Molecular breeding of the basidiomycete Coprinus cinereus strains with high lignin-decolorization and -degradation activities using novel heterologous protein expression vectors

Two chromosome-integrating vectors, pLC1 and pLC2, were used. The former is the pUC19-based vector carrying the Lentinus edodes ras gene promoter and priA gene terminator, and the latter is the pBR322-based vector carrying the promoter and terminator of the priA gene. The manganese (II) peroxidase (MnP) cDNA (mnpc) derived from Pleurotus ostreatus was fused between the promoter and terminator of pLC1 and pLC2, yielding the recombinant plasmids pLC1-mnp and pLC2-mnp. These plasmids were introduced into protoplasts of the Coprinus cinereus trp1 strain with the C. cinereus TRP1-containing plasmid pCc1001 by co-transformation. Two Trp⁺ transformants for each plasmid, showing clearly higher lignin-decolorization activities, were obtained through introduction of pLC1-mnp and pLC2-mnp. Southern-blot analysis revealed that the four transformants all possess the mnpc sequence on their chromosomes. One Trp⁺ MnP⁺ transformant (named TF2-7), which was derived from the introduction of pLC2-mnp and carried the highest number of copies (approx. 10) of mnpc, showed remarkably high lignin-decolorization and -degradation activities; at the time of cultivation when only 35%–40% of the lignin was decolored and degraded by the control Trp⁺ transformant obtained by the introduction of pCc1001 alone, almost all of the lignin was decolored and degraded by TF2-7. Received: 20 October 1997 / Accepted: 31 October 1997     

Spontaneous amplification of yeast CEN ARS plasmids

Summary Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-4 allele (as well as the URA3 and TRP1 markers) yielded His⁺ transformants at 0.1%–50% the frequency of Ura⁺ Trp⁺ transformants. Additional His⁺ derivatives arose on continuous growth of transformants originally scored as His^- Ura⁺ Trp⁺. In all cases, the His⁺ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His⁺ phenotype was lost more readily than the Ura⁺ Trp⁺ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura⁺ Trp⁺ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.     

Effect of NAD+-dependent formate dehydrogenase on anaerobic respiration of Shewanella oneidensis MR-1

An expression plasmid was constructed in order to carry out heterologous expression of the gene of the NAD⁺-dependent formate dehydrogenase (FDH) from methylotrophic bacterium Moraxella sp. in the cells of Shewanella oneidensis MR-1 under aerobic and anaerobic conditions. In both modes of cell cultivation, recombinant FDH activity was revealed in the cell lysate of the transformants. In the medium with la? tate as a carbon source, the rate of anaerobic respiration determined as the rate of conversion of fumarate (the electron acceptor) to succinate was higher in the transformant with recombinant FDH. Anaerobic cultivation of the FDH-containing transformant of S. oneidensis MR-1 in a microbial fuel cell (MFC) revealed increased current density.     

Localization-controlled specificity of FAD:threonine flavin transferases in Klebsiella pneumoniae and its implications for the mechanism of Na-translocating NADH:quinone oxidoreductase

The Klebsiella pneumoniae genome contains genes for two putative flavin transferase enzymes (ApbE1 and ApbE2) that add FMN to protein Thr residues. ApbE1, but not ApbE2, has a periplasm-addressing signal sequence. The genome also contains genes for three target proteins with the Dxx(s/t)gAT flavinylation motif: two subunits of Na⁺-translocating NADH:quinone oxidoreductase (Na⁺-NQR), and a 99.5 kDa protein, KPK_2907, with a previously unknown function. We show here that KPK_2907 is an active cytoplasmically-localized fumarate reductase. K. pneumoniae cells with an inactivated kpk_2907 gene lack cytoplasmic fumarate reductase activity, while retaining this activity in the membrane fraction. Complementation of the mutant strain with a kpk_2907-containing plasmid resulted in a complete recovery of cytoplasmic fumarate reductase activity. KPK_2907 produced in Escherichia coli cells contains 1 mol/mol each of covalently bound FMN, noncovalently bound FMN and noncovalently bound FAD. Lesion in the ApbE1 gene in K. pneumoniae resulted in inactive Na⁺-NQR, but cytoplasmic fumarate reductase activity remained unchanged. On the contrary, lesion in the ApbE2 gene abolished the fumarate reductase but not the Na⁺-NQR activity. Both activities could be restored by transformation of the ApbE1- or ApbE2-deficient K. pneumoniae strains with plasmids containing the Vibrio cholerae apbE gene with or without the periplasm-directing signal sequence, respectively. Our data thus indicate that ApbE1 and ApbE2 bind FMN to Na⁺-NQR and fumarate reductase, respectively, and that, contrary to the presently accepted view, the FMN residues are on the periplasmic side of Na⁺-NQR. A new, “electron loop” mechanism is proposed for Na⁺-NQR, involving an electroneutral Na⁺/electron symport. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Role of post-replication and excision repair mechanism in the induction ofTrp + revenants of UV-irradiatedEscherichia coli

