Concomitant Production of Erythritol and ß-Carotene by Engineered Yarrowia lipolytica.

While the expansion of the erythritol production industry has resulted in unprecedented production of yeast cells, it also suffers from a lack of effective utilization. ß-Carotene is a value-added compound that can be synthesized by engineered Yarrowia lipolytica. Here, we first evaluated the production performance of erythritol-producing yeast strains under two different morphologies and then successfully constructed a chassis with yeast-like morphology by deleting Mhy1 and Cla4 genes. Subsequently, ß-carotene synthesis pathway genes, CarRA and CarB from Blakeslea trispora, were introduced to construct the ß-carotene and erythritol coproducing Y. lipolytica strain ylmcc. The rate-limiting genes GGS1 and tHMG1 were overexpressed to increase the ß-carotene yield by 45.32-fold compared with the strain ylmcc. However, increased ß-carotene accumulation led to prolonged fermentation time; therefore, transporter engineering through overexpression of YTH1 and YTH3 genes was used to alleviate fermentation delays. Using batch fermentation in a 3 L bioreactor, this engineered Y. lipolytica strain produced erythritol with production, yield, and productivity values of 171 g/L, 0.56 g/g glucose, and 2.38 g/(L·h), respectively, with a concomitant ß-carotene yield of 47.36 ± 0.06 mg/g DCW. The approach presented here improves the value of erythritol-producing cells and offers a low-cost technique to obtain hydrophobic terpenoids.

Membrane-bound sorbitol dehydrogenase is responsible for the unique oxidation of D-galactitol to L-xylo-3-hexulose and D-tagatose in Gluconobacter oxydans.

BACKGROUND: Gluconobacter oxydans, is used in biotechnology because of its ability to oxidize a wide variety of carbohydrates, alcohols, and polyols in a stereo- and regio-selective manner by membrane-bound dehydrogenases located in periplasmic space. These reactions obey the well-known Bertrand-Hudson's rule. In our previous study (BBA-General Subjects, 2021, 1865:129740), we discovered that Gluconobacter species, including G. oxydans and G. cerinus strain can regio-selectively oxidize the C-3 and C-5 hydroxyl groups of D-galactitol to rare sugars D-tagatose and L-xylo-3-hexulose, which represents an exception to Bertrand Hudson's rule. The enzyme catalyzing this reaction is located in periplasmic space or membrane-bound and is PQQ (pyrroloquinoline quinine) and Ca2+-dependent; we were encouraged to determine which type of enzyme(s) catalyze this unique reaction. METHODS: Enzyme was identified by complementation of multi-deletion strain of Gluconobacter oxydans 621H with all putative membrane-bound dehydrogenase genes. RESULTS AND CONCLUSIONS: In this study, we identified this gene encoding the membrane-bound PQQ-dependent dehydrogenase that catalyzes the unique galactitol oxidation reaction in its 3'-OH and 5'-OH. Complement experiments in multi-deletion G. oxydans BP.9 strains established that the enzyme mSLDH (encoded by GOX0855-0854, sldB-sldA) is responsible for galactitol's unique oxidation reaction. Additionally, we demonstrated that the small subunit SldB of mSLDH was membrane-bound and served as an anchor protein by fusing it to a red fluorescent protein (mRubby), and heterologously expressed in E. coli and the yeast Yarrowia lipolytica. The SldB subunit was required to maintain the holo-enzymatic activity that catalyzes the conversion of D-galactitol to L-xylo-3-hexulose and D-tagatose. The large subunit SldA encoded by GOX0854 was also characterized, and it was discovered that its 24 amino acids signal peptide is required for the dehydrogenation activity of the mSLDH protein. GENERAL SIGNIFICANCE: In this study, the main membrane-bound polyol dehydrogenase mSLDH in G. oxydans 621H was proved to catalyze the unique galactitol oxidation, which represents an exception to the Bertrand Hudson's rule, and broadens its substrate ranges of mSLDH. Further deciphering the explicit enzymatic mechanism will prove this theory.

Proteomics and phosphoproteomics analysis of tissues for the reoccurrence prediction of colorectal cancer.

