Journal of Microbiology

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Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha: Jin Ho Choo , Su-Bin Lee , Hye Yun Moon , Kun Hwa Lee , Su Jin Yoo , Keun Pil Kim , Hyun Ah Kang; J. Microbiol. 2021;59(2):151-163. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0646-2

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Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein‒coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heatshock‒ resistant yeast host strains.

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A comprehensive review and comparison of L-tryptophan biosynthesis in Saccharomyces cerevisiae and Escherichia coli
Xinru Ren, Yue Wei, Honglu Zhao, Juanjuan Shao, Fanli Zeng, Zhen Wang, Li Li
Frontiers in Bioengineering and Biotechnology.2023;[Epub] CrossRef
Heat shock in Cronobacter sakazakii induces direct protection and cross-protection against simulated gastric fluid stress
Hongmei Niu, MingzheYang, Yonghua Qi, Yangtai Liu, Xiang Wang, Qingli Dong
Food Microbiology.2022; 103: 103948. CrossRef
A review of yeast: High cell-density culture, molecular mechanisms of stress response and tolerance during fermentation
Dongxu Shen, Xiaoli He, Peifang Weng, Yanan Liu, Zufang Wu
FEMS Yeast Research.2022;[Epub] CrossRef

Development of a strategy for the screening of α-glucosidase-producing microorganisms: Bo Zhou+ , Nan Huang+ , Wei Zeng+ , Hao Zhang , Guiguang Chen , Zhiqun Liang; J. Microbiol. 2020;58(2):163-172. Published online January 29, 2020; DOI: https://doi.org/10.1007/s12275-020-9267-4

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Abstract

α-Glucosidase is a crucial enzyme for the production of isomaltooligosaccharide. In this study, a novel method comprising eosin Y (EY) and α-D-methylglucoside (AMG) in glass plates was tested for the primary screening of α-glucosidaseproducing strains. First, α-glucosidase-producing Aspergillus niger strains were selected on plates containing EY and AMG based on transparent zone formation resulting from the solubilization of EY by the hydrolyzed product. Conventional
methods
that use trypan blue (TB) and p-nitrophenyl-α-Dglucopyranoside (pPNP) as indicators were then compared with the new strategy. The results showed that EY-containing plates provide the advantages of low price and higher specificity for the screening of α-glucosidase-producing strains. We then evaluated the correlation between the hydrolytic activity of α-glucosidase and diffusion distance, and found that good linearity could be established within a 6–75 U/ml enzyme concentration range. Finally, the hydrolytic and transglycosylation activities of α-glucosidase obtained from the target isolates were determined by EY plate assay and 3,5- dinitrosalicylic acid-Saccharomyces cerevisiae assay, respectively. The results showed that the diameter of the transparent zone varied among isolates was positively correlated with α-glucosidase hydrolytic activity, while good linearity could also be established between α-glucosidase transglycosylation activity and non-fermentable reducing sugars content. With this strategy, 7 Aspergillus niger mutants with high yield of α-glucosidase from 200 obvious single colonies on the primary screen plate were obtained.

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Purification, characterization of a novel α-glucosidase from Debaryomyces hansenii strain MCC 0202 and chromatographic separation for high purity isomalto-oligosaccharides production
Saravanan Rengarajan, Rameshthangam Palanivel
Process Biochemistry.2024; 136: 109. CrossRef
Development of a PMA‐LAMP visual detection assay for viable Cronobacter sakazakii
Qiming Chen, Yang Yu, Xiaodi Chen, Fangming Tu, Peng Wang, Junyi Huang, Zhanmin Liu
International Journal of Dairy Technology.2024; 77(2): 427. CrossRef
Identification of chitin synthase activator in Aspergillus niger and its application in citric acid fermentation
Chunxu Jiang, Han Wang, Menghan Liu, Li Wang, Ruwen Yang, Peng Wang, Zongmei Lu, Yong Zhou, Zhiming Zheng, Genhai Zhao
Applied Microbiology and Biotechnology.2022; 106(21): 6993. CrossRef
Cloning and characterization of a recombinant α-glucosidase from Ensifer adhaerens NBRC 100388 and evaluation of its glucosyl transfer activity
Tatsuya Suzuki, Miyu Fukaya, Kazuki Takahashi, Michiki Takeuchi, Ryotaro Hara, Jun Ogawa, Makoto Ueda
Biocatalysis and Agricultural Biotechnology.2020; 30: 101837. CrossRef

Research Support, Non-U.S. Gov'ts

Identification of the Genes Involved in 1-Deoxynojirimycin Synthesis in Bacillus subtilis MORI 3K-85: Kyung-Don Kang , Yong Seok Cho , Ji Hye Song , Young Shik Park , Jae Yeon Lee , Kyo Yeol Hwang , Sang Ki Rhee , Ji Hyung Chung , Ohsuk Kwon , Su-Il Seong; J. Microbiol. 2011;49(3):431-440. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-1238-3

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Abstract: 1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation. The genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

Purification and Characterization of the α-Glucosidase Produced by Thermophilic Fungus Thermoascus aurantiacus CBMAI 756: Ana Flávia Azevedo Carvalho , Maurício Boscolo , Roberto da Silva , Henrique Ferreira , Eleni Gomes; J. Microbiol. 2010;48(4):452-459. Published online August 20, 2010; DOI: https://doi.org/10.1007/s12275-010-9319-2

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Abstract: Αn α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0-9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na+, Ba2+, Co2+, Ni2+, Mg2+, Mn2+, Al3+, Zn2+, Ca2+ this enzyme maintained 90-105% of its maximum activity and was inhibited by Cr3+, Ag+, and Hg2+. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The Km measured for the α-glucosidase was 0.07 μM, with a Vmax of 318.0 μmol/min/mg.

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