Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production
Bacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, enginee...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-12-01
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| Series: | Metabolic Engineering Communications |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214030120300316 |
| _version_ | 1818282446085423104 |
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| author | Yanfeng Liu Anqi Su Rongzhen Tian Jianghua Li Long Liu Guocheng Du |
| author_facet | Yanfeng Liu Anqi Su Rongzhen Tian Jianghua Li Long Liu Guocheng Du |
| author_sort | Yanfeng Liu |
| collection | DOAJ |
| description | Bacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered B. subtilis with faster growth phenotype is lacking. Here, fast-growing B. subtilis were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain B. subtilis 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of oppD, hag, and flgD genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting oppD, hag, and flgD genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h-1 in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing B. subtilis strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype. |
| first_indexed | 2024-12-13T00:21:08Z |
| format | Article |
| id | doaj.art-90ec41073d504b2b83a8c03e947e8b7a |
| institution | Directory Open Access Journal |
| issn | 2214-0301 |
| language | English |
| last_indexed | 2024-12-13T00:21:08Z |
| publishDate | 2020-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Metabolic Engineering Communications |
| spelling | doaj.art-90ec41073d504b2b83a8c03e947e8b7a2022-12-22T00:05:35ZengElsevierMetabolic Engineering Communications2214-03012020-12-0111e00141Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein productionYanfeng Liu0Anqi Su1Rongzhen Tian2Jianghua Li3Long Liu4Guocheng Du5Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China; Corresponding author. Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, ChinaScience Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, ChinaScience Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, ChinaScience Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, ChinaScience Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, ChinaBacillus subtilis is a model Gram-positive bacterium, which has been widely used as industrially important chassis in synthetic biology and metabolic engineering. Rapid growth of chassis is beneficial for shortening the fermentation period and enhancing production of target product. However, engineered B. subtilis with faster growth phenotype is lacking. Here, fast-growing B. subtilis were constructed through rational gene knockout and adaptive laboratory evolution using wild type strain B. subtilis 168 (BS168) as starting strain. Specifically, strains BS01, BS02, and BS03 were obtained through gene knockout of oppD, hag, and flgD genes, respectively, resulting 15.37%, 24.18% and 36.46% increases of specific growth rate compared with BS168. Next, strains A28 and A40 were obtained through adaptive laboratory evolution, whose specific growth rates increased by 39.88% and 43.53% compared to BS168, respectively. Then these two methods were combined via deleting oppD, hag, and flgD genes respectively on the basis of evolved strain A40, yielding strain A4003 with further 7.76% increase of specific growth rate, reaching 0.75 h-1 in chemical defined M9 medium. Finally, bioproduction efficiency of intracellular product (ribonucleic acid, RNA), extracellular product (acetoin), and recombinant proteins (green fluorescent protein (GFP) and ovalbumin) by fast-growing strain A4003 was tested. And the production of RNA, acetoin, GFP, and ovalbumin increased 38.09%, 5.40%, 9.47% and 19.79% using fast-growing strain A4003 as chassis compared with BS168, respectively. The developed fast-growing B. subtilis strains and strategies used for developing these strains should be useful for improving bioproduction efficiency and constructing other industrially important bacterium with faster growth phenotype.http://www.sciencedirect.com/science/article/pii/S2214030120300316Bacillus subtilisFast growth phenotypeGene knockoutAdaptive laboratory evolutionBioproduction |
| spellingShingle | Yanfeng Liu Anqi Su Rongzhen Tian Jianghua Li Long Liu Guocheng Du Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production Metabolic Engineering Communications Bacillus subtilis Fast growth phenotype Gene knockout Adaptive laboratory evolution Bioproduction |
| title | Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production |
| title_full | Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production |
| title_fullStr | Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production |
| title_full_unstemmed | Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production |
| title_short | Developing rapid growing Bacillus subtilis for improved biochemical and recombinant protein production |
| title_sort | developing rapid growing bacillus subtilis for improved biochemical and recombinant protein production |
| topic | Bacillus subtilis Fast growth phenotype Gene knockout Adaptive laboratory evolution Bioproduction |
| url | http://www.sciencedirect.com/science/article/pii/S2214030120300316 |
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