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https://www.readbyqxmd.com/read/27812824/development-of-bacillus-subtilis-mutants-to-produce-tryptophan-in-pigs
#1
Karin Bjerre, Mette D Cantor, Jan V Nørgaard, Hanne D Poulsen, Karoline Blaabjerg, Nuria Canibe, Bent B Jensen, Birgitte Stuer-Lauridsen, Bea Nielsen, Patrick M F Derkx
OBJECTIVES: To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. RESULTS: A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l...
November 3, 2016: Biotechnology Letters
https://www.readbyqxmd.com/read/27746324/iteratively-improving-natamycin-production-in-streptomyces-gilvosporeus-by-a-large-operon-reporter-based-strategy
#2
Yemin Wang, Zhengsheng Tao, Hualiang Zheng, Fei Zhang, Qingshan Long, Zixin Deng, Meifeng Tao
Many high-value secondary metabolites are assembled by very large multifunctional polyketide synthases or non-ribosomal peptide synthetases encoded by giant genes, for instance, natamycin production in an industrial strain of Streptomyces gilvosporeus. In this study, a large operon reporter-based selection system has been developed using the selectable marker gene neo to report the expression both of the large polyketide synthase genes and of the entire gene cluster, thereby facilitating the selection of natamycin-overproducing mutants by iterative random mutagenesis breeding...
October 13, 2016: Metabolic Engineering
https://www.readbyqxmd.com/read/27645297/rational-design-of-thermostability-in-bacterial-1-3-1-4-%C3%AE-glucanases-through-spatial-compartmentalization-of-mutational-hotspots
#3
Chengtuo Niu, Linjiang Zhu, Xin Xu, Qi Li
Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures...
September 19, 2016: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/27401975/high-level-of-spinosad-production-in-the-heterologous-host-saccharopolyspora-erythraea
#4
Jun Huang, Zhen Yu, Mei-Hong Li, Ji-Dong Wang, Hua Bai, Jun Zhou, Yu-Guo Zheng
UNLABELLED: Spinosad, a highly effective insecticide, has an excellent environmental and mammalian toxicological profile. Global market demand for spinosad is huge and growing. However, after much effort, there has been almost no improvement in the spinosad yield from the original producer, Saccharopolyspora spinosa Here, we report the heterologous expression of spinosad using Saccharopolyspora erythraea as a host. The native erythromycin polyketide synthase (PKS) genes in S. erythraea were replaced by the assembled spinosad gene cluster through iterative recombination...
September 15, 2016: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/27312879/the-in-vitro-selection-world
#5
REVIEW
Kenan Jijakli, Basel Khraiwesh, Weiqi Fu, Liming Luo, Amnah Alzahmi, Joseph Koussa, Amphun Chaiboonchoe, Serdal Kirmizialtin, Laising Yen, Kourosh Salehi-Ashtiani
Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins...
August 15, 2016: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/27234138/semi-rational-engineering-of-cytochrome-cyp153a-from-marinobacter-aquaeolei-for-improved-%C3%AF-hydroxylation-activity-towards-oleic-acid
#6
Yan Duan, Lina Ba, Jianwei Gao, Xianxing Gao, Dunming Zhu, René M de Jong, Daniel Mink, Iwona Kaluzna, Zhanglin Lin
ω-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved ω-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket...
October 2016: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/27225474/practical-two-step-synthesis-of-enantiopure-styrene-oxide-through-an-optimized-chemoenzymatic-approach
#7
Kai Wu, Hualei Wang, Lifeng Chen, Haiyang Fan, Zhiqiang Zhao, Dongzhi Wei
Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive "1-pot, 2-step" chemoenzymatic approach for producing enantiopure SO with 100 % theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1 % by adding methyl tert-butyl ether (MTBE) as the second phase...
