Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions
<i>Cupriavidus necator</i> is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the <i>phaC1AB1</i> operon) can effectively enhance the production of <i>C. necator</i>, but usually decre...
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MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/25/1/548 |
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| author | Lei Wang Huiying Luo Bin Yao Junhu Yao Jie Zhang |
| author_facet | Lei Wang Huiying Luo Bin Yao Junhu Yao Jie Zhang |
| author_sort | Lei Wang |
| collection | DOAJ |
| description | <i>Cupriavidus necator</i> is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the <i>phaC1AB1</i> operon) can effectively enhance the production of <i>C. necator</i>, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of <i>C. necator</i> in this study by implementing strategies such as blocking the Entner–Doudoroff pathway, completing the phosphopentose pathway by expressing the <i>gnd</i> gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden–Meyerhof–Parnas pathway by expressing the <i>pfkA</i> gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD<sub>600</sub> of the <i>phaC1AB1</i>-knockout strain increased by 44.8% with <i>pfkA</i> gene expression alone, and by 93.1% with <i>gnd</i> and <i>pfkA</i> genes expressing simultaneously. During autotrophic fermentation, <i>gnd</i> and <i>pfkA</i> genes raised the OD<sub>600</sub> of <i>phaC1AB1</i>-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the <i>pfkA</i> gene on the production of <i>C. necator</i>, an alanine-producing <i>C. necator</i> was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the <i>phaC1AB1</i> operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the <i>pfkA</i> gene. The results indicate that completing the Embden–Meyerhof–Parnas pathway by expressing the <i>pfkA</i> gene is an effective method to improve the growth and production of <i>C. necator</i>. |
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| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T15:04:37Z |
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| spelling | doaj.art-540bb58fa2844f27b2ce02d116b862592024-01-10T14:59:55ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-12-0125154810.3390/ijms25010548Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic ConditionsLei Wang0Huiying Luo1Bin Yao2Junhu Yao3Jie Zhang4College of Animal Science and Technology, Northwest A&F University, Xianyang 712100, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, ChinaCollege of Animal Science and Technology, Northwest A&F University, Xianyang 712100, ChinaCollege of Animal Science and Technology, Northwest A&F University, Xianyang 712100, ChinaState Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China<i>Cupriavidus necator</i> is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the <i>phaC1AB1</i> operon) can effectively enhance the production of <i>C. necator</i>, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of <i>C. necator</i> in this study by implementing strategies such as blocking the Entner–Doudoroff pathway, completing the phosphopentose pathway by expressing the <i>gnd</i> gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden–Meyerhof–Parnas pathway by expressing the <i>pfkA</i> gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD<sub>600</sub> of the <i>phaC1AB1</i>-knockout strain increased by 44.8% with <i>pfkA</i> gene expression alone, and by 93.1% with <i>gnd</i> and <i>pfkA</i> genes expressing simultaneously. During autotrophic fermentation, <i>gnd</i> and <i>pfkA</i> genes raised the OD<sub>600</sub> of <i>phaC1AB1</i>-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the <i>pfkA</i> gene on the production of <i>C. necator</i>, an alanine-producing <i>C. necator</i> was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the <i>phaC1AB1</i> operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the <i>pfkA</i> gene. The results indicate that completing the Embden–Meyerhof–Parnas pathway by expressing the <i>pfkA</i> gene is an effective method to improve the growth and production of <i>C. necator</i>.https://www.mdpi.com/1422-0067/25/1/548alanine<i>Cupriavidus necator</i>cell densityhexose utilization pathwaymetabolic engineeringpoly-β-hydroxybutyrate |
| spellingShingle | Lei Wang Huiying Luo Bin Yao Junhu Yao Jie Zhang Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions International Journal of Molecular Sciences alanine <i>Cupriavidus necator</i> cell density hexose utilization pathway metabolic engineering poly-β-hydroxybutyrate |
| title | Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions |
| title_full | Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions |
| title_fullStr | Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions |
| title_full_unstemmed | Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions |
| title_short | Optimizing Hexose Utilization Pathways of <i>Cupriavidus necator</i> for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions |
| title_sort | optimizing hexose utilization pathways of i cupriavidus necator i for improving growth and l alanine production under heterotrophic and autotrophic conditions |
| topic | alanine <i>Cupriavidus necator</i> cell density hexose utilization pathway metabolic engineering poly-β-hydroxybutyrate |
| url | https://www.mdpi.com/1422-0067/25/1/548 |
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