Parameters influencing asymmetric synthesis of (R)-mandelonitrile by a novel (R)-hydroxynitrile lyase from Eriobotrya japonica

(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.     

Simplified high-throughput screening of AOX1-expressed laccase enzyme in Pichia pastoris

The heterologous protein expression in Pichia pastoris under the control of alcohol oxidase (AOX1)promoter comprises two steps, the growth and induction phases, which are time-consuming and technically demanding. Here, we describe an alternate method where expression is carried out directly in the methanol-containing medium. Using this method, we were successful in screening high-activity laccase clones from a library of laccase mutants generated by random mutagenesis. This simplified method not only saves time but also is highly efficient and can be used for screening a large number of clones.     

High throughput,colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z‐factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.     

恒细胞密度发酵策略提高重组毕赤酵母生产碱性果胶酶的表达效率

为了提高重组毕赤酵母生产碱性果胶酶(Alkaline polygalacturonate lyase,PGL)的比速率,开发了一种新的恒细胞密度发酵策略。通过不同的甲醇流加方式,实现发酵过程细胞密度的合理控制。实验结果表明:控制细胞密度为75 g/L的策略为最优,最终单位发酵液体积生产强度和单位菌体生产强度为6.11 U/(mL.h)和81.5 U/(g.h),分别比传统高密度发酵提高了42.1%和191.2%,最终PGL酶活为441.9 U/mL。此外,该策略还具有提高细胞活性和降低蛋白酶降解作用等优势。     

The characterization of Lucilia cuprina acetylcholinesterase as a drug target, and the identification of novel inhibitors by high throughput screening

Acetylcholinesterase (AChE, EC3.1.1.7.) is the molecular target for the carbamate and organophosphate pesticides that are used to combat parasitic arthropods. In this paper we report the functional heterologous expression of AChE from Lucilia cuprina (the sheep blowfly) in HEK293 cells. We show that the expressed enzyme is cell-surface-exposed and possesses a glycosyl-phosphatidylinositol membrane anchor. The substrates acetyl-, propionyl- and butyrylthiocholine (AcTC, PropTC, ButTC), and also 11 further thiocholine and homo-thiocholine derivatives were chemically synthesized to evaluate and compare their substrate properties in L. cuprina AChE and recombinant human AChE. The Michaelis-Menten constants K_M for AcTC, PropTC and ButTC were found to be 3-7-fold lower for the L. cuprina AChE than for the human AChE. Additionally, 2-methoxyacetyl-thiocholine and isobutyryl-thiocholine were better substrates for the insect enzyme than for the human AChE. The AcTC, PropTC and ButTC specificities and the Michaelis-Menten constants for recombinant L. cuprina AChE were similar to those determined for AChE extracted from L. cuprina heads, which are a particularly rich source of this enzyme. The median inhibition concentrations (IC₅₀ values) were determined for 21 organophosphates, 23 carbamates and also 9 known non-covalent AChE inhibitors. Interestingly, 11 compounds were 100- to >4000-fold more active on the insect enzyme than on the human enzyme. The substrate and inhibitor selectivity data collectively indicate that there are structural differences between L. cuprina and human AChE in or near the active sites, suggesting that it may be possible to identify novel, specific L. cuprina AChE inhibitors. To this end, a high throughput screen with 107,893 compounds was performed on the L. cuprina head AChE. This led to the identification of 195 non-carbamate, non-organophosphate inhibitors with IC₅₀ values below 10 ??M. Analysis of the most potent hit compounds identified 19 previously unknown inhibitors with IC₅₀ values below 200 nM, which were up to 335-fold more potent on the L. cuprina enzyme than on the human AChE. Some of these compounds may serve as leads for lead optimization programs to generate fly-specific pesticides.     

