Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor.

Carbonyl reductases are useful for producing optically active alcohols from their corresponding prochiral ketones. Herein, we applied a computer-assisted strategy to increase the thermostability of a previously constructed carbonyl reductase, LsCRM4 (N101D/A117G/F147L/E145A), which showed an outstanding activity in the synthesis of the ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol. The stability changes introduced by mutations at the flexible sites were predicted using the computational tools FoldX, I-Mutant 3.0, and DeepDDG, which demonstrated that 12 virtually screened mutants could be thermally stable; 11 of these mutants exhibited increased thermostability. Then a superior mutant LsCRM4-V99L/D150F was screened out from the library that was constructed by iteratively combining the beneficial sites, which showed a 78% increase in activity and a 17.4°C increase in melting temperature compared to LsCRM4. Our computer-assisted design and combinatorial strategy dramatically increased the efficiency of thermostable enzyme production.

Coevolving stability and activity of LsCR by a single point mutation and constructing neat substrate bioreaction system.

Carbonyl reductase (CR)-catalyzed bioreduction in the organic phase and the neat substrate reaction system is a lasting challenge, placing higher requirements on the performance of enzymes. Protein engineering is an effective method to enhance the properties of enzymes for industrial applications. In the present work, a single point mutation E145A on our previously constructed CR mutant LsCRM3 , coevolved thermostability, and activity. Compared with LsCRM3 , the catalytic efficiency kcat /KM of LsCRM3 -E145A (LsCRM4 ) was increased from 6.6 to 21.9 s-1 mM-1 . Moreover, E145A prolonged the half-life t1/2 at 40°C from 4.1 to 117 h, T m ${T}_{m}$ was increased by 5°C, T 50 30 ${T}_{50}^{30}$ was increased by 14.6°C, and Topt was increased by 15°C. Only 1 g/L of lyophilized Escherichia coli cells expressing LsCRM4 completely reduced up to 600 g/L 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) within 13 h at 45°C, yielding the corresponding (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol ((S)-CFPL) in 99.5% eeP , with a space-time yield of 1.0 kg/L d, the substrate to catalyst ratios (S/C) of 600 g/g. Compared with LsCRM3 , the substrate loading was increased by 50%, with the S/C increased by 14 times. Compared with LsCRWT , the substrate loading was increased by 6.5 times. In contrast, LsCRM4 completely converted 600 g/L CFPO within 12 h in the neat substrate bioreaction system.

Improvement of S-adenosyl-L-methionine production in Saccharomyces cerevisiae by atmospheric and room temperature plasma-ultraviolet compound mutagenesis and droplet microfluidic adaptive evolution.

To improve S-Adenosyl-L-methionine (a compound with important physiological functions, SAM) production, atmospheric and room temperature plasma and ultraviolet-LiCl mutagenesis were carried out with Saccharomyces cerevisiae strain ZY 1-5. The mutants were screened with ethionine, L-methionine, nystatin and cordycepin as screening agents. Adaptive evolution of a positive mutant UV6-69 was further performed by droplet microfluidics cultivation with ethionine as screening pressure. After adaptation, mutant T11-1 was obtained. Its SAM titer in shake flask fermentation reached 1.31 g/L, which was 191% higher than that of strain ZY 1-5. Under optimal conditions, the SAM titer and biomass of mutant T11-1 in 5 L bioreactor reached 10.72 g/L and 105.9 g dcw/L (142.86% and 34.22% higher than those of strain ZY 1-5), respectively. Comparative transcriptome analysis between strain ZY 1-5 and mutant T11-1 revealed the enhancements in TCA cycle and gluconeogenesis/glycolysis pathways as well as the inhibitions in serine and ergosterol synthesis of mutant T11-1. The elevated SAM synthesis of mutant T11-1 may attribute to the above changes. Taken together, this study is helpful for industrial production of SAM.

Directed evolution of a carbonyl reductase LsCR for the enantioselective synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol.

Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase LsCR for the synthesis of (1S)-2-chloro-1-(3,4-difluorophenyl) ethanol ((S)-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a "small and smart" mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant LsCRM3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) LsCR, the catalytic activity of LsCRM3 was increased by 4.7 times, the catalytic efficiency kcat/KM value was increased by 2.9 times, and the half-life t1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% (v/v) isopropanol, LsCRM3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e.) in 11 h with a space-time yield (STY) as high as 809 g/L∙d.

