Tumor Abnormality-Oriented Nanomedicine Design.

Anticancer nanomedicines have been proven effective in mitigating the side effects of chemotherapeutic drugs. However, challenges remain in augmenting their therapeutic efficacy. Nanomedicines responsive to the pathological abnormalities in the tumor microenvironment (TME) are expected to overcome the biological limitations of conventional nanomedicines, enhance the therapeutic efficacies, and further reduce the side effects. This Review aims to quantitate the various pathological abnormalities in the TME, which may serve as unique endogenous stimuli for the design of stimuli-responsive nanomedicines, and to provide a broad and objective perspective on the current understanding of stimuli-responsive nanomedicines for cancer treatment. We dissect the typical transport process and barriers of cancer drug delivery, highlight the key design principles of stimuli-responsive nanomedicines designed to tackle the series of barriers in the typical drug delivery process, and discuss the "all-into-one" and "one-for-all" strategies for integrating the needed properties for nanomedicines. Ultimately, we provide insight into the challenges and future perspectives toward the clinical translation of stimuli-responsive nanomedicines.

Dynamics of a Viscous Droplet in Return Bends of Microfluidic Channels.

Return bends are frequently encountered in microfluidic systems. In this study, a three-dimensional spectral boundary element method for interfacial dynamics in Stokes flow has been adopted to investigate the dynamics of viscous droplets in rectangular return bends. The droplet trajectory, deformation, and migration velocity are investigated under the influence of various fluid properties and operational conditions, which are depicted by the Capillary number, viscosity ratio, and droplet size, as well as the dimensions of the return bend. While the computational results provide information for the design of return bends in microfluidic systems in general, the computational framework shows potential to guide the design and operation of a droplet-based microfluidic delivery system for cell seeding.

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound ß-ionone.

BACKGROUND: ß-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural ß-ionone. To date, chemical extraction from plants remains the main approach for commercial natural ß-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the ß-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural ß-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor). RESULTS: A push-pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards ß-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the ß-carotene strain that achieved > 2.5 g/L ß-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the ß-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L ß-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both ß-carotene medium tests and ß-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck. CONCLUSIONS: We engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma ß-ionone. Although ß-carotene could be produced at high titers by Y. lipolytica, the synthesis of ß-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of ß-carotene. In addition, both ß-carotene and ß-ionone strains showed decreased performances after successive sub-cultures. For industrial application, ß-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.

Synthetic biology for manufacturing chemicals: constraints drive the use of non-conventional microbial platforms.

Genetically modified microbes have had much industrial success producing protein-based products (such as antibodies and enzymes). However, engineering microbial workhorses for biomanufacturing of commodity compounds remains challenging. First, microbes cannot afford burdens with both overexpression of multiple enzymes and metabolite drainage for product synthesis. Second, synthetic circuits and introduced heterologous pathways are not yet as "robust and reliable" as native pathways due to hosts' innate regulations, especially under suboptimal fermentation conditions. Third, engineered enzymes may lack channeling capabilities for cascade-like transport of metabolites to overcome diffusion barriers or to avoid intermediate toxicity in the cytoplasmic environment. Fourth, moving engineered hosts from laboratory to industry is unreliable because genetic mutations and non-genetic cell-to-cell variations impair the large-scale fermentation outcomes. Therefore, synthetic biology strains often have unsatisfactory industrial performance (titer/yield/productivity). To overcome these problems, many different species are being explored for their metabolic strengths that can be leveraged to synthesize specific compounds. Here, we provide examples of non-conventional and genetically amenable species for industrial manufacturing, including the following: Corynebacterium glutamicum for its TCA cycle-derived biosynthesis, Yarrowia lipolytica for its biosynthesis of fatty acids and carotenoids, cyanobacteria for photosynthetic production from its sugar phosphate pathways, and Rhodococcus for its ability to biotransform recalcitrant feedstock. Finally, we discuss emerging technologies (e.g., genome-to-phenome mapping, single cell methods, and knowledge engineering) that may facilitate the development of novel cell factories.