BACKGROUND: Many stage II/III colorectal cancer (CRC) patients may relapse after routine treatments. Aberrant phosphorylation can regulate pathophysiological processes of tumors, and finding characteristic protein phosphorylation is an efficient approach for the prediction of CRC relapse. RESEARCH DESIGN AND METHODS: We compared the tissue proteome and phosphoproteome of stage II/III CRC patients between the relapsed group (n = 5) and the non-relapsed group (n = 5). Phosphopeptides were enriched with Ti4+-IMAC material. We utilized label-free quantification-based proteomics to screen differentially expressed proteins and phosphopeptides between the two groups. Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA) were used for bioinformatics analysis. RESULTS: The immune response of the relapsed group (Z-score -2.229) was relatively poorer than that of the non-relapsed group (Z-score 1.982), while viability of tumor was more activated (Z-score 2.895) in the relapsed group, which might cause increased relapse risk. The phosphorylation degrees of three phosphosites (phosphosite 1362 of TP53BP1, phosphosite 809 of VCL and phosphosite 438 of STK10) might be reliable prognostic biomarkers. CONCLUSIONS: Some promising proteins and phosphopeptides were discovered to predict the relapse risk in postoperative follow-ups.

Bioprospecting Kluyveromyces marxianus as a Robust Host for Industrial Biotechnology.

Kluyveromyces marxianus is an emerging non-conventional food-grade yeast that is generally isolated from diverse habitats, like kefir grain, fermented dairy products, sugar industry sewage, plants, and sisal leaves. A unique set of beneficial traits, such as fastest growth, thermotolerance, and broad substrate spectrum (i.e., hemi-cellulose hydrolysates, xylose, l-arabinose, d-mannose, galactose, maltose, sugar syrup molasses, cellobiose, and dairy industry) makes this yeast a particularly attractive host for applications in a variety of food and biotechnology industries. In contrast to Saccharomyces cerevisiae, most of the K. marxianus strains are apparently Crabtree-negative or having aerobic-respiring characteristics, and unlikely to endure aerobic alcoholic fermentation. This is a desirable phenotype for the large-scale biosynthesis of products associated with biomass formation because the formation of ethanol as an undesirable byproduct can be evaded under aerobic conditions. Herein, we discuss the current insight into the potential applications of K. marxianus as a robust yeast cell factory to produce various industrially pertinent enzymes, bioethanol, cell proteins, probiotic, fructose, and fructo-oligosaccharides, and vaccines, with excellent natural features. Moreover, the biotechnological improvement and development of new biotechnological tools, particularly CRISPR-Cas9-assisted precise genome editing in K. marxianus are delineated. Lastly, the ongoing challenges, concluding remarks, and future prospects for expanding the scope of K. marxianus utilization in modern biotechnology, food, feed, and pharmaceutical industries are also thoroughly vetted. In conclusion, it is critical to apprehend knowledge gaps around genes, metabolic pathways, key enzymes, and regulation for gaining a complete insight into the mechanism for producing relevant metabolites by K. marxianus.

Bioprospecting and biotechnological insights into sweet-tasting proteins by microbial hosts-a review.

Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar consumption practices, worldwide. Artificial sweeteners are considered potential substitutes in several products, e.g., sugar alcohols (polyols), high-fructose corn syrup, powdered drink mixes, and other beverages. Nevertheless, their long-standing health effects continue to be debatable. Consequently, growing interest has been shifted in producing non-caloric sweetenersfrom renewable resources to meet consumers' dietary requirements. Except for the lysozyme protein, various sweet proteins including thaumatin, mabinlin, brazzein, monellin, miraculin, pentadin, and curculin have been identified in tropical plants. Given the high cost and challenging extortion of natural resources, producing these sweet proteins using engineered microbial hosts, such as Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis represents an appealing choice. Engineering techniques can be applied for large-scale biosynthesis of proteins, which can be used in biopharmaceutical, food, diagnostic, and medicine industries. Nevertheless, extensive work needs to be undertaken to address technical challenges in microbial production of sweet-tasting proteins in bulk. This review spotlights historical aspects, physicochemical properties (taste, safety, stability, solubility, and cost), and recombinant biosynthesis of sweet proteins. Moreover, future opportunities for process improvement based on metabolic engineering strategies are also discussed.

Proteomics analysis of tissue small extracellular vesicles reveals protein panels for the reoccurrence prediction of colorectal cancer.