[Biosynthesis and regulatory mechanism of acarbose and its structural analogs: a review].

Acarbose is widely used as α-glucosidase inhibitor in the treatment of type Ⅱ diabetes. Actinoplanes sp. is used for industrial production of acarbose. As a secondary metabolite, the biosynthesis of acarbose is quite complex. In addition to acarbose, a few acarbose structural analogs are also accumulated in the culture broth of Actinoplanes sp., which are hard to remove. Due to lack of systemic understanding of the biosynthesis and regulation mechanisms of acarbose and its structural analogs, it is difficult to eliminate or reduce the biosynthesis of the structural analogs. Recently, the advances in omics technologies and molecular biology have facilitated the investigations of biosynthesis and regulatory mechanisms of acarbose and its structural analogs in Actinoplanes sp.. The genes involved in the biosynthesis of acarbose and its structural analogs and their regulatory mechanism have been extensively explored by using bioinformatics analysis, genetic manipulation and enzymatic characterization, which is summarized in this review.

Gene Cascade Shift and Pathway Enrichment in Rat Kidney Induced by Acarbose Through Comparative Analysis.

Acarbose is an effective anti-diabetic drug to treat type 2 diabetes mellitus (T2DM), a chronic degenerative metabolic disease caused by insulin resistance. The beneficial effects of acarbose on blood sugar control in T2DM patients have been confirmed by many studies. However, the effect of acarbose on patient kidney has yet to be fully elucidated. In this study, we report in detail the gene expression cascade shift, pathway and module enrichment, and interrelation network in acarbose-treated Rattus norvegicus kidneys based on the in-depth analysis of the GSE59913 microarray dataset. The significantly differentially expressed genes (DEGs) in the kidneys of acarbose-treated rats were initially screened out by comparative analysis. The enriched pathways for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were further identified. The protein-protein interaction (PPI) analysis for DEGs was achieved through the STRING database mining. Pathway interrelation and hub genes for enriched pathways were further examined to uncover key biological effects of acarbose. Results revealed 44 significantly up-regulated genes and 86 significantly down-regulated genes (130 significant differential genes in total) in acarbose-treated rat kidneys. Lipid metabolism pathways were considerably improved by acarbose, and the physical conditions in chronic kidney disease (CKD) patients were improved possibly through the increase of the level of high-density lipoprotein (HDL) by lecithin-cholesterol acyl-transferase (LCAT). These findings suggested that acarbose may serve as an ideal drug for CKD patients, since it not only protects the kidney, but also may relieve the complications caused by CKD.

Comparative proteome analysis of Actinoplanes utahensis grown on various saccharides based on 2D-DIGE and MALDI-TOF/TOF-MS.

Comparative proteomes of Actinoplanes utahensis ZJB-03852 grown on various saccharides (glucose, maltotriose, maltose, glucose + maltose) were analyzed using 2D-DIGE and MALDI-TOF/TOF-MS. Acarbose was detected in all groups except in the glucose only culture. The abundance of acarbose synthesis proteins AcbV, AcbK, AcbL and AcbN was highest in the medium containing mixed glucose and maltose. The accumulation of Zwf and Xpk1 in acarbose-producing media indicated that the cyclitol moiety of acarbose was derived from pentose phosphate pathway. The elevation of GlnA supported that glutamine was a good nitrogen source of the nitrogen-atom in acarbose synthesis. SIGNIFICANCE: Non-insulin-dependent diabetes mellitus, also known as Type II diabetes, constitutes >90% of the diabetes mellitus worldwide. Acarbose is clinically utilized to treat Type II diabetes, but the fermentation process of acarbose-producing Actinoplanes is usually accompanied with structural analogues of acarbose. In this study, we compared the proteomics of Actinoplanes utahensis ZJB-03852 grown on various saccharides by 2D-DIGE and MALDI-TOF/TOF-MS. Our findings highlighted the importance of key proteins in the formation of acarbose and its analogues when A. utahensis was cultivated in various saccharides. These results revealed fundamental data to elucidate the complexity of formation of acarbose analogues.