红花石蒜苯丙氨酸解氨酶催化合成N-甲基-L-苯丙氨酸

[背景] N-甲基-L-苯丙氨酸是一种N-烷基化芳香氨基酸,是重要的手性合成单元/中间体/组成成分,在医药、农业、食品等领域有重要应用价值的代谢产物中广泛存在。N-烷基化芳香氨基酸的合成与制备仍具有巨大的挑战。[目的] 在研究加兰他敏的生物合成过程中,我们从产加兰他敏的红花石蒜中克隆并表征苯丙氨酸解氨酶LrPAL3。LrPAL3催化区域及对映选择性的氢胺化反应得到L-苯丙氨酸。通过生物信息学分析,推测LrPAL3可能催化反式-肉桂酸的一步N-甲基胺化反应得到N-甲基-L-苯丙氨酸。[方法] 将反式-肉桂酸与甲胺,以及表达LrPAL3的大肠杆菌全细胞一起孵育。HPLC-DAD及HRESIMS分析表明,上述反应产物为N-甲基-苯丙氨酸。为确定该产物的立体构型,将上述催化反应放大,通过分离纯化得到该酶催化反应产物。[结果] 该化合物的氢谱数据及比旋光数据与N-甲基-L-苯丙氨酸标准品的数据一致。由此说明,LrPAL3能够催化反式-肉桂酸和甲胺发生N-烷基胺化反应,区域和立体专一性地生成N-甲基-L-苯丙氨酸。[结论] 本研究为手性N-烷基氨基酸的不对称合成提供了一种全新的绿色、高效生物催化剂。通过对LrPAL3的蛋白质定向进化及代谢工程,将会进一步扩展LrPAL3的催化反应范围,以多种N-烷基胺类及取代的苯基丙烯酸为底物,实现手性N-烷基-芳基氨基酸的高效区域及立体选择性生物合成。     

Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC5

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX-5 system is unable to fully complement secretion in an M. marinum esx-5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC₅, possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creating M. marinum-M. tuberculosis EccC₅ chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC₅. This region connects NBD1 and NBD2 of EccC₅ and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC₅, showed a distinct secretion profile. These results indicate that linker 2 is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC₅.     

Enhanced production of l‐lactic acid by Lactobacillus thermophilus SRZ50 mutant generated by high‐linear energy transfer heavy ion mutagenesis

The aim of this study was to improve l ‐lactic acid production of Lactobacillus thermophilus SRZ50. For this purpose, high efficient heavy‐ion mutagenesis technique was performed using SRZ50 as the original strain. To enhance the screening efficiency for high yield l ‐lactic acid producers, a scale‐down from shake flask to microtiter plate was developed. The results showed that 24‐well U‐bottom MTPs could well alternate shake flasks for L. thermophilus cultivation as a scale‐down tool due to its a very good comparability to the shake flasks. Based on this microtiter plate screening method, two high l ‐lactic acid productivity mutants, A59 and A69, were successfully screened out, which presented, respectively, 15.8 and 16.2% higher productivities than that of the original strain. Based on fed‐batch fermentation, the A69 mutant can accumulate 114.2 g/L l ‐lactic acid at 96 h. Hence, the proposed traditional microbial breeding method with efficient high‐throughput screening assay was proved to be an appropriate strategy to obtain lactic acid‐overproducing strain.     

An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.     

高通量测序分析一只临床死亡阿拉伯狒狒的胃肠道菌群

运用高通量测序分析一只临床死亡的阿拉伯狒狒胃肠道菌群的组成及多样性,结合其临床症状和检测结果进一步分析该动物的死亡原因,为野生动物的饲养管理和物种健康提供基础资料。

以2017年12月20日成都动物园一只死亡的幼龄雌性阿拉伯狒狒为研究对象,收集其胃、十二指肠、空肠、回肠、盲肠和结肠的内容物及粪便,利用高通量测序技术对细菌16S rRNA基因的V4区进行测序,分析细菌菌群的组成和多样性差异。

十二指肠的菌群有效序列数较高,而盲肠和粪便菌群的alpha多样性较高;胃肠道菌群丰度不同,但都主要来自变形菌门、厚壁菌门和拟杆菌门,而大肠具有较多的梭杆菌门;海尔曼螺杆菌和大肠埃希菌分别在胃和小肠富集,嗜肺巴氏杆菌和具核梭杆菌在结肠富集,并且结肠具有较多来自变形菌门的特有细菌;菌群的功能预测提示胃和小肠具有腹泻和胃肠炎风险,结肠具有菌群失调风险。

