Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination

 For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA ^- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu⁺ transformants (μg pRPLeu10 DNA)^-1 (1×10⁶ viable protoplasts)^-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus. Received: 2 October 1995/Received last revision: 5 December 1995/Accepted: 11 December 1995     

Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbx r Carboxine-Resistance Gene

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu⁺ Cbx^r) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx^r(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 μ plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.     

A multicopy vector system for genetic studies in<Emphasis Type="Italic"> Mucor circinelloides</Emphasis> and other zygomycetes

Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu^- phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.Communicated by E. Cerdà-OlmedoOn 1 January, 2004, the Biotechnological Institute became Bioneer A/S ()     

Characterization of α-isopropylmalate synthases containing different copy numbers of tandem repeats in Mycobacterium tuberculosis

Background  

Alpha-isopropylmalate synthase (α-IPMS) is the key enzyme that catalyzes the first committed step in the leucine biosynthetic pathway. The gene encoding α-IPMS in Mycobacterium tuberculosis, leuA, is polymorphic due to the insertion of 57-bp repeat units referred to as Variable Number of Tandem Repeats (VNTR). The role of the VNTR found within the M. tuberculosis genome is unclear. To investigate the role of the VNTR in leuA, we compared two α-IPMS proteins with different numbers of amino acid repeats, one with two copies and the other with 14 copies. We have cloned leuA with 14 copies of the repeat units into the pET15b expression vector with a His₆-tag at the N-terminus, as was previously done for the leuA gene with two copies of the repeat units.     

Two Origins for the Gene Encoding α-Isopropylmalate Synthase in Fungi

Background

The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy.

Methodology/Principal Findings

Here we identified the leuA gene encoding α-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3⁺ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently.

Conclusions/Significance

The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events.     

Codon optimization of the calf prochymosin gene and its expression in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Chymosin as an important industrial enzyme widely used in cheese manufacture. The yeast Kluyveromyces lactis is a promising host strain for expression of the chymosin gene. However, low yields (80 U/ml in shake flask cultures) were obtained when the K. lactis strain GG799 was used to express chymosin. We hypothesized that the codon-usage bias of the host may have resulted in inefficient translation and chymosin production. To improve expression efficiency of recombinant calf chymosin in K. lactis strain GG799, we designed and synthesized a DNA sequence encoding calf prochymosin using optimized codons, while keeping the G + C content relatively low. We altered 333 nucleotides to optimize codons encoding 315 amino acids. In shaking flask culture, chymosin activity was 575 U/ml in the strain expressing the optimized gene, a sevenfold higher expression level compared with the non-optimized control. SDS–PAGE analysis revealed that the purified recombinant calf chymosin had a molecular mass of 35.6 kDa, the same as the molecular weight of native calf chymosin. Alpha-casein, beta-casein, and kappa-casein were incubated with the recombinant calf chymosin from K. lactis strain GG799 or chymosin from calf stomach and the breakdown products were analyzed by SDS–PAGE. Both the recombinant calf chymosin and the native calf chymosin specifically hydrolyzed kappa-casein. Our results show that codon optimization of the calf chymosin gene improves expression in K. lactis strain GG799. Genetic manipulation to optimize codon usage has important applications for industrial chymosin production.     

Introduction of genes for leucine biosynthesis from Clostridium pasteurianum into C. acetobutylicum by cointegrate conjugal transfer

Summary A Clostridium pasteurianum gene bank was constructed in Escherichia coli, using plasmid pAT153, and several chromosomal fragments found which complemented both leuB and leuC mutations in auxotrophic E. coli K12 strains. No fragments capable of complementing leuA or leuD mutations were identified. Conjugal transfer of the LeuB/leuC genes from Bacillus subtilis into two different Leu^- C. acetobutylicum auxotrophic strains was elicited by their incorporation into a large plasmid cointegrate composed of the conjugal plasmid pAM1 and a specially constructed gram-positive, replication-deficient plasmid, pMTL21 EC. Inheritance of the cointegrate plasmid restored one of the auxotrophic C. acetobutylicum strains to prototrophy. The second strain remained Leu^-.     

