The antibiotic tetracycline (TC) is an emerging pollutant frequently detected in various environments. Although enzymatic remediation is a promising strategy for mitigating TC contamination, the availability of effective TC-degrading enzymes remains limited, and their mechanisms and applications are not fully understood. This study developed a comprehensive TC-degrading enzyme library from the gut microbiome of the highly TC-resistant saprophagous insect, black soldier fly larvae (BSFL), using an integrated metagenomic and comparative metatranscriptomic approach, identifying 105 potential novel TC-degradation genes. Bioinformatics analysis of 10 selected genes underscored the novelty of the identified enzymes. Among these, Trg2 demonstrated strong binding affinity and significant degradation capacity for TC. Key functional amino acid residues, including Thr231, Ala64, Ala82, Gly68, Gly79, and Ser81, were identified as essential for the interaction between TC and Trg2. Six TC degradation pathways were proposed, involving the transformation of TC into 19 metabolites through de-grouping, ring opening, oxidation, reduction, and addition reactions, effectively reducing TC toxicity. Furthermore, Trg2 exhibited resilience under harsh conditions, maintaining the capacity to remove about 45 % of the total TC in mariculture wastewater across eight successive batches. This study advances the understanding of TC degradation mechanisms and highlights the potential application of novel enzymes for bioremediation purposes.

The antibiotic tetracycline (TC) is an emerging pollutant frequently detected in various environments. Biodegradation is a crucial approach for eliminating TC contamination. However, only a few efficient TC-degrading bacteria have been isolated, and the molecular mechanisms of TC degradation, as well as their application potential, remain poorly understood. This study isolated a novel TC-degrading bacterium, Providencia stuartii TX2, from the intestine of black soldier fly larvae. TX2 exhibited remarkable performance, degrading 72.17 % of 400 mg/L TC within 48 h. Genomic analysis of TX2 unveiled the presence of antibiotic resistance genes and TC degradation enzymes. Transcriptomic analysis highlighted the roles of proteins related to efflux pumps, enzymatic transformation, adversity resistance, and unknown functions. Three TC degradation pathways were proposed, with TC being transformed into 27 metabolites through epimerization, hydroxylation, oxygenation, ring opening, and de-grouping, reducing TC toxicity. Additionally, TX2 significantly enhanced TC biodegradation in four TC-contaminated environmental samples and reduced antibiotic resistance genes and mobile genetic elements in chicken manure. This research provides insights into the survival and biodegradation mechanisms of Providencia stuartii TX2 and evaluates its potential for environmental bioremediation.

The widely used ET recombination requires an ssDNA product degraded by Rac phage protein E588 from dsDNA for strand invasion. However, proof of the ssDNA product is still elusive. The study provided three levels of proof sequentially. The probable ssDNAs degraded by E588 from the fluorescent plus-, minus-, or double-stranded dsDNA pET28a-xylanase exhibited a half fluorescence intensity of the corresponding dsDNAs, equivalent to the E588 degradation nucleotides half that of the total nucleotides degraded from the corresponding dsDNA. The ssDNA product degraded by E588 from the fluorescent minus-stranded dsDNA was confirmed by gradient gel-electrophoresis and two nuclease degradation reactions. Degraded by E588 from the dsDNA pET28a-xylanase that had a phosphorothioated plus-stranded 5'-terminus, the plus-stranded ssDNA product was separated via gel electrophoresis and recovered via a DNAclean kit. The recovered ssDNA product was proven to have intact 5'- and 3'-ends by DNA sequencing analysis. This study provides a solid foundation for the mechanism of ssDNA invasion.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Developing for almost half a century, plasmid-construction has explored more than 37 methods. Some methods have evolved into new versions. From a global and evolutionary viewpoint, a review will make a clear understand and an easy practice for plasmid-construction. The 37 methods employ three principles as creating single-strand overhang, recombining homology arms, or serving amplified insert as mega-primer, and are classified into three groups as single strand overhang cloning, homologous recombination cloning, and mega-primer cloning. The methods evolve along a route for easy, efficient, or/and seamless cloning. Mechanism of plasmid-construction is primer annealing or/and primer invasion. Scar junction is a must-be faced scientific problem in plasmid-construction.