阿拉伯狒狒的胃肠道细菌菌群的高通量测序分析和临床诊断及器官组织病理检测结果表明该动物患有的间质性肺炎与小肠富集的大肠埃希菌关系密切,其直接死亡可能与嗜肺巴氏杆菌有关,这些症状进一步导致其结肠菌群失调而加剧了死亡,提示饲养管理中应加强对动物园阿拉伯狒狒等野生动物肠道大肠埃希菌和嗜肺巴氏杆菌的监测,提高野生动物的饲养管理和物种健康。

     

Discovery of novel pyrazolo[1,5-a]pyrimidines as potent pan-Pim inhibitors by structure- and property-based drug design

Pim kinases are promising targets for the development of cancer therapeutics. Among the three Pim isoforms, Pim-2 is particularly important in multiple myeloma, yet is the most difficult to inhibit due to its high affinity for ATP. We identified compound 1 via high throughput screening. Using property-based drug design and co-crystal structures with Pim-1 kinase to guide analog design, we were able to improve potency against all three Pim isoforms including a significant 10,000-fold gain against Pim-2. Compound 17 is a novel lead with low picomolar potency on all three Pim kinase isoforms.     

途径工程及Tn5转座子介导突变提高大肠杆菌丙酮酸生产

为了开发丙酮酸高产菌株,以大肠杆菌MG1655为出发菌株,通过基因敲除阻断副产物途径构建了产丙酮酸大肠杆菌工程菌KLPP。进一步利用p UT Mini-Tn5载体进行转座子随机突变,构建了含有7 197个单克隆的突变体文库。使用基于丙酮酸的二硝基苯肼显色法,建立了96孔板-酶标仪快速筛选方法,经过两轮的筛选,成功筛选到了6个突变体菌株,比KLPP丙酮酸产量提高了38%、31%、19%、28%、44%和14%。利用全基因组重测序确定了其转座子插入的位置,进而确定了可能影响丙酮酸产量的基因位点,为后续菌株改造工作奠定了基础。     

A high-throughput screen for hyaluronic acid accumulation in recombinant Escherichia coli transformed by libraries of engineered sigma factors

Hyaluronic acid (HA) is an important biomaterial with functional medical and cosmetic applications. As its synthesis has been recently reported in recombinant bacteria, it is of interest to develop a high throughput screening method for the rapid isolation of HA accumulating strains transformed by combinatorial libraries. Here we report a novel two-step screening strategy to select for better HA-producing recombinant Escherichia coli strains transformed by mutation libraries of rpoD and rpoS, coding for the sigma(D) and sigma(S) factors of the RNA polymerase, respectively. The first screen, based on translucent colony morphology identification, was used to qualitatively distinguish HA-producing strains on agar plates from non-HA producing strains that exhibit dense colony morphology. The second screen was based on the photometric measurement of an alcian blue staining solution that precipitates with HA, creating an inverse relationship between HA concentration and alcian blue absorbance. The color attenuation fitted a second-order polynomial between HA concentration and OD(540) absorbance. Using the alcian blue absorbance quantification, 74 translucent colonies from the HA-rpoD library and 78 translucent colonies from the HA-rpoS library were isolated and cultured for optimal strain selection. Three representative superior recombinants with high, medium and low increase of HA accumulation, respectively, were identified by the screen from the HA-rpoD and HA-rpoS mutant library. Further flask culture confirmed that results of the library screen were reliable and the superior recombinant D72 highly accumulated HA of 561.4 mg/L with a productivity of approximately 265 mg HA/g dry cell. Sequencing results showed that the mutant rpoD gene in D72 is in a truncated protein that lacks the conserved regions 3 and 4 of the sigma(D). Generally, this two-step high throughput screen presents a promising strategy for selecting superior HA-producing strains from large scale mutation libraries.     

Identification by high throughput screening of small compounds inhibiting the nucleic acid destabilization activity of the HIV-1 nucleocapsid protein

Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem–loop sequence complementary to the transactivation response element, doubly labelled at its 5′ and 3′ ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An “in-house” chemical library of 4800 molecules was screened and five compounds with IC₅₀ values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.     