October 2016: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/27216910/combinatorial-evolution-of-phosphotriesterase-toward-a-robust-malathion-degrader-by-hierarchical-iteration-mutagenesis
#8
Xiao-Jing Luo, Jian Zhao, Chun-Xiu Li, Yun-Peng Bai, Manfred T Reetz, Hui-Lei Yu, Jian-He Xu
Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling...
November 2016: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/27094289/combined-and-iterative-use-of-computational-design-and-directed-evolution-for-protein-ligand-binding-design
#9
Meng Wang, Huimin Zhao
The advantages of computational design and directed evolution are complementary, and only through combined and iterative use of both approaches, a daunting task such as protein-ligand interaction design, can be achieved efficiently. Here, we describe a systematic strategy to combine structure-guided computational design, iterative site saturation mutagenesis, and yeast two-hybrid system (Y2H)-based phenotypic screening to engineer novel and orthogonal interactions between synthetic ligands and human estrogen receptor α (hERα) for the development of novel gene switches...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/26914401/new-concepts-for-increasing-the-efficiency-in-directed-evolution-of-stereoselective-enzymes
#10
Zhoutong Sun, Ylva Wikmark, Jan-E Bäckvall, Manfred T Reetz
Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site...
April 4, 2016: Chemistry: a European Journal
https://www.readbyqxmd.com/read/26804479/solubilization-and-iterative-saturation-mutagenesis-of-%C3%AE-1-3-fucosyltransferase-from-helicobacter-pylori-to-enhance-its-catalytic-efficiency
#11
Yun Hee Choi, Jong Hoon Kim, Bum Seok Park, Byung-Gee Kim
α1,3-Fucosyltransferase (α1,3-FucT) is essential for the biosynthesis of biologically active α1,3-fucosyloligosacchairdes (3-FOs) from human milk oligosaccharides (HMO), particularly 3-fucosyllactose (3-FL) trisaccharide. α1,3-FucT from Helicobacter pylori 26695 (FutA) accepts lactose and LacNAc as glycan acceptors and has a very low level of expression in Escherichia coli, and it shows a low catalytic activity for lactose in the large-scale synthesis of 3-FL. To overcome the poor solubility of FutA, codon optimization, and systematic truncation of the protein at the C-terminus with only one heptad repeat remaining (Δ52 FutA) were conducted to yield 150-200 mg/L of soluble protein of FutA and resulting in more than an 18-fold increase in the 3-FL yield...
August 2016: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/26563000/-screening-for-mutants-with-thermostabe-lipase-a-from-burkholderia-sp-zyb002
#12
Yanru Liu, Liqing Qiu, Jianzhong Huang, Bingchun Zhao, Zuozhen Wang, Xiaolan Zhu, Yuanyuan Gao, Zhengyu Shu
OBJECTIVE: We improved the thermostability of LipA from Burkholderia cecapia ZYB002 by protein engineering technology to expand the application of lipase LipA. METHOD: On the basis of B-factor value of lipase LipA, series of potential mutation hotspots were selected for iterative saturation mutagenesis and the corresponding small mutation gene libraries were then constructed to screen the hyperthermal variants. RESULTS: From the above mutation libraries, we obtained a series of mutants whose enzyme half-life at 55 degrees C increased by 1...
June 4, 2015: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
https://www.readbyqxmd.com/read/26435512/mutational-mapping-of-the-transmembrane-binding-site-of-the-g-protein-coupled-receptor-tgr5-and-binding-mode-prediction-of-tgr5-agonists
#13
Christoph G W Gertzen, Lina Spomer, Sander H J Smits, Dieter Häussinger, Verena Keitel, Holger Gohlke
TGR5 (Gpbar-1, M-Bar) is a class A G-protein coupled bile acid-sensing receptor predominately expressed in brain, liver and gastrointestinal tract, and a promising drug target for the treatment of metabolic disorders. Due to the lack of a crystal structure of TGR5, the development of TGR5 agonists has been guided by ligand-based approaches so far. Three binding mode models of bile acid derivatives have been presented recently. However, they differ from one another in terms of overall orientation or with respect to the location and interactions of the cholane scaffold, or cannot explain all results from mutagenesis experiments...
