Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis
<i>Leuconostoc</i><i>citreum</i>, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first...
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MDPI AG
2020-08-01
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| Series: | International Journal of Molecular Sciences |
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| author | Jaewoo Son Seung Hoon Jang Ji Won Cha Ki Jun Jeong |
| author_facet | Jaewoo Son Seung Hoon Jang Ji Won Cha Ki Jun Jeong |
| author_sort | Jaewoo Son |
| collection | DOAJ |
| description | <i>Leuconostoc</i><i>citreum</i>, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering <i>L. citreum</i>. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of <i>Streptococcus pyogenes</i> (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine–Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of <i>ribF</i> and <i>folE</i> genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the <i>rib</i> operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain. |
| first_indexed | 2024-03-10T17:55:24Z |
| format | Article |
| id | doaj.art-dff8cfa586ce4a439fd5c07c70c2750d |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T17:55:24Z |
| publishDate | 2020-08-01 |
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| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-dff8cfa586ce4a439fd5c07c70c2750d2023-11-20T09:12:42ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-08-012116561410.3390/ijms21165614Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin BiosynthesisJaewoo Son0Seung Hoon Jang1Ji Won Cha2Ki Jun Jeong3Department of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, KoreaDepartment of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, KoreaDepartment of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, KoreaDepartment of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea<i>Leuconostoc</i><i>citreum</i>, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering <i>L. citreum</i>. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of <i>Streptococcus pyogenes</i> (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine–Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of <i>ribF</i> and <i>folE</i> genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the <i>rib</i> operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.https://www.mdpi.com/1422-0067/21/16/5614lactic acid bacteria<i>Leuconostoc citreum</i>CRISPRisynthetic biologyriboflavin |
| spellingShingle | Jaewoo Son Seung Hoon Jang Ji Won Cha Ki Jun Jeong Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis International Journal of Molecular Sciences lactic acid bacteria <i>Leuconostoc citreum</i> CRISPRi synthetic biology riboflavin |
| title | Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis |
| title_full | Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis |
| title_fullStr | Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis |
| title_full_unstemmed | Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis |
| title_short | Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of <i>Leuconostoc citreum</i> and Its Application for Engineering Riboflavin Biosynthesis |
| title_sort | development of crispr interference crispri platform for metabolic engineering of i leuconostoc citreum i and its application for engineering riboflavin biosynthesis |
| topic | lactic acid bacteria <i>Leuconostoc citreum</i> CRISPRi synthetic biology riboflavin |
| url | https://www.mdpi.com/1422-0067/21/16/5614 |
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