Intermolecular recE4-independent recombination in Bacillus subtilis: formation of plasmid pKBT1

Summary The plasmid pKBT1 was derived by in vivo recE4-independent recombinational event(s) yielding a structure containing regions of plasmid and chromosomal origin. BamHI digests of plasmid pUB110 (Kan^r/Neo^r) and Bg/II digests of pTL12 (Tmp^r, leuA) were mixed, ligated and used to transform competent cells of a recE4 strain of Bacillus subtilis. Kanamycin-resistant transformants were electrophoretically screened for hybrid plasmids. Plasmid pKBT1 (8.0 kb) was smaller than pTL12 (10.4 kb) but larger than monomeric pUB110 (4.5 kb). Plasmid PKBT1 was stably maintained in recE4 strains of B. subtilis and conferred kanamycin resistance but did not specify trimethoprim resistance or leucine prototrophy. At least 86% of the pUB110 monomer length was present in pKBT1 and was completely contained within a single 5.58 kb HindIII fragment. The other segment of pKBT1 was of chromosomal origin as evidenced by lack of homology to pTL12 and strong hybridization to B. subtilis chromosomal DNA. At least one of the in vivo recE4-independent event(s) which produced pKBT1 must have involved intermolecular recombination between transforming and chromosomal DNA. This finding differs from previous reports in which recE4-independent recombination involving pUB110 sequences was a strictly intramolecular event.     

Direct Effect of Sodium Nitrite on Extracted Histones

Irpex lacteus milk-clotting enzyme hydrolyzed the Phe(105)-Met(106) bond of κ-casein, causing the precipitation of para-κ-casein along with other casein fractions in the presence of calcium ions, with a mechanism similar to other milk-clotting enzymes. Furhtermore, Irpex enzyme hydrolyzed at the positions Leu(79)-Ser(80) and Tyr(30)-Val(31) of para-κ-casein.

Degradation patterns of β-casein by Irpex and Mucor miehei enzymes were almost the same by polyacrylamide gel electrophoresis, but Endothia parasitica enzyme showed a different degradation pattern. Under the conditions employed, β-casein appeared to be scarcely hydrolyzed by chymosin.

Comparing the specificity of Irpex enzyme on β-casein with that of chymosin, the common cleaving points were Leu(165)-Ser(166), Ala(189)-Phe(190), and Tyr(192)-Glu(193). The difference in the specificity between the enzymes was exhibited in the cleavage at the Leu(139)-Leu(140) bond by chymosin and of the Ser(142)-Trp(143) bond by Irpex enzyme. Although the cleaving points of β-casein by both enzymes resembled each other, each enzyme exhibited different degradation patterns of β-casein because of thier different order of cleavage.     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.     

Constitutive expression,purification and characterization of bovine prochymosin in <Emphasis Type="Italic">Pichia pastoris</Emphasis> GS115

Chymosin can specifically break down the Phe105–Met106 peptide bond of milk κ-casein to form insoluble para-κ-casein, resulting in milk coagulation, a process that is used in making cheese. In this study, in order to obtain an alternative milk coagulant which is safe and efficient, and simultaneously can produce cheese with a good taste, bovine prochymosin B was chosen and constitutively expressed to a high level in Pichia pastoris. The recombinant chymosin was expressed mainly as a secretory form, and it exhibited milk-clotting activity. It was purified by ammonium sulfate fractionation, anion exchange, followed by cation exchange chromatography. A final yield of 24.2% was obtained for the purified enzyme, which appeared as a single band in SDS–PAGE having a molecular mass of approximate 36 kDa. Proteolysis assay showed that it specifically hydrolyzed κ-casein. It was stable at 25–50°C and had optimal activity at 37°C and pH 4.0. The activity of the recombinant chymosin was activated by cations such as Mn²⁺, Fe³⁺, Mg²⁺ and Na⁺, but inhibited by K⁺, Co²⁺, Zn²⁺, Ni²⁺, and to a lesser extent by Cu²⁺. These results suggested that recombinant bovine chymosin is an acid milk coagulant, and it could be considered as a safe and efficient enzyme suitable for use in cheese production.     