A plant malonyl-CoA synthetase enhances lipid content and polyketide yield in yeast cells.

Malonyl-CoA is the essential building block of natural products such as fatty acids, polyketides, and flavonoids. Engineering the biosynthesis of fatty acids is important for biofuel production while that of polyketides provides precursors of medicines and nutritional supplements. However, microorganisms maintain a small amount of cellular malonyl-CoA, which could limit production of lipid and polyketides under certain conditions. Malonyl-CoA concentration is regulated by multiple pathways and signals, and changes in intracellular malonyl-CoA often lead to complex alterations in metabolism. In the present work, overexpression of a plant malonyl-CoA synthetase gene (AAE13) in Saccharomyces cerevisiae resulted in 1.6- and 2.4-fold increases in lipid and resveratrol accumulation simultaneously. We also demonstrated that AAE13 partially complemented the temperature-sensitive acc1 mutant, replacing this key enzyme in central metabolism. Mechanistic analysis by CoA quantification and transcriptomic measurement suggested that increases in malonyl-CoA concentration were coupled with drastic reductions in other major CoA compounds and clear suppression of tricarboxylic acid cycle-related genes. These results suggest that malonyl-CoA is a critical target for fatty acid and polyketide engineering and that overexpression of malonyl-CoA synthetic enzymes needs to be combined with upregulation of CoA synthesis to maintain metastasis of central metabolism.

Sulfoxide-containing polymers conjugated prodrug micelles with enhanced anticancer activity and reduced intestinal toxicity.

Poly(ethylene glycol) (PEG) is widely utilized as a hydrophilic coating to extend the circulation time and improve the tumor accumulation of polymeric micelles. Nonetheless, PEGylated micelles often activate complement proteins, leading to accelerated blood clearance and negatively impacting drug efficacy and safety. Here, we have crafted amphiphilic block copolymers that merge hydrophilic sulfoxide-containing polymers (psulfoxides) with the hydrophobic drug 7-ethyl-10-hydroxylcamptothecin (SN38) into drug-conjugate micelles. Our findings show that the specific variant, PMSEA-PSN38 micelles, remarkably reduce protein fouling, prolong blood circulation, and improve intratumoral accumulation, culminating in significantly increased anti-cancer efficacy compared with PEG-PSN38 counterpart. Additionally, PMSEA-PSN38 micelles effectively inhibit complement activation, mitigate leukocyte uptake, and attenuate hyperactivation of inflammatory cells, diminishing their ability to stimulate tumor metastasis and cause inflammation. As a result, PMSEA-PSN38 micelles show exceptional promise in the realm of anti-metastasis and significantly abate SN38-induced intestinal toxicity. This study underscores the promising role of psulfoxides as viable PEG substitutes in the design of polymeric micelles for efficacious anti-cancer drug delivery.

Neutrophil Extracellular Traps-Inhibiting and Fouling-Resistant Polysulfoxides Potently Prevent Postoperative Adhesion, Tumor Recurrence, and Metastasis.

Peritoneal metastasis (PM) is considered one of the most dreaded forms of cancer metastases for both patients and physicians. Aggressive cytoreductive surgery (CRS) is the primary treatment for peritoneal metastasis. Unfortunately, this intensive treatment frequently causes clinical complications, such as postoperative recurrence, metastasis, and adhesion formation. Emerging evidence suggests that neutrophil extracellular traps (NETs) released by inflammatory neutrophils contribute to these complications. Effective NET-targeting strategies thus show considerable potential in counteracting these complications but remain challenging. Here, one type of sulfoxide-containing homopolymer, PMeSEA, with potent fouling-resistant and NET-inhibiting capabilities, is synthesized and screened. Hydrating sulfoxide groups endow PMeSEA with superior nonfouling ability, significantly inhibiting protein/cell adhesion. Besides, the polysulfoxides can be selectively oxidized by ClO- which is required to stabilize the NETs rather than H2O2, and ClO- scavenging effectively inhibits NETs formation without disturbing redox homeostasis in tumor cells and quiescent neutrophils. As a result, PMeSEA potently prevents postoperative adhesions, significantly suppresses peritoneal metastasis, and shows synergetic antitumor activity with chemotherapeutic 5-Fluorouracil. Moreover, coupling CRS with PMeSEA potently inhibits CRS-induced tumor metastatic relapse and postoperative adhesions. Notably, PMeSEA exhibits low in vivo acute and subacute toxicities, implying significant potential for clinical postoperative adjuvant treatment.

