Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine
The <i>amyE</i> gene encoding α-amylase from <i>Bacillus subtilis</i> 168 was fused to several genes, the products of which are membrane proteins to express AmyE extracellularly. Genes of CapA, a subunit of the capsular poly-γ-glutamate synthetase of <i>Bacillus subtili...
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MDPI AG
2023-04-01
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| Series: | Applied Microbiology |
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| Online Access: | https://www.mdpi.com/2673-8007/3/2/26 |
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| author | Hideyuki Suzuki Nana Iwamoto Manami Nishimura |
| author_facet | Hideyuki Suzuki Nana Iwamoto Manami Nishimura |
| author_sort | Hideyuki Suzuki |
| collection | DOAJ |
| description | The <i>amyE</i> gene encoding α-amylase from <i>Bacillus subtilis</i> 168 was fused to several genes, the products of which are membrane proteins to express AmyE extracellularly. Genes of CapA, a subunit of the capsular poly-γ-glutamate synthetase of <i>Bacillus subtilis</i> subsp. <i>natto</i>; YiaT<sup>R232</sup>, the first 232 residues of YiaT, an outer membrane protein of <i>Escherichia coli</i> K-12; and PgsA, cytidine 5′-diphosphate-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase of <i>B. subtilis</i> subsp. <i>natto</i>, were used. The <i>Escherichia coli</i> strain harboring the plasmid carrying the <i>pgsA-amyE</i> fusion expressed a very high level of amylase not only on the extracellular surface of the cells but also in the medium and the intracellular space and solubilized steamed rice in two days. The N-terminal amino acid sequence of the amylase purified from the culture medium (Ser-Ala-Glu-Thr-Ala) indicated that it was cleaved at the signal peptide cleavage site of AmyE. The strain SH2204, transformed with pKN11 carrying <i>speA</i> (arginine decarboxylase gene), <i>speB</i> (agmatinase gene) and <i>argA</i><sup>ATG Y19C</sup> (<i>N</i>-acetylglutamate synthase gene) and pMAN63 carrying <i>pgsA-amyE</i> produced 1.2 mM putrescine from 5 mM arginine by adding 0.5 mM IPTG in eight days. Discarding steamed rice is not only a food loss, but also a waste of renewable, biogenerated resources due to the burning of it with petroleum, increasing carbon dioxide in the atmosphere. The <i>E. coli</i> strain developed in this study can help solve this problem because it can produce an important chemical using steamed rice as the sole carbon and energy source. |
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| language | English |
| last_indexed | 2024-03-11T02:49:21Z |
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| spelling | doaj.art-1703ba31972348b2baf0239576370f212023-11-18T09:05:15ZengMDPI AGApplied Microbiology2673-80072023-04-013237538710.3390/applmicrobiol3020026Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of PutrescineHideyuki Suzuki0Nana Iwamoto1Manami Nishimura2Division of Applied Microbiology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, JapanDivision of Applied Microbiology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, JapanDivision of Applied Microbiology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, JapanThe <i>amyE</i> gene encoding α-amylase from <i>Bacillus subtilis</i> 168 was fused to several genes, the products of which are membrane proteins to express AmyE extracellularly. Genes of CapA, a subunit of the capsular poly-γ-glutamate synthetase of <i>Bacillus subtilis</i> subsp. <i>natto</i>; YiaT<sup>R232</sup>, the first 232 residues of YiaT, an outer membrane protein of <i>Escherichia coli</i> K-12; and PgsA, cytidine 5′-diphosphate-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase of <i>B. subtilis</i> subsp. <i>natto</i>, were used. The <i>Escherichia coli</i> strain harboring the plasmid carrying the <i>pgsA-amyE</i> fusion expressed a very high level of amylase not only on the extracellular surface of the cells but also in the medium and the intracellular space and solubilized steamed rice in two days. The N-terminal amino acid sequence of the amylase purified from the culture medium (Ser-Ala-Glu-Thr-Ala) indicated that it was cleaved at the signal peptide cleavage site of AmyE. The strain SH2204, transformed with pKN11 carrying <i>speA</i> (arginine decarboxylase gene), <i>speB</i> (agmatinase gene) and <i>argA</i><sup>ATG Y19C</sup> (<i>N</i>-acetylglutamate synthase gene) and pMAN63 carrying <i>pgsA-amyE</i> produced 1.2 mM putrescine from 5 mM arginine by adding 0.5 mM IPTG in eight days. Discarding steamed rice is not only a food loss, but also a waste of renewable, biogenerated resources due to the burning of it with petroleum, increasing carbon dioxide in the atmosphere. The <i>E. coli</i> strain developed in this study can help solve this problem because it can produce an important chemical using steamed rice as the sole carbon and energy source.https://www.mdpi.com/2673-8007/3/2/26α-amylasestarchrenewablebiogenerated resourceputrescine |
| spellingShingle | Hideyuki Suzuki Nana Iwamoto Manami Nishimura Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine Applied Microbiology α-amylase starch renewable biogenerated resource putrescine |
| title | Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine |
| title_full | Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine |
| title_fullStr | Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine |
| title_full_unstemmed | Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine |
| title_short | Construction of an <i>Escherichia coli</i> Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine |
| title_sort | construction of an i escherichia coli i strain capable of utilizing steamed rice as the sole carbon and energy source by extracellular expression of amylase and its use for the production of putrescine |
| topic | α-amylase starch renewable biogenerated resource putrescine |
| url | https://www.mdpi.com/2673-8007/3/2/26 |
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