One-step eantioselective bioresolution for (S)-2-chlorophenylglycine methyl ester catalyzed by the immobilized Protease 6SD on multi-walled carbon nanotubes in a triphasic system.

(S)-2-chlorophenylglycine methyl ester ((S)-1) is a key chiral building block of clopidogrel, which is a widely administered antiaggregatory and antithrombotic drug. Herein, Protease 6SD was covalently immobilized on multi-walled carbon nanotubes (MWCNT), and the as-prepared immobilizate P-6SD@NH2-MWCNT was applied in the enantioselective resolution of (R,S)-1 to yield (S)-1. In order to overcome the poor solubility of (R,S)-1 in aqueous solution, a novel triphasic reaction system constituting P-6SD@NH2-MWCNT, aqueous phase and methyl tert-butyl ether (MTBE) as the organic phase was constructed, which simultaneously improved the substrate solubility and the immobilizate recyclability. Under the optimized reaction conditions, P-6SD@NH2-MWCNT catalyzed 10 mM (R,S)-1 for 2 h, yielding optically pure (S)-1 (>99.0 % ees) with 70.74 % conversion of the (R,S)-1. Moreover, P-6SD@NH2-MWCNT can be reused for 15 batches, displaying an exquisite recycling performance. It is for the first time that enantiomerically pure (S)-1 was successfully synthesized by protease-catalyzed one-step resolution.

Transcriptome analysis of Actinoplanes utahensis reveals molecular signature of saccharide impact on acarbose biosynthesis.

Different carbon sources lead to differential acarbose production in Actinoplanes. To uncover the underlying differentiation in the context of genes and pathways, we performed transcriptome sequencing of Actinoplanes utahensis ZJB-03852 grown on different saccharides, such as glucose, maltose, or the saccharide complex consisting of glucose plus maltose. The differentially expressed genes were classified into GO (gene ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways for functional annotations. Key enriched modules were uncovered. Our data revealed that both maltose and its complex with glucose gave improved acarbose titer. Sugar transportation, cytochrome oxidase, protein synthesis and amino acid metabolism modules were enriched under the saccharide complex condition, while ferritin metabolism gene expressions were enriched in the glucose medium. Our results provided the foundation for uncovering the mechanism of carbon source on acarbose production in A. utahensis.

Efficient degradation of ivermectin by newly isolated Aeromonas taiwanensis ZJB-18,044.

Ivermectin (IVM) is a widely used antiparasitic agent and acaricide. Despite its high efficiency against nematodes and arthropods, IVM may pose a threat to the environment due to its ecotoxcity. In this study, degradation of IVM by a newly isolated bacterium Aeromonas taiwanensis ZJB-18,044 was investigated. Strain ZJB-18,044 can completely degrade 50 mg/L IVM in 5 d with a biodegradation ability of 0.42 mg/L/h. Meanwhile, it exhibited high tolerance (50 mg/L) to doramectin, emamectin, rifampicin, and spiramycin. It can also efficiently degrade doramectin, emamectin, and spiramycin. The IVM degradation of strain ZJB-18,044 can be inhibited by erythromycin, azithromycin, spiramycin or rifampicin. However, supplement of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, can partially recover the IVM degradation. Moreover, strain ZJB-18,044 cells can pump out excess IVM to maintain a low intracellular IVM concentration. Therefore, the IVM tolerance of strain ZJB-18,044 may be due to the regulation of the intracellular IVM concentration by the activated macrolide efflux pump(s). With the high IVM degradation efficiency, A. taiwanensis ZJB-18,044 may serve as a bioremediation agent for IVM and other macrolides in the environment.

Genome sequencing and analysis of fungus Hirsutella sinensis isolated from Ophiocordyceps sinensis.