November 2, 2015: European Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/26423948/differentiation-coupled-induction-of-human-cytomegalovirus-replication-by-union-of-the-major-enhancer-retinoic-acid-cyclic-amp-and-nf-%C3%AE%C2%BAb-response-elements
#14
Jinxiang Yuan, Ming Li, Yasaira Rodriguez Torres, Courtney S Galle, Jeffery L Meier
UNLABELLED: Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed...
December 2015: Journal of Virology
https://www.readbyqxmd.com/read/26355458/cell-cycle-regulates-nuclear-stability-of-aid-and-determines-the-cellular-response-to-aid
#15
Quy Le, Nancy Maizels
AID (Activation Induced Deaminase) deaminates cytosines in DNA to initiate immunoglobulin gene diversification and to reprogram CpG methylation in early development. AID is potentially highly mutagenic, and it causes genomic instability evident as translocations in B cell malignancies. Here we show that AID is cell cycle regulated. By high content screening microscopy, we demonstrate that AID undergoes nuclear degradation more slowly in G1 phase than in S or G2-M phase, and that mutations that affect regulatory phosphorylation or catalytic activity can alter AID stability and abundance...
September 2015: PLoS Genetics
https://www.readbyqxmd.com/read/26266754/improvement-in-the-thermostability-of-a-type-a-feruloyl-esterase-aufaea-from-aspergillus-usamii-by-iterative-saturation-mutagenesis
#16
Xin Yin, Jian-Fang Li, Chun-Juan Wang, Die Hu, Qin Wu, Ying Gu, Min-Chen Wu
Feruloyl or ferulic acid esterase (Fae, EC 3.1.1.73) catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris, 15 promising variants were obtained...
December 2015: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/26254469/the-ribosome-sec61-translocon-complex-forms-a-cytosolically-restricted-environment-for-early-polytopic-membrane-protein-folding
#17
Melissa A Patterson, Anannya Bandyopadhyay, Prasanna K Devaraneni, Josha Woodward, LeeAnn Rooney, Zhongying Yang, William R Skach
Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments...
November 27, 2015: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/26206151/structural-plasticity-of-green-fluorescent-protein-to-amino-acid-deletions-and-fluorescence-rescue-by-folding-enhancing-mutations
#18
Shu-su Liu, Xuan Wei, Xue Dong, Liang Xu, Jia Liu, Biao Jiang
BACKGROUND: Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs...
2015: BMC Biochemistry
https://www.readbyqxmd.com/read/26177455/a-crispr-cas9-system-for-genetic-engineering-of-filamentous-fungi
#19
Christina S Nødvig, Jakob B Nielsen, Martin E Kogle, Uffe H Mortensen
The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi...
2015: PloS One
https://www.readbyqxmd.com/read/26115233/engineered-hydrophobic-pocket-of-s-selective-arylmalonate-decarboxylase-variant-by-simultaneous-saturation-mutagenesis-to-improve-catalytic-performance
#20
Shosuke Yoshida, Junichi Enoki, Robert Kourist, Kenji Miyamoto
A bacterial arylmalonate decarboxylase (AMDase) catalyzes asymmetric decarboxylation of unnatural arylmalonates to produce optically pure (R)-arylcarboxylates without the addition of cofactors. Previously, we designed an AMDase variant G74C/C188S that displays totally inverted enantioselectivity. However, the variant showed a 20,000-fold reduction in activity compared with the wild-type AMDase. Further studies have demonstrated that iterative saturation mutagenesis targeting the active site residues in a hydrophobic pocket of G74C/C188S leads to considerable improvement in activity where all positive variants harbor only hydrophobic substitutions...
2015: Bioscience, Biotechnology, and Biochemistry
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