A structural basis for the biosynthesis of the major chlorogenic acids found in coffee.

Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.

Centipeda minima: An update on its phytochemistry, pharmacology and safety.

ETHNOPHARMACOLOGICAL RELEVANCE: Centipeda minima (CM), the dried whole plant of Centipeda minima (L.) A. Braun and Aschers, has been used as a traditional Chinese medicinal herb for thousands of years for the treatments of rhinitis, sinusitis, cough and asthmatic diseases. This review aimed to evaluate the therapeutic potential of CM by summarizing its phytochemistry, pharmacology, clinical application and safety. METHODS: This review summarizes the published studies on CM in the Chinese Pharmacopoeia and literature databases including PubMed, Web of Science, Baidu Scholar, Wiley and China Knowledge Resource Integrated Database (CNKI), as well as the research articles on the phytochemistry, pharmacology, clinical application and safety of CM. RESULTS: A total of 191 compounds have been isolated and identified from CM, including terpenes, flavonoids, sterols, phenols, organic acids and volatile oils. In addition, the pharmacological effects of CM, such as anti-cancer, anti-inflammatory and anti-bacterial activities, have also been evaluated by both in vitro and in vivo studies. The signaling pathways and mechanisms of action underlying the anti-cancer effects of CM have been revealed. Clinical applications of CM mainly include rhinitis and sinusitis, gynecological inflammation, cough, as well as asthma. CONCLUSION: CM is a medicinal herb that possesses many therapeutic effects. Cutting-edge technology and system biology could provide us a more comprehensive understanding of the therapeutic effects, constituting components and toxicity of CM, which are the prerequisites for its translation into therapeutics for various disease treatments.

Discovery of a novel anti-obesity meroterpenoid agent targeted subcutaneous adipose tissue.

BACKGROUND: Meroterpenoid furanasperterpene A (T2-3) with a novel 6/6/6/6/5 pentacyclic skeleton was isolated from the Aspergillus terreus GZU-31-1. Previously, we showed that T2-3 possessed significant lipid-lowering effects in 3T3-L1 adipocytes at 5 µM concentration. However, its therapeutic effect in metabolic disease and the underlying mechanisms of action remain unclear. METHODS: High fat diet-induced obesity (DIO) mouse model and 3T3-L1 cell model were used to assess the anti-obesity effects of T2-3. Lipids in the adipocytes were examined by Oil Red O staining. ß-catenin expression was examined by immunofluorescence and Western blotting, its activity was assessed by TOPflash/FOPflash assay. RESULTS: T2-3 possessed potent anti-obesity effects in DIO mice, it significantly reduced body weight and subcutaneous adipose tissue (SAT) mass. Mechanistic studies showed that T2-3 significantly inhibited 3T3-L1 preadipocyte differentiation as indicated by the reduced number of mature adipocytes. The treatments also reduced the expressions of critical adipogenic transcription factors CEBP-α and PPAR-γ in both 3T3-L1 adipocytes and SAT in DIO mice. Interestingly, T2-3 increased the cytoplasmic and nuclear expressions of ß-catenin and the transcriptional activity of ß-catenin in 3T3-L1 adipocytes; the elevated ß-catenin expression was also observed in SAT of the T2-3-treated DIO mice. Indeed, upregulation of ß-catenin activity suppressed adipogenesis, while ß-catenin inhibitor JW67 reversed the anti-adipogenic effect of T2-3. Taken together, our data suggest that T2-3 inhibits adipogenesis by upregulating ß-catenin activity. CONCLUSIONS: Our study is the first report demonstrating meroterpenoid furanasperterpene A as a novel 6/6/6/6/5 pentacyclic skeleton (T2-3) that possesses potent anti-adipogenic effect by targeting ß-catenin signaling pathway. Our findings drive new anti-obesity drug discovery and provide drug leads for chemists and pharmacologists.