Because indel results in frame-shift mutations, seamless repair of double-stranded break (DSB)s plays a pivotal role in synthetic biology, molecular biology, and genome integrity. However, DSB repair is not well documented. T4 DNA ligase (T4lig) served to ligate intra-molecularly a zero bp break-apart DSB linear plasmid DNA pET22b(28a)-xylanase. An ATP T4lig ligation reaction joined one single-stranded break (SSB) into a phosphodiester-bond, whereas the opposite SSB into an abortive ligation intermediate blocking the DSB sequential repair. The intermediate proved to be fluorescent Cy5-AMP-SSB by a T4lig ligation reaction in the aid of Alexa Fluor 647 ATP having Cy5-AMP fluorescence. The fluorescent Cy5-AMP-SSB was de-adenylated into SSB by an ATP-free T4lig or Mg2+-free T4ligL159L reaction. The de-adenylated SSB was re-joined into another phosphodiester-bond by a sequential ATP T4lig re-ligation reaction. Thereby, DSB repair proceeds an abortive ligation, a reverse de-adenylation, and a sequential re-ligation reaction. The result has a potential usage in synthetic biology, molecular biology, and cancer-curing.

Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5′- and 3′-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-AdD. The generality was checked by constructing plasmid pET21a-AdD and pET22b-AdD in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.

5'端/3'端间同源长度对分子内同源重组质粒构建的影响还不明确.本研究以与GFO对应的五种反向引物CR1、CR2、CR3、CR4、CR5,分别扩增5'端/3'端间同源长度有10、20、30、40、50 bp的5.9 kb线性质粒pET20b-C2-GH10-C2作为模式DNA,经重组酶Exnase II 37℃体外重组30 min,42℃热激诱导转化BL21(DE3)感受态细胞,比较转化率、质粒位置正确率、接口正确率.结果显示,同源长度30 bp DNA重组后转化子正确质粒最多,正确接口转化率3.4个/ng DNA,其次是同源长度20 bp DNA.电泳检测显示DNA重组后产生nick质粒,是转化子的来源,胞内酶修饰nick可产生基因-载体接口异常.

Used as model for sandwich fusion, a mesophilic Aspergillus niger GH11 xylanase (Xyn) was fused into C2-Xyn-C2 with a thermophilic Thermotaga maritima GH10 xylanase carbohydrate-binding module CBM9_2 (C2). Linearized plasmids C2-pET20b-C2-Xyn were amplified from template pET20b-Xyn-C2 with a 4.3 kb C2-pET20b megaprimer, ligated into circular plasmids in blunt-end ligation, and transformed into E. coli BL21 (DE3) cells. The C2-Xyn-C2 had optimum activity at 45 °C and pH 4.2, a 2.85 h thermal inactivation half-life at 80 °C and a 8.69 h at 50 °C, with the 8.69 h value 24.8-, 7.5-, and 7.1-fold longer than the Xyn and single terminal fusion enzymes Xyn-C2, and C2-Xyn. Thermodynamics showed that the enzyme had a 1.8 °C higher melting temperature, lower values ΔS, ΔΔG, and a denser structure than the Xyn. Kinetics showed that the C2-Xyn-C2 catalytic efficiency was 1.2-~6-fold and 2.7-~7.9-fold higher on beechwood and oat-spelt xylan than those of the enzymes Xyn, Xyn-C2, and C2-Xyn. The sandwich fusion evolved the xylanase with "armor-hands" to enhance simultaneously thermostability and activity in quality.

To analyze the quantity effect of homologous primers on DNA amplification,forward primer GFO having 45%,90%,100%,64%,9% complementation with reverse primer CR1,CR2,CR3,CR4,CR5 were designed to amplify a 5.9 kb model linearized plasmid DNA pET20b-C2-G10-C2 containing an Aspergillus niger GH10 xylanase gene.Compared with non-producing target DNAs by equal quantities of the primers CR3 and CR4 with the GFO,the target DNA bands were amplified by 10-fold decreased primers CR3 and CR4.All target DNA bands were amplified by the five primers with 10-fold decreased primer GFO,verifying that equal quantities of homologous primers inhibited DNA amplifications.Maximal DNAs were amplified with the 100-fold decreased CR3 and the 10-fold decreased CR4. Maximal DNAs of 1.4-2.2 μg were amplified by the decreased quantities five homologous primers after 15-20 cycles,similar to that of normal PCR amplification. Different from single-strand DNA amplification of asymmetric non-homologous primer PCR,the unequal quantity primer PCR amplified double-strand DNAs by decreasing complementation effect of homologous primers.

