A practical random mutagenesis system for probiotic Lactobacillus casei using Tn5 transposition complexes

Aims: Despite the fact that the entire genome sequence of probiotic Lactobacillus casei has recently been available, their mechanisms of beneficial effects are poorly clarified, probably because of the lack of an efficient mutagenesis system. The aim of this study was to establish a practical random mutagenesis system of L. casei using the Tn5 transposome complexes. Methods and Results: We optimized the conditions for transformation using a plasmid pUCYIT356‐1‐Not2 and then transposition reaction using Tn5 transposome system for L. casei ATCC 27139. Tn5 insertion library of this strain being consisted of 9408 mutants was constructed by repeating the mutagenesis procedure. To examine the utility of this mutagenesis system, we screened a panel of insertion mutants for nutrient requirements. Six auxotrophic mutants were isolated and their Tn5 insertion sites were determined by inverse PCR, which demonstrated that insertions occur randomly throughout the whole bacterial genome. Conclusions: Tn5 transposome system functioned efficiently to generate transposon insertion mutants of L. casei and enabled to construct useful L. casei Tn5 insertion library at optimized conditions for transformation and transposition. Significance and Impact of the Study: The availability of this system facilitates the study of the mechanisms of beneficial effects of L. casei for human health.     

Further Thymol Derivatives from Ageratina cylindrica

From the leaves of Ageratina cylindrica, in addition to the described [(2S)‐2‐{4‐formyl‐5‐hydroxy‐2‐[(2‐methylpropanoyl)oxy]phenyl}oxiran‐2‐yl]methyl benzoate (cylindrinol A, 8 ), seven new thymol derivatives were isolated and named cylindrinols B – H ( 1 – 7 ). The structures of these compounds were established as (2‐{4‐(hydroxymethyl)‐2‐[(2‐methylpropanoyl)oxy]phenyl}oxiran‐2‐yl)methyl benzoate ( 1 ), (2‐{4‐formyl‐2‐[(2‐methylpropanoyl)oxy]phenyl}oxiran‐2‐yl)methyl benzoate ( 2 ), (2‐{4‐[(acetyloxy)methyl]‐2‐[(2‐methylpropanoyl)oxy]phenyl}oxiran‐2‐yl)methyl benzoate ( 3 ), [2‐(2‐[(2‐methylpropanoyl)oxy]‐4‐{[(2‐methylpropanoyl)oxy]methyl}phenyl)oxiran‐2‐yl]methyl benzoate ( 4 ), [2‐(5‐hydroxy‐2‐[(2‐methylpropanoyl)oxy]‐4‐{[(2‐methylpropanoyl)oxy]methyl}phenyl)oxiran‐2‐yl]methyl benzoate ( 5 ), 2‐{4‐(hydroxymethyl)‐2‐[(2‐methylpropanoyl)oxy]phenyl}prop‐2‐en‐1‐yl benzoate ( 6 ), and 2‐hydroxy‐2‐[2‐hydroxy‐4‐(hydroxymethyl)‐phenyl]‐3‐[(2‐methylpropanoyl)oxy]propyl benzoate ( 7 ), by spectroscopic means. Compounds 1 showed moderate antiprotozoal activity on both protozoa. Compounds 4 and 5 showed selectivity on Giardia lamblia trophozoites. All isolated compounds were less active than two antiprotozoal drugs, metronidazole and emetine, used as positive controls. Compound 5 exhibited a high inhibitory effect on hyperpropulsive movement of the small intestine in rats; its effect was best than loperamide, antidiarrheal drug used as a positive control.     

Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets

Cross‐strand disulfides bridge two cysteines in a registered pair of antiparallel β‐strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross‐strand disulfides. Seventy‐six cross‐strand disulfides were found of which 75 and 1 occurred at non‐hydrogen‐bonded (NHB) and hydrogen‐bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T _m . All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG ⁰ = ?3.3 to ?6.7 kcal/mol). The data demonstrate that introduction of cross‐strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Physiological control of amonabactin biosynthesis inAeromonas hydrophila

Summary Amonabactin is a siderophore fromAeromonas hydrophila which is produced in two biologically active forms composed of the phenolate 2,3-dihydroxybenzoic acid (DHB), lysine, glycine, and either trytophan (amonabactin T) or phenylalanine (amonabactin P). Amonabactin biosynthetic mutants (generated by chemical mutagenesis) that either produced no amonabactin or overproduced the siderophore were isolated and identified on chrome azurol S siderophore detection agar. Amonabactin-negative mutants were of two categories. One type produced no phenolates and used exogenous DHB to synthesize amonabactin (both forms) while the other type excreted DHB but not amonabactin. This suggests an amonabactin biosynthetic pathway composed of two segments, one producing DHB and the other assembling amonabactin from DHB and the amino acids. Overproduction mutants used amonabactin poorly or not at all, indicating that they contained lesions in amonabactin utilization. Adding the analogd-tryptophan to wild-typeA. hydrophila cultures reduced synthesis of both amonabactin T and amonabactin P and lengthened the lag phase in iron restricted medium. The tryptophan and phenylalanine forms of amonabactin may be synthesized by a single assembly pathway that contains a novel enzyme (sensitive tod-tryptophan) which inserts either tryptophan or phenylalanine into amonabactin.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.     

Biochemical and genetic characterization of dehydrobiotin resistant mutants of Escherichia coli

Summary Dehydrobiotin (DHB) resistant mutants were isolated from strains of Escherichia coli K-12 and were classified into two groups; dhbA and dhbB.In dhbB mutants the structural genes for enzymes of the biotin pathway are expressed constitutively at a high rate. The dhbB gene is co-transducible with argE at a frequency of about 50% by P1 transduction and maps on the chromosome between arg EC BH and rif. The dhbB ⁺ gene is trans-dominant over the mutant allele indicating that the dhbB ⁺ gene controls the production of a diffusible substance such as a repressor molecule.The dhbA mutants show biotin biosynthetic activity comparable to the wild type and are as sensitive to repression by biotin as the parent strain. The mutants appear to be deficient in DHB transport as suggested by the findings that the ability of the mutants to take up biotin is reduced significantly and that DHB, a competitive inhibitor of biotin uptake, is much less inhibitory to biotin uptake in the mutants than in the wild type.     

An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.     

The σE stress response is required for stress‐induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σ^E acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ³², which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σ^E promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σ^E, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.     

Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase

For the synthesis of polylactic acid (PLA) and its copolymers by one‐step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (Pct_Cp) and Pseudomonas sp. MBEL 6‐19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1_Ps6‐19) were introduced into Escherichia coli for the generation of lactyl‐CoA endogenously and incorporation of lactyl‐CoA into the polymer, respectively. Since the wild‐type PhaC1_Ps6‐19 did not efficiently accept lactyl‐CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild‐type Pct_Cp was not able to efficiently convert lactate to lactyl‐CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error‐prone PCR was carried out. By employing engineered PhaC1_Ps6‐19 and Pct_Cp, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), containing 20–49 mol% lactate could be produced up to 62 wt% from glucose and 3HB. By controlling the 3HB concentration in the medium, PLA homopolymer and P(3HB‐co‐LA) containing lactate as a major monomer unit could be synthesized. Also, P(3HB‐co‐LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Fed‐batch cultures were performed to produce P(3HB‐co‐LA) copolymers having 9–64 mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined. Biotechnol. Bioeng. 2010; 105: 150–160. © 2009 Wiley Periodicals, Inc.     

High‐throughput fluorescence‐activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low‐lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence‐activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high‐lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid‐induction periods.     