Structural and kinetic analysis of the unnatural fusion protein 4-coumaroyl-CoA ligase::stilbene synthase.

To increase the biochemical efficiency of biosynthetic systems, metabolic engineers have explored different approaches for organizing enzymes, including the generation of unnatural fusion proteins. Previous work aimed at improving the biosynthesis of resveratrol, a stilbene associated a range of health-promoting activities, in yeast used an unnatural engineered fusion protein of Arabidopsis thaliana (thale cress) 4-coumaroyl-CoA ligase (At4CL1) and Vitis vinifera (grape) stilbene synthase (VvSTS) to increase resveratrol levels 15-fold relative to yeast expressing the individual enzymes. Here we present the crystallographic and biochemical analysis of the 4CL::STS fusion protein. Determination of the X-ray crystal structure of 4CL::STS provides the first molecular view of an artificial didomain adenylation/ketosynthase fusion protein. Comparison of the steady-state kinetic properties of At4CL1, VvSTS, and 4CL::STS demonstrates that the fusion protein improves catalytic efficiency of either reaction less than 3-fold. Structural and kinetic analysis suggests that colocalization of the two enzyme active sites within 70 Å of each other provides the basis for enhanced in vivo synthesis of resveratrol.

Berry skin development in Norton grape: distinct patterns of transcriptional regulation and flavonoid biosynthesis.

BACKGROUND: The complex and dynamic changes during grape berry development have been studied in Vitis vinifera, but little is known about these processes in other Vitis species. The grape variety 'Norton', with a major portion of its genome derived from Vitis aestivalis, maintains high levels of malic acid and phenolic acids in the ripening berries in comparison with V. vinifera varieties such as Cabernet Sauvignon. Furthermore, Norton berries develop a remarkably high level of resistance to most fungal pathogens while Cabernet Sauvignon berries remain susceptible to those pathogens. The distinct characteristics of Norton and Cabernet Sauvignon merit a comprehensive analysis of transcriptional regulation and metabolite pathways. RESULTS: A microarray study was conducted on transcriptome changes of Norton berry skin during the period of 37 to 127 days after bloom, which represents berry developmental phases from herbaceous growth to full ripeness. Samples of six berry developmental stages were collected. Analysis of the microarray data revealed that a total of 3,352 probe sets exhibited significant differences at transcript levels, with two-fold changes between at least two developmental stages. Expression profiles of defense-related genes showed a dynamic modulation of nucleotide-binding site-leucine-rich repeat (NBS-LRR) resistance genes and pathogenesis-related (PR) genes during berry development. Transcript levels of PR-1 in Norton berry skin clearly increased during the ripening phase. As in other grapevines, genes of the phenylpropanoid pathway were up-regulated in Norton as the berry developed. The most noticeable was the steady increase of transcript levels of stilbene synthase genes. Transcriptional patterns of six MYB transcription factors and eleven structural genes of the flavonoid pathway and profiles of anthocyanins and proanthocyanidins (PAs) during berry skin development were analyzed comparatively in Norton and Cabernet Sauvignon. Transcriptional patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin. CONCLUSIONS: This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties.

Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering.