Many stage II/III colorectal cancer (CRC) patients might relapse after routine treatment and there is a great need of reliable biomarkers for predicting its reoccurrence risk. Small extracellular vesicles (sEVs) could regulate many pathophysiological processes of diseases, which are promising source for biomarker discovery. In this study, we implemented a MS-based workflow that utilizes data-dependent acquisition (DDA) for discovery and parallel reaction monitoring (PRM) for validation of high relapse risk related biomarkers. We compared the protein profiling of sEVs from CRC tissues and paired adjacent tissues in relapsed group (n = 5) and non-relapsed group (n = 5). 417 and 1140 proteins were differentially expressed between the tumor tissues and adjacent tissues in relapsed group and non-relapsed group, respectively. Bioinformatics analysis showed that immunity of the relapsed patients (Z-score - 0.69) was relatively poorer than the non-relapsed patients (Z-score 2.59), while chronic inflammatory response was activated (Z-score 3.0), which might enhance the reoccurrence risk. Four proteins (HLA-DPA1, S100P, NUP205, PCNA) showed significant expressions in the adjacent tissues of the relapsed group by PRM validation. ROC analysis of HLA-DPA1 (AUC = 0.96) achieved the best classification accuracy in separating the relapsed group and the non-relapsed group. Our data demonstrate that tissue-derived sEVs harbor prognostic proteomic signatures of CRC. SIGNIFICANCE: In this research, our proteomics analysis of tissue sEVs revealed that poor immunity as well as chronic inflammatory of the CRC relapsed patient likely lead to poor prognosis and high risk of reoccurrence. The significant expression levels of four proteins (HLA-DPA1, S100P, NUP205, PCNA) in the adjacent tissues of the relapsed group might be used to predict the risk of relapse in postoperative follow-ups.

Comprehensive Map of the Artemisia annua Proteome and Quantification of Differential Protein Expression in Chemotypes Producing High versus Low Content of Artemisinin.

Artemisia annua is well known for biosynthesizing the antimalarial drug artemisinin. Here, a global proteomic profiling of A. annua is conducted with identification of a total of 13 403 proteins based on the genome sequence annotation database. Furthermore, a spectral library is generated to perform quantitative proteomic analysis using data independent acquisition mass spectrometry. Specifically, proteins between two chemotypes that produce high (HAP) and low (LAP) artemisinin content, respectively, are comprehensively quantified and compared. 182 proteins are identified with abundance significantly different between these two chemotypes means after the statistic use the p-value and fold change it is found 182 proteins can reach the demand conditions which represent the expression are significantly different between the high artemisnin content plants (HAPs) and the low artemisnin content plants (LAPs). Data are available via ProteomeXchange with identifier PXD015547. Overall, this current study globally identifies the proteome of A. annua and quantitatively compares the targeted sub-proteomes between the two cultivars of HAP and LAP, providing systematic information on metabolic pathways of A. annua.

Affinity chromatography assisted comprehensive phosphoproteomics analysis of human saliva for lung cancer.

Affinity chromatography is a powerful technology for phosphopeptide enrichment from body fluids. Saliva is a non-invasive body fluid for disease diagnosis, while few studies applied affinity enrichment for saliva phosphoproteome. In this study, we tested two kinds of affinity chromatography materials, Ti4+-IMAC (immobilized metal affinity chromatography) and CaTiO3, for the enrichment of phosphopeptides. Through comparison, Ti4+-IMAC method was demonstrated as the superior one, which was utilized for the comprehensive analysis of salivary phosphoproteome. More than 360 phosphoproteins were specifically extracted and identified from human saliva. Ti4+-IMAC method was further applied to compare the phosphoprotein profiling in the saliva of lung cancer group and normal control group through label-free quantification. Accordingly, 477 and 699 phosphopeptides were enriched, respectively, which corresponded to 339 and 466 proteins. In total, 796 unique phosphopeptides were revealed for 517 saliva phosphoproteins. In particular, 709 phosphorylation sites were identified, among which 26 were up-regulated (>1.5) and 149 were down-regulated (<0.66) in lung cancer. Their corresponding proteins were mainly associated with cancer promotion, system disorder, and organismal injury. Our data collectively demonstrated that salivary phosphopeptides can be comprehensively characterized through Ti4+-IMAC method. These discovered phosphoprotein candidates might be used for lung cancer detection through salivary diagnostics.

Comparative Proteomic Analysis of Exosomes and Microvesicles in Human Saliva for Lung Cancer.

