations, an IPP-bypass pathway was previously developed using the promiscuous activity of diphosphomevalonate decarboxylase, and enabled the production of isoprenol at a comparable yield and titer to the original pathway. In this study, we optimized this pathway, substantially improving isoprenol production. A titer of 3.7 g/L (0.14 g isoprenol per g glucose) was achieved in batch conditions using minimal medium by pathway optimization, and a further optimization of the fed-batch fermentation process enabled an isoprenol titer of 10.8 g/L (yield of 0.105 g/g and maximum productivity of 0.157 g L-1 h-1), which is the highest reported titer for this compound. The substantial increase in isoprenol titer via the IPP-bypass pathway in this study will facilitate progress toward commercialization.

Lessons from Two Design-Build-Test-Learn Cycles of Dodecanol Production in Escherichia coli Aided by Machine Learning.

The Design-Build-Test-Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and biobased products. Here, we report on implementation of two DBTL cycles to optimize 1-dodecanol production from glucose using 60 engineered Escherichia coli MG1655 strains. The first DBTL cycle employed a simple strategy to learn efficiently from a relatively small number of strains (36), wherein only the choice of ribosome-binding sites and an acyl-ACP/acyl-CoA reductase were modulated in a single pathway operon including genes encoding a thioesterase (UcFatB1), an acyl-ACP/acyl-CoA reductase (Maqu_2507, Maqu_2220, or Acr1), and an acyl-CoA synthetase (FadD). Measured variables included concentrations of dodecanol and all proteins in the engineered pathway. We used the data produced in the first DBTL cycle to train several machine-learning algorithms and to suggest protein profiles for the second DBTL cycle that would increase production. These strategies resulted in a 21% increase in dodecanol titer in Cycle 2 (up to 0.83 g/L, which is more than 6-fold greater than previously reported batch values for minimal medium). Beyond specific lessons learned about optimizing dodecanol titer in E. coli, this study had findings of broader relevance across synthetic biology applications, such as the importance of sequencing checks on plasmids in production strains as well as in cloning strains, and the critical need for more accurate protein expression predictive tools.

Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism.

Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research.IMPORTANCEP. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.

A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction.

BACKGROUND: Single guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established. RESULTS: We developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the HCT target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of HCT. For the targeting of the second gene, Golgi-localized nucleotide sugar transporter2 (GONST2), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation. CONCLUSIONS: This screening method is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.

Structural insights into dehydratase substrate selection for the borrelidin and fluvirucin polyketide synthases.

Engineered polyketide synthases (PKSs) are promising synthetic biology platforms for the production of chemicals with diverse applications. The dehydratase (DH) domain within modular type I PKSs generates an α,ß-unsaturated bond in nascent polyketide intermediates through a dehydration reaction. Several crystal structures of DH domains have been solved, providing important structural insights into substrate selection and dehydration. Here, we present two DH domain structures from two chemically diverse PKSs. The first DH domain, isolated from the third module in the borrelidin PKS, is specific towards a trans-cyclopentane-carboxylate-containing polyketide substrate. The second DH domain, isolated from the first module in the fluvirucin B1 PKS, accepts an amide-containing polyketide intermediate. Sequence-structure analysis of these domains, in addition to previously published DH structures, display many significant similarities and key differences pertaining to substrate selection. The two major differences between BorA DH M3, FluA DH M1 and other DH domains are found in regions of unmodeled residues or residues containing high B-factors. These two regions are located between α3-ß11 and ß7-α2. From the catalytic Asp located in α3 to a conserved Pro in ß11, the residues between them form part of the bottom of the substrate-binding cavity responsible for binding to acyl-ACP intermediates.

Methyl ketone production by Pseudomonas putida is enhanced by plant-derived amino acids.

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).

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.

Mass Spectrometry-Based Microbial Metabolomics: Techniques, Analysis, and Applications.

The demand for understanding the roles genes play in biological systems has steered the biosciences into the direction the metabolome, as it closely reflects the metabolic activities within a cell. The importance of the metabolome is further highlighted by its ability to influence the genome, transcriptome, and proteome. Consequently, metabolomic information is being used to understand microbial metabolic networks. At the forefront of this work is mass spectrometry, the most popular metabolomics measurement technique. Mass spectrometry-based metabolomic analyses have made significant contributions to microbiological research in the environment and human disease. In this chapter, we break down the technical aspects of mass spectrometry-based metabolomics and discuss its application to microbiological research.

Liquid Chromatography and Mass Spectrometry Analysis of Isoprenoid Intermediates in Escherichia coli.

Isoprenoids are a highly diverse group of natural products with broad application as high value chemicals and advanced biofuels. They are synthesized using two primary building blocks, namely, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that are generated via the mevalonate (MVA) or deoxy-D-xylulose-5-phosphate (DXP) pathways. Isoprenoid biosynthetic pathways are prevalent in eukaryotes, archaea, and bacteria. Measurement of isoprenoid intermediates via standard liquid chromatography-mass spectrometry (LC-MS) protocols is generally challenging because of the hydrophilicity and complex physicochemical properties of the molecules. In addition, there is currently no reliable analytical method that can simultaneously measure metabolic intermediates from MVA and DXP pathways, including the prenyl diphosphates. Therefore, we describe a robust hydrophilic interaction liquid chromatography time-of-flight mass spectrometry (HILIC-TOF-MS) method for analyzing isoprenoid intermediates from metabolically engineered Escherichia coli strains.