Resveratrol is a unique, natural polyphenolic compound with diverse health benefits. In the present study, we attempted to improve resveratrol biosynthesis in yeast by different methods of metabolic engineering. We first mutated and then re-synthesized tyrosine ammonia lyase (TAL) by replacing the bacteria codons with yeast-preferred codons, which increased translation and improved p-coumaric acid and resveratrol biosynthesis drastically. We then demonstrated that low-affinity, high-capacity bacterial araE transporter could enhance resveratrol accumulation, without transporting resveratrol directly. Yeast cells carrying the araE gene produced up to 2.44-fold higher resveratrol than control cells. For commercial applications, resveratrol biosynthesis was detected in sucrose medium and fresh grape juice using our engineered yeast cells. In collaboration with the Chaumette Winery of Missouri, we were able to produce resveratrol-containing white wines, with levels comparable to the resveratrol levels found in most red wines.

Metabolic engineering of flavonoids in plants and microorganisms.

Over 9,000 flavonoid compounds have been found in various plants, comprising one of the largest families of natural products. Flavonoids are an essential factor in plant interactions with the environment, often serving as the first line of defense against UV irradiation and pathogen attacks. Flavonoids are also major nutritional compounds in foods and beverages, with demonstrated health benefits. Some flavonoids are potent antioxidants, and specific flavonoid compounds are beneficial in many physiological and pharmacological processes. Therefore, engineering of flavonoid biosynthesis in plants or in microorganisms has significant scientific and economical importance. Construction of biosynthetic pathways in heterologous systems offers promising results for large-scale flavonoid production by fermentation or bioconversion. Genomics and metabolomics now offer unprecedented tools for detailed understanding of the engineered transgenic organism and for developing novel technologies to further increase flavonoid production yields. We summarize some of the recent metabolic engineering strategies in plants and microorganisms, with a focus on applications of metabolic flux analysis. We are confident that these engineering approaches will lead to successful industrial flavonoid production in the near future.

Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT-PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.

Development and Processing of Novel Heparin Binding Functionalized Modified Spider Silk Coating for Catheter Providing Dual Antimicrobial and Anticoagulant Properties.

Tailored surface coatings have been used for decades to improve material performance in blood. Among different approaches, heparin based biomedical coatings have found great success in the commercial catheter market. However, they have their own limitations. Coating of a vascular device with a heparin binding peptide (HBP), which can sequester the circulating heparin, presents numerous advantages over both systemic heparin therapy and direct heparin bound surfaces. Embedding HBP in a silk biopolymer provides the mechanical integrity necessary under dynamic flow conditions to both insert the catheter and maintain proper blood flow. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property, a critical and unique aspect to combat catheter related blood stream infections and extend the longevity of hemodialysis catheters. To assess this hypothesis, a recombinant fusion protein (S4H4) containing both silk amino acid motifs and HBP was assessed as a coating on a silicone surface. After validating that, the protein was deposited on the surface via XPS, Raman spectroscopy, ATR and SEM imaging, antimicrobial and anticoagulant activities were evaluated. The coating was able to prevent not only planktonic bacterial growth but also prevented the growth of a biofilm. Finally, the coating had both antibacterial and anticoagulant effect simultaneously. This study proves the successful production of a silk-based biopolymer that can be embedded with a heparin-binding functionality to create a dual functional device coating that can prevent infection and thrombosis together.

Application of Stable Isotope Tracing to Elucidate Metabolic Dynamics During Yarrowia lipolytica α-Ionone Fermentation.