Extracellular vesicles (EVs) are cell-derived microparticles present in most body fluids, mainly including microvesicles and exosomes. EV-harbored proteins have emerged as novel biomarkers for the diagnosis and prediction of different cancers. We successfully isolated microvesicles and exosomes from human saliva, which were further characterized comprehensively. Salivary EV protein profiling in normal subjects and lung cancer patients was systematically compared through utilizing LC-MS/MS-based label-free quantification. 785 and 910 proteins were identified from salivary exosomes and microvesicles, respectively. According to statistical analysis, 150 and 243 proteins were revealed as dysregulated candidates in exosomes and microvesicles for lung cancer. Among them, 25 and 40 proteins originally from distal organ cells were found in the salivary exosomes and microvesicles of lung cancer patients. In particular, 5 out of 25 and 9 out of 40 are lung-related proteins. Six potential candidates were selected for verification by Western blot, and four of them, namely, BPIFA1, CRNN, MUC5B, and IQGAP, were confirmed either in salivary microvesicles or in exosomes. Our data collectively demonstrate that salivary EVs harbor informative proteins that might be used for the detection of lung cancer through a noninvasive way.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.

Novel ruthenium complexes ligated with 4-anilinoquinazoline derivatives: synthesis, characterisation and preliminary evaluation of biological activity.

The ruthenium DMSO complexes cis-Ru(II)C12(DMSO)4 and [(DMSO)2H][trans-Ru(III)Cl4(DMSO)2] reacted with 4-(3'-chloro-4'-fluoroanilino)-6-(2-(2-aminoethyl)aminoethoxy)-7-methoxyquinazoline (L1), 4-(3'-chloro-4'-fluoroanilino)-6-(2-(1H-imidazol-1-yl)ethoxy)-7-methoxy quinazoline (L2), N-(benzo[d]imidazol-4-yl)-6,7-dimethoxyquinazolin-4-amine hydrochloride (L3), 5-(6,7-dimethoxyquinazolin-4-ylamino)quinolin-8-ol hydrochloride (L4), respectively, to afford [Ru(II)Cl2(DMSO)2(L1)] (1), [Ru(III)Cl3(DMSO)(L1)] (2), [Ru(III)Cl4(DMSO)(H-L2)] (3), [Ru(III)Cl4(DMSO)(H-L3)] (4), and [Ru(III)Cl3(DMSO)(H-L4)] (5), which were characterised by mass spectrometry, NMR, elementary analysis and single crystal X-ray diffraction (complex 1). Experimental screening (ELISA) showed that complexes 1, 2 and 3 are remarkably inhibitory towards epidermal growth factor receptor (EGFR) with IC50 values at submicromolar or nanomolar level. Docking studies indicated that complexation with ruthenium has little interference with the formation of the two essential H-bonds between the N3 of the quinazoline ring in L1 and L2 and O-H of Thr766 through a water molecule, and the N1 of the quinazoline ring and N-H of Met769 in EGFR. Moreover, complex 2 was shown to be more active against the EGF-stimulated proliferation of human breast cancer cell line MCF-7 than the better EGFR inhibitor 4-(3'-chloro-4'-fluoroanilino)-6,7-dimethoxyquinazoline, being more potential to induce early-stage apoptosis than gefitinib. These imply that apart from inhibiting EGFR, complex 2 may involve in regulating other biological events related to the proliferation of MCF-7, implicating a novel type of multi-targeting metal-based anticancer agents.

Complexation with organometallic ruthenium pharmacophores enhances the ability of 4-anilinoquinazolines inducing apoptosis.

The complexation with organoruthenium fragments confers 4-anilinoquinazoline pharmacophores with higher potential for inducing cellular apoptosis while the highly inhibitory activity of 4-anilinoquinazolines against EGFR and the reactivity of the ruthenium centre to 9-ethylguanine are well preserved.

Development of an improved three-dimensional in vitro intestinal mucosa model for drug absorption evaluation.

Human epithelial cell culture models of monolayer Caco-2 cells have been widely employed to assess the absorption of drug molecules across intestinal mucosa. However, cautions should be taken when interpreting the conclusions from those models due to their undesirable phenotype and functionality when compared with the native intestinal tissue. In the present study, an improved, more physiologically relevant three-dimensional (3D) culture model of the intestinal mucosa was developed to study drug absorption, in which a coculture of epithelial cells, including Caco-2 cells and HT29-methotrexate cells, was indirectly seeded on a Transwell filter insert with collagen gel and stromal cells (fibroblasts and immunocytes) incorporation. This setting-up provided a compatible environment to improve the phenotype and functionality of the epithelial cells. Compared with the monolayer culture of Caco-2 cells, the reconstructed 3D model displayed more physiologically relevant characteristics evidenced by its decreased TEER value and mucus-like layer formation. A decreased expression of P-gp and an increased expression of BCRP were also observed in the current 3D culture model, leading to a changed secretory permeability of their substrates. More importantly, an improved correlation (R(2)=0.843) was obtained between the absorptive permeability across the 3D coculture model and the human absorption fraction especially for those model compounds with moderate or high permeability. Thus, this engineered 3D coculture model presents a unique, improved opportunity to evaluate drug permeability in vitro.