[Objective] Instead of standard PCR setting a single annealing temperature (S-Tm),we studied double annealing temperature PCR (D-Tm PCR) setting respective annealing temperature for forward and reverse primers from higher to lower.[Methodsl A 4.3 kb pET20b-Xyn (Aspergillus niger xylanase gene) model DNA was amplified with Q5 DNA polymerase by using PxF61 and VPel as forward and reverse primers.The PCR procedure was:pre-denaturation at 98 ℃ for 3 min,and 30 cycles of denaturation at 98 ℃ for 30 s,annealing at Tm1 70 ℃ (PxF61) for 15 s and at Tm2 62 ℃ (VPel) for 15 s,extension at 72 ℃ for 130 s.[Results] The 4.3 kb target DNA band of D-Tm PCR was a little brighter,whereas non-specific DNA bands were two less than those of the S-Tm PCR (Tm=61 ℃).Twenty-five cycles of amplification created the brightest target DNA band in the D-Tm PCR.A 5.3 kb recombinant plasmid DNA was clearly amplified in the D-Tm PCR than the S-Tm PCR.[Conclusion] The D-Tm PCR amplified directly target DNA band without demanding for investigation of an optimal annealing temperature and for setting closer annealing temperatures between forward and reverse primers.Moreover,two respective annealing steps were clearly elucidated from theoretical viewpoint.

T4 DNA ligase(T4Lig)is important in gene recombination. Because of wide usage and being provided by company,its property is not thoroughly understood,which leads to low efficiency,sometimes even failure,in blunt-end DNA ligation. In order to recombine gene efficiently in blunt end DNA ligation and to further elucidate ligation mechanism of blunt-end DNA,the paper summarized such properties as enzyme size,fidelity,ligation mechanism of nick DNA,assay methods of nick-sealing,and rational engineering of enzyme properties. Some unsolved problems in blunt end DNA ligation were also provided. Therefore,to understand thoroughly T4Lig properties is useful for genetic recombination in vitro.

There exist significant differences between the 2 main types of xylanases, family F10 and G11. A clear understanding of the expression pattern of microbial F10 and G11 under different culture conditions would facilitate better production and industrial application of xylanase. In this study, the fungal xylanase producer Aspergillus niger A09 was systematically investigated in terms of induced expression of xylanase F10 and G11. Results showed that carbon and nitrogen sources could influence xylanase F10 and G11 transcript abundance, with G11 more susceptible to changes in culture media composition. The most favorable carbon and nitrogen sources for high G11 and low F10 production by A. niger A09 were xylan (2%) and (NH4)2C2O4 (0.3%), respectively. Following cultivation at 33 °C for 60 h, the highest xylanase activity (1132 IU per gram of wet mycelia) was observed. On the basis of differential gene expression of F10 and G11, as well as their different properties, we deduced that the F10 protein initially targeted xylan and hydrolyzed it into fragments including xylose, after which xylose acted as the inducer of F10 and G11 gene expression. These speculations also accounted for our failure to identify conditions favoring the high production of F10 but a low production of G11.

Journal: Canadian Journal of Microbiology Manuscript ID cjm-2015-0394.R4 Manuscript Type: Article Date Submitted by the Author: 21-Apr-2016 Complete List of Authors: Cui, Shixiu; Henan Agricultural University, College of Life Sciences Wang, Tianwen; East China University of Science and Technology, School of Biotechnology Hu, Hong; Henan Academy of Sciences, Institute of Biology Co., Ltd Liu, Liangwei; Henan Agricultural University, College of Life Sciences Song, An-Dong; Henan Agricultural University, College of Life Sciences Chen, Hongge; Henan Agricultural University, College of Life Sciences Keyword: Aspergillus niger, qPCR, F10 xylanase, G11 xylanase, Expression pattern

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module.