Selective activity of <Emphasis Type="Italic">Mucor plumbeus</Emphasis> reductase towards (−)-camphorquinone

The biotransformation of 1R-(−)-camphorquinone, achieved by growing cells of four fungi species isolated from soil (Mucor plumbeus, Lecanicillium muscarium, Thamnostylum sp. and Syncephalastrum racemosum), was investigated in optimized culture media for each species. Fungi were grown aerobically under shaking and their activities with respect to camphorquinone were monitored for 20 days by gas chromatography coupled to mass spectrometry (GCMS). Camphorquinone was found to be stable in control flasks throughout the experiment. The most interesting results were found for M. plumbeus, which was only able to perform monoreduction of camphorquinone when cultivated on a glucose–peptone–yeast extract medium. Large-scale experiments were set up and the camphorquinone biotransformation products formed by M. plumbeus were purified by column chromatography and identified by ¹H and ¹³C nuclear magnetic resonance (NMR). Theoretical calculations were employed as a complementary technique to unambiguously identify the biotransformation products. These findings suggest that M. plumbeus could be of great use for the selective reduction of camphorquinone and related compounds.     

A large-scale<Emphasis Type="Italic"> Agrobacterium</Emphasis>-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10^-3 to 10^-5 compared with random integration by non-homologous end joining. We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10³ stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10^-2 using the gene for diphtheria toxin A fragment as a negative marker. The established transformation procedure provides a basis for efficient gene targeting in rice.Abbreviations AS: Acetosyringone - 5-FU: 5-Fluorouracil - FW: Fresh weight - GT: Gene targeting - HR: Homologous recombination - NHEJ: Non-homologous end joining Communicated by H. Ebinuma     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Production of Chymosin in Escherichia coli Cells and Its Enzymatic Properties

The production of prochymosin directed by a cloned cDNA under the control of a trp promoter was examined in E. coli C600 and HB101. The latter host exhibited a higher degree of expression as to the production of prochymosin in the form of inclusion bodies, which accounted for more than 15 ~ 20% of the total cellular protein. The conditions for the processing of prochymosin in the inclusion bodies to active chymosin were determined. Several enzymatic properties of the processed bacterial chymosin, such as its specific activities as to milk-clotting and proteolysis, heat stability and Ca^{2 +} dependence of the clotting activity, were almost identical to those of authentic chymosin. However, a slight difference was observed with regard to the immunological reactivity with anti-prochymosin antibody.     

A genetic map of Phycomyces blakesleeanus

Summary Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Malic enzyme: its purification and characterization fromMucor circinelloides and occurrence in other oleaginous fungi

Malic enzyme was purified 43-fold from Mucor circinelloides. The enzyme was dependent on Mg²⁺ or Mn²⁺ for activity, was not active with Dmalate and had a pH optimum at 7.8. The apparent K_m values for malate and NADP⁺ were 488 ΜM and 41 Μm respectively. The M_r of the native enzyme was 160 kDa. Five metabolic analogues of malate: oxaloacetate, tartronic acid, 1-methylenecyclopropane trans-2,3-dicarboxyIic acid, malonic acid and glutaric acid, were found to inhibit malic enzyme activity at 10 mM. Four oleaginous fungi, Mucor circinelloides, Mortierella alpina, Mortierella elongata and Pythium ultimum, were also examined, all possessed a soluble malic enzyme, two also possessed a microsomal malic enzyme.     

Efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) from stem explants using a two-step kanamycin-hygromycin selection method

Summary To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l⁻¹ of Km for 6 wk and then transfer to 30 mg l⁻¹ of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mgl⁻¹ cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16h photoperiod of 30 μmol m⁻²s⁻¹. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf dises 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.