Synthesis, characterization, screening and docking analysis of 4-anilinoquinazoline derivatives as tyrosine kinase inhibitors.

We report here the design and synthesis of a series of 4-anilinoquinazoline derivatives, of which 7 compounds were crystallographically characterized, as epidermal growth factor receptor (EGFR) inhibitors by modifications on the aniline ring or at the 6-alkoxy site of the 6,7-dimethoxy-4-anilinoquinazoline pharmacophore. The relative inhibition efficiency on EGFR of all as-prepared compounds were measured and ordered, and the IC50 values of nine highly active compounds were determined by ELISA. Docking studies indicated that all 4-anilinoquinazoline derivatives could be inserted into the ATP-binding pocket of the EGFR via indirect docking, and that the modifications at the 3'-position of the anilino group and 6-alkoxy site of the quinazoline ring have little interference with the formation of the two essential H-bonds between the N3 of the quinazoline ring and Thr766 through a water molecule, and the N1 of the quinazoline ring and N-H of Met769. The displacing of the phenyl at 4-position with pyridinyl dramatically reduces the activity of the quinazoline pharmacophore, the resulting derivative (10) being the least active compound. The docking results also showed that the formation of new H-bonds between the N-H of the ethylenediamine group linked to the 6-alkoxy site and Asp776/Cys773 in the binding pocket of EGFR makes compounds 19 (IC50=12.1±1.6 nM) and 20 (IC50=13.6±0.8 nM) the most potent EGFR inhibitors in this class and worthy of further modification to obtain more potent anticancer compounds.

Quantitative mass spectrometry combined with separation and enrichment of phosphopeptides by titania coated magnetic mesoporous silica microspheres for screening of protein kinase inhibitors.

We describe herein the development of a matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) approach for screening of protein kinase inhibitors (PKIs). MS quantification of phosphopeptides, the kinase-catalyzed products of nonphosphorylated substrates, is a great challenge due to the ion suppression effect of highly abundant nonphosphorylated peptides in enzymatic reaction mixtures. To address this issue, a novel type of titania coated magnetic hollow mesoporous silica spheres (TiO(2)/MHMSS) material was fabricated for capturing phosphopeptides from the enzymatic reaction mixtures prior to MS analysis. Under optimized conditions, even in the presence of 1000-fold of a substrate peptide of tyrosine kinase epidermal growth factor receptor (EGFR), the phosphorylated substrates at the femtomole level can be detected with high accuracy and reproducibility. With a synthetic nonisotopic labeled phosphopeptide, of which the sequence is similar to that of the phosphorylated substrate, as the internal standard, the MS signal ratio of the phosphorylated substrate to the standard is linearly correlated with the molar ratio of the two phosphopeptides in peptide mixtures over the range of 0.1 to 4 with r(2) being 0.99. The IC(50) values of three EGFR inhibitors synthesized in our laboratory were then determined, and the results are consistent with those determined by an enzyme-linked immunosorbent assay (ELISA). The developed method is sensitive, cost/time-effective, and operationally simple and does not require isotope/radioative-labeling, providing an ideal alterative for screening of PKIs as therapeutic agents.

Synthesis and docking study of 2-phenylaminopyrimidine Abl tyrosine kinase inhibitors.

Six analogs of imatinib, an Abl kinase inhibitor clinically used as a first-line therapeutic agent for chronic myeloid leukaemia (CML), have been synthesized and characterized. And their potency as Abl kinase inhibitors have been screened by a robust virtual screening method developed based on the crystal structure (PDB code 2hyy) of Abl-imatinib complex using Surflex-Docking. The docking results are consistent with the inhibitory potency of the compounds characterized by MS method. And the H-bonds between imatinib analogs and Thr315 and Met318 residues in Abl kinase are shown to be crucial for achieving accurate poses and high binding affinities for the ATP-competitive kinase inhibitors.