Targeted metabolite analysis in combination with 13C-tracing is a convenient strategy to determine pathway activity in biological systems; however, metabolite analysis is limited by challenges in separating and detecting pathway intermediates with current chromatographic methods. Here, a hydrophilic interaction chromatography tandem mass spectrometry approach was developed for improved metabolite separation, isotopologue analysis, and quantification. The physiological responses of a Yarrowia lipolytica strain engineered to produce ∼400 mg/L α-ionone and temporal changes in metabolism were quantified (e.g., mevalonate secretion, then uptake) indicating bottleneck shifts in the engineered pathway over the course of fermentation. Dynamic labeling results indicated limited tricarboxylic acid cycle label incorporation and, combined with a measurable ATP shortage during the high ionone production phase, suggested that electron transport and oxidative phosphorylation may limit energy supply and strain performance. The results provide insights into terpenoid pathway metabolic dynamics of non-model yeasts and offer guidelines for sensor development and modular engineering.

Biosynthesis of terpene compounds using the non-model yeast Yarrowia lipolytica: grand challenges and a few perspectives.

Yarrowia lipolytica has emerged as an important non-model host for terpene production. However, three main challenges remain in industrial production using this yeast. First, considerable knowledge gaps exist in metabolic flux across multiple compartments, cofactor generation, and catabolism of non-sugar carbon sources. Second, many enzymatic steps in the complex-terpene synthesis pathway can pose rate-limitations, causing accumulation of toxic intermediates and increased metabolic burdens. Third, metabolic shifts, morphological changes, and genetic mutations are poorly characterized under industrial fermentation conditions. To overcome these challenges, systems metabolic analysis, protein engineering, novel pathway engineering, model-guided strain design, and fermentation optimization have been attempted with some successes. Further developments that address these challenges are needed to advance the Yarrowia lipolytica platform for industrial-scale production of high-value terpenes, including those with highly complex structures such as anticancer molecules withanolides and insecticidal limonoids.

Analysis of Yarrowia lipolytica growth, catabolism, and terpenoid biosynthesis during utilization of lipid-derived feedstock.

This study employs biomass growth analyses and 13C-isotope tracing to investigate lipid feedstock utilization by Yarrowia lipolytica. Compared to glucose, oil-feedstock in the minimal medium increases the yeast's biomass yields and cell sizes, but decreases its protein content (<20% of total biomass) and enzyme abundances for product synthesis. Labeling results indicate a segregated metabolic network (the glycolysis vs. the TCA cycle) during co-catabolism of sugars (glucose or glycerol) with fatty acid substrates, which facilitates resource allocations for biosynthesis without catabolite repressions. This study has also examined the performance of a ß-carotene producing strain in different growth mediums. Canola oil-containing yeast-peptone (YP) has resulted in the best ß-carotene titer (121 ±â€¯13 mg/L), two-fold higher than the glucose based YP medium. These results highlight the potential of Y. lipolytica for the valorization of waste-derived lipid feedstock.

Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense.

Gossypol, a type of plant defence sesquiterpenoid phytoalexin, is synthesized from the MEP (2C-methyl-D-erythritol 4-phosphate) and MVA (mevalonate) pathway in the isoprenoid biosynthetic system. The key step is the isomerization of IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate), which is catalysed by IPI (IPP isomerase; EC 5.3.3.2). A full-length cDNA encoding IPI (designated GbIPI) was cloned from Gossypium barbadense by RACE (rapid amplification of cDNA ends). The full-length cDNA of GbIPI was 1205 bp and contained a 906 bp ORF (open reading frame) encoding a protein of 302 amino acids, with a predicted molecular mass of 34.39 kDa and an isoelectric point of 6.07. Amino acid sequence analysis revealed that the GbIPI has a high level of similarity to other IPIs. Southern-blot analysis revealed that GbIPI belongs to a small gene family. Expression analysis indicated that GbIPI expression is highest in stems, followed by leaves, and is lowest in roots, and that the expression of GbIPI could be induced by Verticillium dahliae Kleb, MeJA (methyl jasmonate) and SA (salicylic acid). The functional colour assay indicated that GbIPI could accelerate the accumulation of beta-carotene in Escherichia coli transformants. The cloning and functional analysis of GbIPI will be useful in increasing understanding of the role of IPI in isoprenoid biosynthesis at the molecular level.