Both the post-replication and the excision repair mechanism participate in the induction ofTrp ⁺ revertants inEscherichia coli B/rHer ⁺ thy trp after a UV-irradiation. At low radiation doses (surviving cell fraction > 10^?) mostTrp ⁺ reversions are due to post-replication repair mechanism while at high doses (surviving cell fraction « 10^?1) theTrp ⁺ reversions arise probably as the result of an inaccurate excision repair. The absolute accuracy of repair processes decreases with increasing radiation dose.     

A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair

We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNA^Trp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNA^Trp pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNA^Trp pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.     

Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila

Summary The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8⁺. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8⁺ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.     

Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris

A full-length cDNA sequence of Aoxyn11A, a mesophilic xylanase-encoding gene from Aspergillus oryzae, was obtained from total RNA, using 3′ and 5′ rapid amplification of cDNA ends methods. The cDNA sequence is 1,086 base pairs in length, containing 5′-untranslated and 3′-untranslated regions and an open reading frame encoding a 20 amino acid (aa) signal peptide, a 24 aa propeptide and a 188 aa mature peptide (designated AoXyn11A). Multiple alignments verified that AoXyn11A belongs to glycoside hydrolase family 11. Its three-dimensional structure was predicted by multiple templates–based homology modeling. In addition, an AoXyn11A-encoding cDNA gene was extracellularly expressed in Pichia pastoris GS115, mediated by the modified pPIC9K vector. One P. pastoris transformant, numbered as GSAorX4-3 and having the highest recombinant AoXyn11A (reAoXyn11A) activity of 98.0 U/ml, was chosen. The reAoXyn11A showed maximum activity at pH 5.5 and 50 °C. It was highly stable at a pH range of 4.0–8.0 and at 40 °C. Its activity was not significantly affected by metal ions that were tested or EDTA, but was strongly inhibited by Mn²⁺ and Ag⁺. The K _m and V _max of the reAoXyn11A were 1.85 mg/ml and 3,018 U/mg, respectively.     

A single mutational modification of a tryptophan-specific transfer RNA permits aminoacylation by glutamine and translation of the codon UAG

In this work we show that the wild-type (su^?7) progenitor of the recessivelethal suppressors of UAG (su⁺7(UAG)) and of UAA/G (su⁺7(UAA/G)) is the structural gene for transfer RNA^Trp, the adaptor for translating the codon UGG. The su⁺7(UAG) suppressor form of the tRNA has a C for U substitution in the middle base of the anticodon; in the su⁺7(UAA/G) suppressor tRNA both C residues of the anticodon are replaced by U. Our data establish that the mutational change altering the tRNA^Trp to a UAG suppressor is accompanied by a loss of tryptophan-accepting specificity and the acquisition of glutamine-acceptor activity.     

Transformation of Aspergillus nidulans using the argB gene

Transformation of Aspergillus nidulans has been achieved using a chimeric vector comprising Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans DNA. Protoplasts of argB^? strains (defective for the ornithine carbamoyl transferase [carbamoylphosphate: l-ornithine carbamoyltransferase, EC 2.1.3.3] gene) of A. nidulans were incubated with plasmid pSal43 containing the cloned argB⁺ gene in the presence of poly(ethylene glycol) and CaCl₂. Transformant progeny was of three types; the majority were small slow-growing colonies which were non-viable when transferred to MM. The remaining large colonies, which were recovered at a frequency of 50 μg^?1 DNA in the best experiments, made up the other two types. One group were mitotically stable, showing no evidence of instability; the other comprised unstable types which segregated apparent transformant and parental phenotypes. The apparent transformants showed similar segregational properties. Southern hybridizations with a stable transformant suggested that it arose following integration of the argB⁺ at the arg locus. Analysis of an unstable transformant suggested that possibly more than one copy of the plasmid was integrated and then subjected to rearrangement.     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

