• Journal of Microbiology and Biotechnology
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• 제34권5호
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• pp.1178-1187
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• 2024

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.110-113
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• 1995

Transposon Tn5 was used to induce random insertional mutations in Bradyrhizobium japonicum, a soybean endosymbiont. By genomic Southern blot analysis, transposition events were found to have occurred randomly throughout the B. japonicum genome. After screening 3, 626 mutants by auxotrophy test, a histidine auxotroph was isolated. Upon plant infection test, the His mutant showed a 3~4 day delay in nodule formation.

• Journal of Microbiology
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• 제33권2호
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• pp.142-145
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• 1995

The pumb gene encoding a basic amino acid transport protein in Neurospora crassa could be cloned by using a mutant strain defective in pmb gene as a host strain, using a negative selection on the media containing amino acid analogue canavanine. To select positive transformants of the genes for cloning, an auxotrophic marker (his-2) was added to a pmb mutant strain by mating ; a triple mutant (pmn : pmb : his-2) was constructued by crossing a strain defective in basic amino acid transport system (# 1683-bat um 535 "A") to a double mutant strain defective in neutral amino acid transport and histidine production (mitrol : his-2 "a"). Crossing was performed on synthetic crossing (SC) media containing histidine. The pmn : pmb and pmn :pmb : his-2 strains were selected among the progeny colonies from crosses on plates containing 5- .mu.g/ml para-fluoro-phenylalanine (PFPA), 200 .mu.g/ml canavanine, and 500 .mu.g/ml histidine. The selected colonies were cultured on minimal media with or without histidine for discarding pmn : pmb strain, because the pmn : pmb : his -2 strain grows only on histidine containing media. The pmn :pmb : his-2 strain selected can be used as a host strain for the cloning of the pmb and the pmn genes from a Neurospora genomic library by means of positive selections.

• The Plant Pathology Journal
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• 제23권2호
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• pp.51-56
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• 2007

A plant pathogenic fungus, Gibberella zeae (anamorph: Fusarium graminearum), not only generates economic losses by causing disease on cereal grains, but also leads to severe toxicosis in human and animals through the production of mycotoxins in infected plants. Here, we characterized a histidine auxotrophic mutant of G. zeae, designated Z43R1092, which was generated using a restriction enzyme-mediated integration (REMI) procedure. The mutant exhibited pleiotropic phenotypic changes, including a reduction in mycelial growth and virulence and loss of sexual reproduction. Outcrossing analysis confirmed that the histidine auxotrophy is linked to the insertional vector in Z43R1092. Molecular analysis showed that the histidine requirement of Z43R1092 is caused by a disruption of an open reading frame, designated GzHIS7. The deduced product of GzHIS7 encodes a putative enzyme with an N-terminal glutamine amidotransferase and a C-terminal cyclase domain, similar to the Saccharomyces cerevisiae HIS7 required for histidine biosynthesis. The subsequent gene deletion and complementation analyses confirmed the functions of GzHIS7 in G. zeae. This is the first report of the molecular characterization of histidine auxotrophy in G. zeae, and our results demonstrate that correct histidine biosynthesis is essential for virulence, as well as sexual development, in G. zeae. In addition, our results could provide a G. zeae histidine auxotroph as a recipient strain for genetic transformation using this new selectable marker.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.426-429
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• 2001

The continuous production of L -proline was studied using L-histidine auxotrophic mutant of Corynebacterium acetoacidophilum under various substrate limited conditions. Among the $NH_4\;^+$ $PO_4\;^3$ and L -histidine limited conditions, the highest production of L -proline was observed under the L-histidine limited condition. Under $NH_4\;^+$ and $PO_4\;^3$ limited conditions, no or poor L-proline production was observed, respectively. For the kinetic parameters under L -histidine limitation the specific rate of L -proline production was increased with dilution rate upto $0.1hr^{-1}$ but decreased above $0.1hr^{-1}$. The volumetric rate of L -proline production was showed similar pattern with specific rate. The dried cell weight was gradually increased according to decrease the dilution rate. Specific rate of glucose consumption was proportionally increased with dilution rate. The results of continuous culture (higher production of L-proline at dilution rate $0.1hr^{-1}$) will be used in fed-batch culture for the control of cell growth rate and mass production of L-proline.

• 미생물학회지
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• 제25권1호
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• pp.46-51
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• 1987

In order to increase the production of tryptophan by maximizing expression of recombinant trp plasmid, Klebsiella pneumoniae KC 105(pheA tyrA trpE trpR tyrR) was genetically modified. KC 107, inosine monophospate(IMP) auxotroph from KC 105 and KC 108, histidine(His) auxotroph from KC 107 were also derived respectively to increase phosphoribosylpyrophosphate(PRPP) production which is required for tryptophan biosynthesis. From KC 107 phosphoribosylpyrophosphate consumption which is required for tryptophan biosynthesis. From KC 107 and KC 108, KC 109 and KC 110, both arginine auxotrophs were derived respectively. To investigate the expression of recombinant trp plasmid in the selected K. pneumoniae mutants, the auxotrophic mutants were transformed with recombinant trp plasmids pSC 101-$trpE^{FBR}$, pSC 101-trpL(.DELTA.att) $trpE^{FBR}$ (pSC 101-trp-AF). Amount of tryptophan produced and activities of tryptophan synthase of $trpR^{-}$ mutant (KC 100) and $tyrR^{-}$ mutnat(KC 105) containing recombinant plasmid pSC 101-trp operon were increased by 30-40% as compared with KC 99(pheA tyrA trpE) containing recombinant plasmid pSC 101-trp operon. Activities of tryptophan synthase and production of tryptophan of KC 108 ($His^{-}$) and KC 109($Arg^{-}$) containing recombinant plasmid pSC 101-trp operon were increase by two-fold as compared with KC 107 containing pSC 101-trp operon.

• 미생물학회지
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• 제24권3호
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• pp.243-250
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• 1986

Candida pseudotropicalis CBS 607에서의 야생형과 영양요구성 돌연변이 균주에 대한 원형질체 형성 및 재생과 상보적 영양요구성 균주간의 융합에 관해서 연구하였다. 야생형과 his tidine 혹은 adenine요구성 균주에서 원형질체 형성율은 96-100%이였으나 methionine과 tryptophan요구성 균주는 각각 52%와 72%였다. Myoinositol (0.5 mg/ml)을 첨가한 배지에서 전배양한 methionine과 tryptophan조 구성 균주는 원형질체 형성시 bovine serum albumin (BSA, 4 mg/ml)을 첨가하면 96-98%의 원형질체 형성율이 얻어졌다. 원형질체 재생 빈도는 18-20% 였으나. BSA와 myoinositol을 첨가하면 adenine요구성 균주(약 20%)을 제외한 나머지 영양요구성 균주에서는 재생율이 20% 이상 증가했다. 원형질체 융합에 대한 PEG와 $CaCl_2$,최적농도는 20%와 100mM이었고 최적 pH와 반 응시간은 각각 6.0과 30분이 있다. 상보적 영양요구성 균수의 융합빈도는 $1.5{\times}10^{-3}\;to\;8.8{\times}10^{-3}$ 였으며 원행점체의 재생 증진으로 인해 융합율이 현저히 증가했다. Histidine요구성 균주와 tryptophan 요구성 균주을 융합시켰을 때 몇개의 융합세포를 얻었는데 측정된 DNA양과 액상의 차이로 보아 aneuploid나 diploid상태임을 알 수 있었다.