It is difficult to construct recombinant plasmid containing repeated DNA sequences,because multimer would be produced in PCR amplification. Megaprimer reverse PCR was used to construct recombinant plasmid pET20b-C1-Xyn-C1,which contained a xylanase(Xyn)DNA flanked by repeated DNA sequence,a Carbohydrate binding module C1. Firstly,megaprimer C1-pET20b was amplified using forward primer LF for C1,reverse primer VRP for pET20b,and pET20b-Xyn-C1 template. Secondly,the linear recombinant plasmid C1-pET20b-C1-Xyn DNA was amplified by using reverse primer RX for Xyn,pET20b-C1-Xyn template,and the amplified C1-pET20b as forward megaprimer. The forward megaprimer annealed to pET20b instead of region of the template. The linear recombinant plasmid was ligated with T4 DNA ligase and transformed to DH5α competent cell. The recombinant pET20b-C1-Xyn-C1 containing repeated sequences C1 was constructed.

PEG能够提高连接酶连接效率,但是对转化率的影响被忽视,特别是基因工程操作进入到精细定量阶段.以BL21(DE3)为宿主细胞,探讨32%PEG200、75℃30min处理对pET20b质粒转化率的影响.以不经任何处理的1 ng pET20b转化率100%作为对照,高温处理后转化率为82.3%,PEG处理后转化率为8%,PEG且高温处理后转化率为0%,利用PCR产物回收试剂盒去除PEG可恢复15.3%转化率,说明75℃高温处理对转化率影响很小,PEG则严重影响转化率,PEG且75℃高温处理导致转化失败.通过研究发现PEG严重影响转化率,提出部分恢复转化率的解决方案,并探讨了PEG影响转化率的机理.

为提高外源蛋白表达量,以pET20b载体、BL21(DE3) E.coli宿主细胞,比较了乳糖和IPTG对CBM2(碳水化合物结合结构域)蛋白表达量的诱导效果.ZYM-5052自诱导培养基菌体OD600是IPTGLB培养基菌体的4.4倍.SDS-PAGE显示200 μL ZYM-5052菌体细胞含有82.3 μg CBM2,占细胞总蛋白55.3％;等量IPTG LB菌体细胞含有16.6 μg CBM2,占细胞总蛋白25.7％.取不同培养时间等量等同OD600的ZYM-5052菌体进行SDS-PAGE,结果显示单位细胞中蛋白量随着培养时间而增加.所以高密度自诱导不仅提高了细胞生长密度、而且提高了单位细胞中蛋白表达量.自诱导方案用于表达葡聚糖酶,与IPTG LB菌体相比,自诱导菌体OD600是其2.3倍,酶活性是其4.5倍.该研究为提高外源蛋白表达量提供了较为简单、普适的高密度自诱导方案.

A simple restriction enzyme-free method,step-reverse PCR,was developed to construct recombinant plasmid in two steps. An Aspergillus niger xylanase gene(Xyn),for example,was amplified in the first step with a forward primer(IF)and a chimeric reverse primer(IR). IR 3’-end was complementary to Xyn gene,and its 5’-end contained a 19 bp fragment of one end of pET20b(+). In the second reverse PCR step,the amplified Xyn 19 bp 3’-end served as the forward primer to anneal to and extend along circular pET20b template,and the recombinant plasmid was amplified by cooperation with an inverse primer(VR)complementary to pET20b at the other end. After treatment with T4 DNA ligase and restriction enzyme DpnI,the PCR product was directly transformed home-made competent BL21(DE3)E. coli cell. Generality was checked by cloning various sizes of genes(622,639,1125,1209 bp)into pET20b.

12 F /10 xylanase structures were selected from PDB,and optimum temperature was used as xylanase thermostability indicator. Amino-acid contents were calculated for secondary structures: alpha-helix,beta-strand,and irregular areas,and correlation with xylanase thermostability was analyzed. The results showed thermostability was positively correlated with the residual content of helix, with a coefficient of 3. 1,but negatively correlated with the residual content of irregular regions,with a coefficient of 2. 2,and negatively correlated with the residual content of strand,although with a low coefficient of 0. 2. This result is comparable with F /10 xylanase ( β / α) 8 structure. Helixes locate in the molecular outside,strands locate in the internal part and form the active center region of enzyme, and irregular area connects strand with helix. The result is useful for the research of protein thermostability,and can be used to increase enzyme thermostability by increasing helix content.