Nucleotide sequence of an endo-1,4-β-glucanase gene (celA) from Clostridium josui

The nucleotide sequence of a DNA fragment containing an endo-1,4-β-glucanase (EG-1) gene of Clostridium josui was determined by the dideoxy-chain termination method. The EG-1 coding sequence was an open reading frame encoding 369 amino acids, and a putative promoter sequence was located in the upstream region of the open reading frame. The N-terminal amino acid sequence of the endoglucanase (EG-1C) purified from the Escherichia coli transformant (JM103/pUCJ1) was consistent with the deduced sequence from ³⁰Val to ⁴⁴Lys. The estimated molecular weights of the precursor and the mature enzymes were 41,774 and 38,352, respectively. The region of amino acids from 61st to 335th of the enzyme revealed high homology with those of Bacillus sp. and Clostridium acetobutylicum endoglucanases.     

Synthetic ColE1 plasmids carrying genes for penicillin-binding proteins in Escherichia coli

Clarke and Carbon's collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA⁺-aroB⁺ (pLC29-47), ponB⁺-tonA⁺ (pLC4-43, pLC4-44, and pLC19-19), and argG⁺-dacB⁺ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation (Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977)Plasmid1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA⁺ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB⁺ led the host cell to overproduce the respective PBP.     

荧光蛋白标签对gD囊膜蛋白在BHK-21细胞亚细胞定位的影响

【目的】探究荧光蛋白标签对马疱疹病毒I型(Equine herpes virus type 1,EHV-1)gD囊膜蛋白亚细胞定位的影响。【方法】以EHV-1基因组为模板利用PCR扩增gD全基因,分别克隆至pAcGFP1-C1和p Ds Red2-N1质粒,构建p Ac-GFP-gD(GFP-gD)和p Ds-gD-Red(gD-Red)重组质粒;将GFP基因插入gD基因信号肽序列之后并克隆至PVAX-1质粒,构建PVAX-S-GFP-gD’(S-GFP-gD’)重组质粒;将Flag标签序列与gD囊膜蛋白N端序列融合后并克隆至p VAX-1表达载体,构建p VAX-Flag-gD(Flag-gD)重组质粒。将4种不同重组真核表达质粒分别转染BHK-21细胞,通过激光共聚焦显微镜对不同融合蛋白gD进行亚细胞定位。【结果】成功构建4种不同的融合蛋白gD真核表达载体;在BHK-21细胞单独表达时,不同融合蛋白gD绝大部分都定位于高尔基体,极少量定位于细胞核内。【结论】不同插入位点的荧光蛋白标签对gD囊膜蛋白亚细胞定位无明显影响,这对今后研究其它蛋白亚细胞定位提供参考。     

Thrombin Specificity: Further Evidence for the Importance of the β-Insertion Loop and Trp96. Implications of the Hydrophobic Interaction Between Trp96 and Pro60B Pro60C for the Activity of Thrombin

A number of thrombin mutants have been constructed to investigate the role of Trp⁹⁶ and the β-insertion loop for the specificity of thrombin. Thrombin(60D) consists of the replacement of the β-insertion loop (14 amino acid residues from 59 to 63, including a 9-residue insertion at position 60) with the corresponding four residues in trypsin, Tyr-Lys-Ser-Gly; thrombin(GGG) is a smaller loop mutation in which the residues Tyr^60APro^60BPro^60CTrp^60D Asp^60ELys^60F of the β-insertion loop were replaced by Gly-Gly-Gly; thrombin(96S) consists of a point mutation Trp⁹⁶→Ser; and thrombin(GGG/96S) is the double mutant incorporating both changes. Thrombin(96S) clots fibrinogen ~3 times more slowly than thrombin, with the two β-insertion loop mutants, thrombin(GGG) and thrombin(GGG/96S), reacting ~3000- and 1300-fold more slowly, respectively. The specificity constant k _cat/K _m for the cleavage of fibrinopeptide A and fibrinopeptide B by thrombin(96S) was 2.6 and 0.35 μM^?1 s^?1 respectively, compared to 10 and 2.5 μM^?1 s^?1 for wild-type recombinant thrombin, respectively. Kinetic constants were determined for the hydrolysis of H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroaniline. The Michaelis constant K _m increased ~6-fold for thrombin(96S) and >200-fold for thrombin(GGG) and thrombin(GGG/96S) when compared to wild-type recombinant thrombin, while the catalytic constant k _cat remained approximately the same. All mutants were more susceptible to inhibition by BPTI than wild-type recombinant thrombin. Clearly, the β-insertion loop is important for thrombin activity. But the mutation of Trp⁹⁶→Ser can compensate somewhat for the loss of binding at the β-insertion loop. The deletion of the hydrophobic interaction between Trp⁹⁶ and Pro^60BPro^60C appears to decrease the stability of the β-insertion loop, thereby causing a decrease in binding efficiency.     