利用机器学习提高M. jannaschii酪氨酰tRNA合成酶底物特异性分子建模预测的准确度

设计结合不同化学结构底物的酶结合袋是一个巨大的挑战. 传统的湿实验要筛选成千上万甚至上百万个突变体来寻找对特定配体结合的突变体,此过程需要耗费大量的时间和资源. 为了加快筛选过程,我们提出了一种新的工作流程,将分子建模和数据驱动的机器学习方法相结合,生成具有高富集率的突变文库,用于高效筛选能识别特定底物的蛋白质突变体. M. jannaschii酪氨酰tRNA合成酶（Mj. TyrRS）能识别特定的非天然氨基酸并催化形成氨酰tRNA,其不同的突变体能够识别不同结构的非天然氨基酸,并且已经有了许多报道和数据的积累,因此我们使用TyrRS作为一个例子来进行此筛选流程的概念验证. 基于已知的多个Mj. TyrRS的晶体结构及分子建模的结果,我们发现D158G/P是影响残基158~163位α螺旋蛋白骨架变化的关键突变. 我们的模拟结果表明,在含有687个突变体的测试数据中,与随机突变相比,分子建模和打分函数计算排序可以将目标突变体的富集率提高2倍,而使用已知突变体和对应的非天然氨基酸数据训练的机器学习模型进行校准后,筛选富集率可提高11倍. 这种分子建模和机器学习相结合的计算和筛选流程非常有助于Mj.TyrRS的底物特异性设计,可以大大减少湿实验的时间和成本. 此外,这种新方法在蛋白质计算设计领域具有广泛的应用前景.     

Structural basis for the transcriptional repressor NicR2 in nicotine degradation from Pseudomonas

Nicotine is an environmental toxicant in tobacco wastes, imposing severe hazards for the health of human and other mammalians. NicR2, a TetR‐like repressor from Pseudomonas putida S16, plays a critical role in regulating nicotine degradation. Here, we determined the crystal structures of NicR2 and its complex with the inducer 6‐hydroxy‐3‐succinoyl‐pyridine (HSP). The N‐terminal domain of NicR2 contains a conserved helix‐turn‐helix (HTH) DNA‐binding motif, while the C‐terminal domain contains a cleft for its selective recognition for HSP. Residues R91, Y114 and Q118 of NicR2 form hydrogen bonds with HSP, their indispensable roles in NicR2's recognition with HSP were confirmed by structure‐based mutagenesis combined with isothermal titration calorimetry analysis. Based on sequence alignment and structure comparison, Tyr67, Tyr68 and Lys72 of HTH motif were corroborated to take the major responsibility for DNA‐binding using site‐directed mutants. The 30‐residue N‐terminal extension of NicR2, especially residues 21–30 in the TFR arm, is required for the association with the operator DNA. Finally, we proposed that either NicR2 or the DNA would undergo a conformational change upon their association. Altogether, our structural and biochemical investigations unravel how NicR2 selectively recognizes HSP and DNA, and provide new insights into the TetR family of repressors.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

A Simple Chiral Cu(II) Complex as an Effective Phase‐Transfer Catalyst for the Enantioselective Alkylation of Dissymmetric Glycinate Ketimines

Catalytic asymmetric benzylation of a dissymmetric tert‐butylglycinate ketimine, incorporating 1‐naphthyl and phenyl groups as the Schiff base substituents, under phase‐transfer conditions was investigated. It was interesting to note that the sense of asymmetric induction of the alkylation of Z‐imine stereoisomer is opposite to that of the corresponding E stereoisomer with a similar degree of enantioselectivity. More interestingly, the chiral Cu(II) complex of the Schiff base derived from (R)‐2‐phenylglycinol and 2‐hydroxy‐1‐naphthaldehyde was found to catalyze the same reaction under solid‐liquid conditions with comparable enantioselectivity (up to 60% ee) with respect to known cinchona alkaloid catalysts. The solvent/base‐system parameter was shown to control the optimal catalytic activity. Chirality 27:944–950, 2015. © 2015 Wiley Periodicals, Inc.