Ophiocordyceps sinensis has been used as a traditional medicine or healthy food in China for thousands of years. Hirsutella sinensis was reported as the only correct anamorph of O. sinensis. It is reported that the laboratory-grown H. sinensis mycelium has similar clinical efficacy and less associated toxicity compared to the wild O. sinensis. The research of the H. sinensis is becoming more and more important and urgent. To gain deeper insight into the biological and pharmacological mechanisms, we sequenced the genome of H. sinensis. The genome of H. sinensis (102.72 Mb) was obtained for the first time, with > 99% coverage. 10,200 protein-encoding genes were predicted based on the genome sequence. A detailed secondary metabolism analysis and structure verification of the main ingredients were performed, and the biosynthesis pathways of seven ingredients (mannitol, cordycepin, purine nucleotides, pyrimidine nucleotides, unsaturated fatty acid, cordyceps polysaccharide and sphingolipid) were predicted and drawn. Furthermore, infection process and mechanism of H. sinensis were studied and elaborated in this article. The enzymes involved in the infection mechanism were also predicted, cloned and expressed to verify the mechanism. The genes and proteins were predicted and annotated based on the genome sequence. The pathways of several active components in H. sinensis were predicted and key enzymes were confirmed. The work presented here would improve the understanding of the genetic basis of this organism, and contribute to further research, production and application of H. sinensis.

Integrated bioinformatics analyses identified SCL3-induced regulatory network in Arabidopsis thaliana roots.

OBJECTIVES: To uncover key genes and pathways regulated by SCL3, a GRAS transcription factor, in the context of gibberellin (GA) in the roots of the model plant Arabidopsis thaliana. RESULTS: Gene expression profiles of ga1-3 mutant and ga1-3 and scl3 double mutant are considerably similar to each other, revealed by Principal Component Analysis (PCA). More than 400 significantly Differentially Expressed Genes (DEGs) among the Arabidopsis thaliana roots of ga1-3 mutant, ga1-3 and scl3 double mutant and GA loss/SCL3 gain mutant were uncovered by comprehensive bioinformatics analyses. Protein synthesis pathway, including RPL proteins, RPS proteins, etc., and flavonoid biosynthesis pathway, including TT4, F3H, TT5, CHIL, etc. were significantly increased when SCL3 expression was higher than normal by means of pathway enrichment analysis and protein-protein interaction analysis, which is further supported by comparison analyses between wild type samples and SCL3 overexpressed roots. CONCLUSION: Protein synthesis and flavonoid biosynthesis were regulated by SCL3 in the context of GA in Arabidopsis thaliana root system identified by comprehensive bioinformatic analyses.

Integrated analysis of gene expression, alteration and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 1 in cancer.

Even though cell-cell adhesion molecule carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is extensively studied since the discovery, the role of CEACAM1 in different cancers is not completely clarified. In the present study, we examined CEACAM1 expression and its association with patient survival in various cancers by analysis of multiple databases. Oncomine database analysis revealed that CEACAM1 expression was upregulated in lung and pancreatic cancers, but downregulated in colorectal and head and neck cancers. PrognoScan and Kaplan­Meier analyses showed that colorectal cancer patients as well as head and neck cancer patients with high CEACAM1 expression exhibited a higher overall survival rate. STRING analysis identified CEACAM3, CEACAM8, FN1, etc. as CEACAM1 interactors. Gene alteration analysis showed that CEACAM1 mutation predominantly occurred in the N-terminal. Coexpression analysis demonstrated that CEACAM1 had distinct coexpressed genes in different cancers, but KRT protein was consistently coexpressed with CEACAM1 in diverse cancer types. All the observations supported that CEACAM1 can serve as a diagnostic marker for some cancers, such as pancreatic cancer. And high CEACAM1 expression provides a better prognosis for some cancers, such as colorectal and head and neck cancers.

Enhancing Catalytic Efficiency of an Actinoplanes utahensis Echinocandin B Deacylase through Random Mutagenesis and Site-Directed Mutagenesis.

Echinocandin B deacylase (EBDA), from Actinoplanes utahensis ZJB-08196, is capable of cleaving the linoleoyl group from echinocandin B (ECB), forming the echinocandin B nucleus (ECBN), which is a key precursor of semisynthetic antifungal antibiotics. In the present study, molecular evolution of AuEBDA by random mutagenesis combined with site-directed mutagenesis (SDM) and screening was performed. Random mutagenesis on the wild-type (WT) AuEBDA generated two beneficial substitutions of G287Q, R527V. The "best" variant AuEBDA-G287Q/R527V was obtained by combining G287Q with R527V through SDM, which was most active at 35 °C, pH 7.5, with Km and vmax values of 0.68 mM and 395.26 U/mg, respectively. Mutation of G287Q/R527V markedly increased the catalytic efficiency kcat/Km by 290% compared with the WT-AuEBDA.

miRNA-342 Regulates CEACAM1-induced Lumen Formation in a Three-dimensional Model of Mammary Gland Morphogenesis.