A missense mutation in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase suppresses a mitochondrial tRNA(Asp) mutation

The nuclear suppressor allele NSM3 in strain FF1210-6C/170-E22 (E22), which suppresses a mutation of the yeast mitochondrial tRNA^Asp gene in Saccharomyces cerevisiae, was cloned and identified. To isolate the NSM3 allele, a genomic DNA library using the vector YEp13 was constructed from strain E22. Nine YEp13 recombinant plasmids were isolated and shown to suppress the mutation in the mitochondrial tRNA^Asp gene. These nine plasmids carry a common 4.5-kb chromosomal DNA fragment which contains an open reading frame coding for yeast mitochondrial aspartyl-tRNA synthetase (AspRS) on the basis of its sequence identity to the MSD1 gene. The comparison of NSM3 DNA sequences between the suppressor and the wild-type version, cloned from the parental strain FF1210-6C/170, revealed a G to A transition that causes the replacement of amino acid serine (AGU) by an asparagine (AAU) at position 388. In experiments switching restriction fragments between the wild type and suppressor versions of the NSM3 gene, the rescue of respiratory deficiency was demonstrated only when the substitution was present in the construct. We conclude that the base substitution causes the respiratory rescue and discuss the possible mechanism as one which enhances interaction between the mutated tRNA^Asp and the suppressor version of AspRS.     

Incompatibility between Flac, R386, and F:pSC101 recombinant plasmids: the specificity of F incompatibility genes.

The specificity of F incompatibility genes (inc⁺) has been studied with the Flac and R386 plasmids, members of the IncFI incompatibility group. Recently, two inc⁺ regions, incA (46.4–49.3F) and incB (43.1–46.4F) were identified by cloning these F sequences onto pSC101 and subsequently demonstrating incompatibility of the recombinants with Flac. It is shown here that the FincA⁺ recombinant is incompatible with both Flac and R386 while the FincB⁺ recombinant is incompatible only with Flac. Also, a plasmid mutant is described that has reduced incompatibility against Flac and R386. The mutation is located on the BamHI restriction fragment that contains the FincA region. These genetic findings are consistent with the deduction of Palchaudhuri and Maas, based on heteroduplex analysis of IncFI plasmids, that placed the IncFI determinant in the 46.4–48.6F region. The findings also indicate that the FincB⁺ gene product, which has been implicated in negative control of F copy number, is specific for the F replicon.     

A new way of stabilizing recombinant plasmids

A new method to stabilize recombinant plasmids extremely well was exploited using Escherichia coli Tna (trpAEI trpR tnaA) and pSC101trpI15-14 (tetracycline resistance, whole trp operon) as a model system. We mutagenized the Tna strain carrying pSC101trpI15-14 and isolated a mutant 6F484 that stably maintained the recombinant plasmid for 100 generations. From 6F484, plasmid-free cells (tetracycline sensitive) were screened for on selective agar plates containing fusaric acid. The host strain FA14 was found to have lost the ability for active transport of tryptophan, in addition to the phenotype of Trp⁻. Therefore, strain FA14 could not grow normally even in a complete medium. However, when the strain was transformed with the trp operon recombinant plasmid, its growth rate was almost restored to the original level. These results suggest that the recombinant plasmid is indispensable for the normal growth of host cells like FA14. Even if plasmid-free segregants appear during the cultivation, they cannot grow so rapidly and are diluted as a minority in total population. Consequently, owing to the deficiency of both the biosynthesis and uptake of tryptophan in host strain, the trp operun recombinant plasmid can be stably maintained.     

Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS⁺). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS⁺, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway. [BMB Reports 2012; 45(12): 748-753]