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Glycosylation is a ubiquitous modification present across all of biology, affecting many things such as physicochemical properties, cellular recognition, subcellular localization, and immunogenicity. Nucleotide sugars are important precursors needed to study glycosylation and produce glycosylated products. Saccharomyces cerevisiae is a potentially powerful platform for producing glycosylated biomolecules, but it lacks nucleotide sugar diversity. Nucleotide sugar metabolism is complex, and understanding how to engineer it will be necessary to both access and study heterologous glycosylations found across biology. This review overviews the potential challenges with engineering nucleotide sugar metabolism in yeast from the salvage pathways that convert free sugars to their associated UDP-sugars to de novo synthesis where nucleotide sugars are interconverted through a complex metabolic network with governing feedback mechanisms. Finally, recent examples of engineering complex glycosylation of small molecules in S. cerevisiae are explored and assessed.

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.

QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

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.

Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria. This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP-D-fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

Corynebacterium glutamicum is a promising host for production of valuable polyketides. Propionate addition, a strategy known to increase polyketide production by increasing intracellular methylmalonyl-CoA availability, causes growth inhibition in C. glutamicum. The mechanism of this inhibition was unclear before our work. Here we provide evidence that accumulation of propionyl-CoA and methylmalonyl-CoA induces growth inhibition in C. glutamicum. We then show that growth inhibition can be relieved by introducing methylmalonyl-CoA-dependent polyketide synthases. With germicidin as an example, we used adaptive laboratory evolution to leverage the fitness advantage of polyketide production in the presence of propionate to evolve improved germicidin production. Whole-genome sequencing revealed mutations in germicidin synthase, which improved germicidin titer, as well as mutations in citrate synthase, which effectively evolved the native glyoxylate pathway to a new methylcitrate pathway. Together, our results show that C. glutamicum is a capable host for polyketide production and we can take advantage of propionate growth inhibition to drive titers higher using laboratory evolution or to screen for production of polyketides.

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.

BackgroundAdvanced spark ignition engines require high performance fuels with improved resistance to autoignition. Biologically derived olefinic alcohols have arisen as promising blendstock candidates due to favorable octane numbers and synergistic blending characteristics. However, production and downstream separation of these alcohols are limited by their intrinsic toxicity and high aqueous solubility, respectively. Bioproduction of carboxylate esters of alcohols can improve partitioning and reduce toxicity, but in practice has been limited to saturated esters with characteristically low octane sensitivity. If olefinic esters retain the synergistic blending characteristics of their alcohol counterparts, they could improve the bioblendstock combustion performance while also retaining the production advantages of the ester moiety.ResultsOptimization of Escherichia coli isoprenoid pathways has led to high titers of isoprenol and prenol, which are not only excellent standalone biofuel and blend candidates, but also novel targets for esterification. Here, a selection of olefinic esters enhanced blendstock performance according to their degree of unsaturation and branching. E. coli strains harboring optimized mevalonate pathways, thioester pathways, and heterologous alcohol acyltransferases (ATF1, ATF2, and SAAT) were engineered for the bioproduction of four novel olefinic esters. Although prenyl and isoprenyl lactate titers were limited to 1.48 ± 0.41 mg/L and 5.57 ± 1.36 mg/L, strains engineered for prenyl and isoprenyl acetate attained titers of 176.3 ± 16.0 mg/L and 3.08 ± 0.27 g/L, respectively. Furthermore, prenyl acetate (20% bRON = 125.8) and isoprenyl acetate (20% bRON = 108.4) exhibited blend properties comparable to ethanol and significantly better than any saturated ester. By further scaling cultures to a 2-L bioreactor under fed-batch conditions, 15.0 ± 0.9 g/L isoprenyl acetate was achieved on minimal medium. Metabolic engineering of acetate pathway flux further improved titer to attain an unprecedented 28.0 ± 1.0 g/L isoprenyl acetate, accounting for 75.7% theoretical yield from glucose.ConclusionOur study demonstrated novel bioproduction of four isoprenoid oxygenates for fuel blending. Our optimized E. coli production strain generated an unprecedented titer of isoprenyl acetate and when paired with its favorable blend properties, may enable rapid scale-up of olefinic alcohol esters for use as a fuel blend additive or as a precursor for longer-chain biofuels and biochemicals.

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

High titer, rate, yield (TRY), and scalability are challenging metrics to achieve due to trade-offs between carbon use for growth and production. To achieve these metrics, we take the minimal cut set (MCS) approach that predicts metabolic reactions for elimination to couple metabolite production strongly with growth. We compute MCS solution-sets for a non-native product indigoidine, a sustainable pigment, in Pseudomonas putida KT2440, an emerging industrial microbe. From the 63 solution-sets, our omics guided process identifies one experimentally feasible solution requiring 14 simultaneous reaction interventions. We implement a total of 14 genes knockdowns using multiplex-CRISPRi. MCS-based solution shifts production from stationary to exponential phase. We achieve 25.6 g/L, 0.22 g/l/h, and ~50% maximum theoretical yield (0.33 g indigoidine/g glucose). These phenotypes are maintained from batch to fed-batch mode, and across scales (100-ml shake flasks, 250-ml ambr®, and 2-L bioreactors).

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

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.

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 β-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.

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.

Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the inherent trade-off between cellular use of carbon towards growth vs. target metabolite production. We hypothesized that being able to strongly couple product formation with growth may lead to improvements across both metrics. In this study, we use elementary mode analysis to predict metabolic reactions that could be targeted to couple the production of indigoidine, a sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting engineered P. putida strain could achieve high TRY. The engineered pairing of product formation with carbon use also shifted production from stationary to exponential phase and the high TRY phenotype was maintained across scale. In one design cycle, we constructed an engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in exponential phase. These desirable phenotypes were maintained from batch to fed-batch cultivation mode, and from 100ml shake flasks to 250 mL ambr® and 2 L bioreactors.

Non-ribosomal peptides (NRPs) constitute a diverse class of valuable secondary metabolites, with potential industrial applications including use as pharmaceuticals, polymers and dyes. Current industrial production of dyes is predominantly achieved via chemical synthesis, which can involve toxic precursors and generate hazardous byproducts. Thus, alternative routes of dye production are highly desirable to enhance both workplace safety and environmental sustainability. Correspondingly, biological synthesis of dyes from renewable carbon would serve an ideal green chemistry paradigm. Therefore, we engineered the fungal host Rhodosporidium toruloides to produce the blue pigment indigoidine, an NRP with potential applications in the dye industry, using various low-cost carbon and nitrogen sources. To demonstrate production from renewable carbon sources and assess process scalability we produced indigoidine in 2 L bioreactors, reaching titers of 2.9 ± 0.8 g L-1 using a sorghum lignocellulosic hydrolysate in a batch process and 86.3 ± 7.4 g L-1 using glucose in a high-gravity fed-batch process. This study represents the first heterologous production of an NRP in R. toruloides, thus extending the range of microbial hosts that can be used for sustainable, heterologous production of NRPs. In addition, this is the first demonstration of producing an NRP using lignocellulose. These results highlight the potential of R. toruloides for the sustainable, and scalable production of NRPs, with the highest reported titer of indigoidine or any heterologously produced NRP to date.

Halophilic Halomonas bluephagenesis , a natural producer of poly-3-hydroxybutyrate (PHB), was metabolically engineered to synthesize ethylene glycol and glycolate from xylose. Xylose utilization was achieved by overexpressing either the xylonate pathway or the ribulose-1-phosphate pathway. The key genes encoding for xylonate dehydratase and 2-keto-3-deoxy-xylonate aldolase in the xylonate pathway were screened. With further overexpressing aldehyde reductase gene yjgB , ethylene glycol accumulation was improved to 0.91 g/L, accompanied with 1.48 g/L of PHB accumulation. The disruption of native glycolate oxidase was found to be essential for glycolate production, and the defective recombinant strain produced 0.80 g/L glycolate with 1.14 g/L PHB in shake flask cultures. These results indicated that H. bluephagenesis has the potential to produce diverse metabolic chemicals from xylose. • H. bluephagenesis was firstly engineered to produce ethylene glycol and glycolate. • Two xylose assimilation pathways were established in H. bluephagenesis. • Ethylene glycol and glycolate titers reached 0.91 g/L and 0.80 g/L in shake flasks, respectively. [ABSTRACT FROM AUTHOR]

A new mode of mechanical entanglement in extended structures is described where 1D organic ribbons of corner-sharing squares are mutually interlocked to form 3D woven covalent organic framework-500, COF-500. Reaction of aldehyde-functionalized tetrahedral Cu(PDB)2PO2Ph2 complexes (PDB = 4,4'-(1,10-phenanthroline-2,9-diyl)dibenzaldehyde) with rectangular tetratopic ETTBA (4',4‴,4''''',4''''‴-(ethene-1,1,2,2-tetrayl)tetrakis([1,1'-biphenyl]-4-amine)) linkers through imine condensation, yielded a crystalline porous metalated COF, COF-500-Cu, with pts topology. Upon removal of the Cu(I) ions, the individual 1D square ribbons in the demetalated form (COF-500) are held together only by mechanical interlocking of rings, which allows their collective movement to produce a narrow-pore form, as evidenced by nitrogen adsorption and solid-state photoluminescence studies. When exposed to tetrahydrofuran vapor, the interlocking ribbons can dynamically move away from each other to reopen up the structure. The structural integrity of COF-500 is maintained during its dynamics because the constituent square ribbons cannot part company due to spatial confinement imparted by their interlocking nature.

The synthesis of new isoreticular non-interpenetrated woven covalent organic frameworks (COFs) was achieved by linking aldehyde-functionalized copper(I) bisphenanthroline complexes with benzidine linkers in the presence of a bulky anion, diphenylphosphinate (PO2Ph2-) to give metalated COF-506-Cu and, upon removal of copper(I), the demetalated COF-506. The structures of these COFs were determined by a combination of powder X-ray diffraction and electron microscopy techniques. Guest-accessibility to the pores of the two frameworks was examined by vapor and dye inclusion studies and compared to the already reported doubly-interpenetrated COF-505-Cu.  Remarkably, COF-506 was found to take up guest molecules that exceed the size of the COF-506-Cu pores, thus giving credence to the notion of a novel mode of motional dynamics in solids we term 'adaptive inclusion'.

Human visual perception system is a key issue of the cognitive researches. It is also an inspiring prototype of thecutting-edge artificial intelligence researches - deep learning. It is interesting to investigate the behaviors of humans and deeplearning machines on vision tasks. In this paper, we focus on the perceptual decision making and object recognition on distortedimages. We found that in a wide range of distortion levels, the recognition rates of human subjects are smoothly increased alongwith the decreases of distortions. Although the deep learning machines perform obviously worse than human subjects, theirrecognition rates vary with the similar trends. It indicates that the deep learning machines make a good simulation to the humanbeing on the perceptual decision making tasks.