Biocatalytic resolution of glycidyl aryl ethers by Trichosporon loubierii: cell/substrate ratio influences the optical purity of (R)-epoxides

Glycidyl aryl ethers were resolved by using lyophilized cells of Trichosporon loubierii ECU1040 having epoxide hydrolase activity. The activity and enantioselectivity depended on the structure of the aryl group. Different cell/substrate ratios also influenced the optical purity of remaining substrate. An additional stability test of the whole-cell enzyme suggests that rapid deactivation of the epoxide hydrolase was the potential reason. (R)-Epoxides were prepared in gram amounts with optical purity of 87% - 99% ee.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Fast identification of thermostable beta‐glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach

Engineering costly cellulases on natural cellulosic substrates is of importance for emerging biomass‐based biorefineries. Directed enzyme evolution is becoming a popular tool, but identification of desired mutants from a large mutant library remains challenging sometimes. In this work, we demonstrated a novel combinatorial selection/screening strategy for finding thermostable beta‐glucosidase on its natural substrate—cellobiose. First, selection was conducted through complementation of beta‐glucosidase for non‐cellobiose‐utilizing Escherichia coli so that only the cells expressing active beta‐glucosidase can grow on a M9 synthetic medium with cellobiose as the sole carbon source (selection plate). Second, the clones on the selection plates were duplicated by using nylon membranes. After heat treatment, the nylon membranes were overlaid on M9/cellobiose screening plates so that remaining activities of thermostable beta‐glucosidase mutants hydrolyzed cellobiose on the screening plates to glucose. Third, the growth of an indicator E. coli strain that can utilize glucose but not cellobiose on the screening plates helped detect the thermostable beta‐glucosidase mutants on the selection plates. Several thermostable mutants were identified from a random mutant library of the Paenibacillus polymyxa beta‐glucosidase. The most thermostable mutant A17S had an 11‐fold increase in the half‐life of thermoinactivation at 50°C. Biotechnol. Bioeng. 2009;103: 1087–1094. © 2009 Wiley Periodicals, Inc.     

High level expression of a recombinant xylanase by Pichia pastoris NC38 in a 5 L fermenter and its efficiency in biobleaching of bagasse pulp

A genetically modified XynA gene from Thermomyces lanuginosus was expressed in Pichia pastoris under the control of GAP promoter. P. pastoris expressed greater levels of xylanase (160 IU ml(-1)) on BMGY medium without zeocin after 56 h. The xylanase production by recombinant P. pastoris was scaled up in a 5L fermenter containing 1% glycerol and the highest xylanase production of 139 IU ml(-1) was observed after 72 h. Further studies carried out in fermenter under controlled pH (5.5) yielded a maximum xylanase production of 177 IU ml(-1) after 72 h. The biobleaching efficacy of crude xylanase was also evaluated on bagasse pulp and a brightness of 47.4% was observed with 50 IU of crude xylanase used per gram of pulp, which was 2.1 points higher in brightness than the untreated samples. Reducing sugars (24.8 mg g(-1)) and UV absorbing lignin-derived compounds values were considerably higher with xylanase treated samples.     

Expression of Azotobacter vinelandii soluble transhydrogenase perturbs xylose reductase-mediated conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae

Pyridine nucleotide transhydrogenase is a metabolic enzyme transferring the reducing equivalent between two nucleotide acceptors such as NAD⁺ and NADP⁺ for balancing the intracellular redox potential. Soluble transhydrogenase (STH) of Azotobacter vinelandii was expressed in a recombinant Saccharomyces cerevisiae strain harboring the Pichia stipitis xylose reductase (XR) gene to study effects of redox potential change on cell growth and sugar metabolism including xylitol and ethanol formation. Remarkable changes were not observed by expression of the STH gene in batch cultures. However, expression of STH accelerated the formation of ethanol in glucose-limited fed-batch cultures, but reduced xylitol productivity to 71% compared with its counterpart strain expressing xylose reductase gene alone. The experimental results suggested that A. vinelandii STH directed the reaction toward the formation of NADH and NADP⁺ from NAD⁺ and NADPH, which concomitantly reduced the availability of NADPH for xylose conversion to xylitol catalyzed by NADPH-preferable xylose reductase in the recombinant S. cerevisiae.