Lumen formation of breast epithelium is rapidly lost during tumorigenesis along with expression of cell adhesion molecule CEACAM1. CEACAM1 induces lumena in a three-dimensional culture of MCF7/CEACAM1 cells that otherwise fail to form lumena. We hypothesized miRNAs may be involved because >400 genes were up- or down-regulated in MCF7/CEACAM1 cells and miRNAs may modify global expression patterns. Comparative analysis of miRNA expression in MCF7 versus MCF7/CEACAM1 cells revealed two miRNAs significantly down-regulated (hsa-miR-30a-3p by 6.73-fold and hsa-miR-342-5p by 5.68-fold). Location of miR-342 within an intron of the EVL gene, hypermethylated and involved in tumorigenesis, suggested that miR-342 overexpression may block lumen formation. In fact, overexpression of miR-342 in MCF7/CEACAM1 cells significantly blocked lumen formation (p < 0.001). ID4, a dominant-negative inhibitor of basic helix-loop-helix transcription factors, up-regulated in MCF7/CEACAM1 cells, down-regulated in breast cancer, and containing a miR-342 binding site, was tested as a potential target of miR-342. The ratio of ID4 to miR-342 increased from 1:2 in MCF7 cells to 30:1 in MCF7/CEACAM1 cells and a miR-342 inhibitor was able to induce 3'-UTR ID4 reporter activity in MCF7 cells. Because 5-methylcytosine methyltransferase DNMT1 is also a potential target of miR-342, we inhibited miR-342 in MCF7 cells and found DNMT1 was up-regulated with no change in EVL expression, suggesting that miR-342 regulates DNMT1 expression but DNMT1 does not affect the EVL expression in these cells. We conclude that the regulation of lumen formation by miR-342 involves at least two of its known targets, namely ID4 and DNMT1.

Identification and characterization of novel amphioxus microRNAs by Solexa sequencing.

BACKGROUND: microRNAs (miRNAs) are endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. While the number of known human and murine miRNAs is continuously increasing, information regarding miRNAs from other species such as amphioxus remains limited. RESULTS: We combined Solexa sequencing with computational techniques to identify novel miRNAs in the amphioxus species B. belcheri (Gray). This approach allowed us to identify 113 amphioxus miRNA genes. Among them, 55 were conserved across species and encoded 45 non-redundant mature miRNAs, whereas 58 were amphioxus-specific and encoded 53 mature miRNAs. Validation of our results with microarray and stem-loop quantitative RT-PCR revealed that Solexa sequencing is a powerful tool for miRNA discovery. Analyzing the evolutionary history of amphioxus miRNAs, we found that amphioxus possesses many miRNAs unique to chordates and vertebrates, and these may thus represent key steps in the evolutionary progression from cephalochordates to vertebrates. We also found that amphioxus is more similar to vertebrates than are tunicates with respect to their miRNA phylogenetic histories. CONCLUSIONS: Taken together, our results indicate that Solexa sequencing allows the successful discovery of novel miRNAs from amphioxus with high accuracy and efficiency. More importantly, our study provides an opportunity to decipher how the elaboration of the miRNA repertoire that occurred during chordate evolution contributed to the evolution of the vertebrate body plan.

In vitro evidence suggests that miR-133a-mediated regulation of uncoupling protein 2 (UCP2) is an indispensable step in myogenic differentiation.

UCP2 and UCP3, two novel uncoupling proteins, are important regulators of energy expenditure and thermogenesis in various organisms. The striking disparity between UCP2 mRNA and protein levels in muscle tissues prompted initial speculation that microRNAs are implicated in the regulatory pathway of UCP2. We found, for the first time, that the repression of UCP2 expression in cardiac and skeletal muscle resulted from its targeting by a muscle-specific microRNA, miR-133a. Moreover, our findings illustrate a novel function of UCP2 as a brake for muscle development. We also show that MyoD can remove the braking role of UCP2 via direct up-regulation of miR-133a during myogenic differentiation. Taken together, our current work delineates a novel regulatory network employing MyoD, microRNA, and uncoupling proteins to fine-tune the balance between muscle differentiation and proliferation during myogenesis.