Identification and Combinatorial Overexpression of Key Genes for Enhancing ε-Poly-L-lysine Biosynthesis in Streptomyces albulus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains, Plasmids, and Media
2.2. Plasmid and Strain Construction
2.3. Shake-Flask, Batch, and Fed-Batch Fermentation of S. albulus
2.4. Fermentation Parameters Analysis
2.5. RNA Sample Preparation and RT–PCR and RT–qPCR Analysis
2.6. Metabolic Network of ε-PL Synthesis and Construction of An Econometric Model
2.7. Statistical Analysis
3. Results
3.1. Fermentation Profiles of S. albulus WG-608 and S. albulus M-Z18
3.2. Comparison of the Metabolic Flux of S. albulus WG-608 and S. albulus M-Z18
3.2.1. Metabolic Flux Analysis of Glucose-6-phosphate Node
3.2.2. Metabolic Flux Analysis of Phosphoenolpyruvate Node
3.2.3. Metabolic Flux Analysis of Pyruvate Node
3.2.4. Metabolic Flux Analysis of Oxaloacetate Node
3.2.5. Metabolic Flux Analysis of Aspartate Node
3.2.6. Metabolic Flux Analysis of α-Ketoglutarate Node
3.3. Screening and Validation of Key Genes of the ε-PL Biosynthetic Pathway
3.3.1. Screening of Key Genes
3.3.2. Construction of Key Gene Overexpression Strains
3.3.3. Effect of Key Gene Overexpression on ε-PL Synthesis
3.4. Co-Expression of Effective Key Genes Further Enhances ε-PL Synthesis
3.4.1. Shake-Flask Fermentation of OE-ppc-pyc-pls Strains
3.4.2. Fed-Batch Fermentation of OE-ppc-pyc-pls Strains
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strains | Breeding Strategies | Fermentation Strategies | Production (g/L) | Reference |
---|---|---|---|---|
Traditional Mutagenesis | ||||
S. albulus 11011A | AEC, Gly resistance screening | Constant pH 4.0 | 20.0 | [14] |
S. albulus S410 | AEC, Gly resistance screening | Two-stage pH control | 48.3 | [21] |
S. albulus SAR14 | ARTP mutagenesis | Shake flask fermentation | 1.1 | [12] |
S. albulus UN2-71 | Nitrite, UV, AEC compound treatment | Shake flask fermentation | 1.6 | [22] |
S. albulus F3-4 | Genome shuffling | Constant pH 3.8 | 13.5 | [15] |
S. albulus FEEL-1 | Ribosome engineering | Constant pH 4.0 | 24.5 | [13] |
S. albulus F4-22 | Genome shuffling | Constant pH 4.0 | 40.0 | [16] |
S. albulus AG3-28 | Gentamicin screening | Constant pH 3.8 | 56.5 | [23] |
S. albulus M-Z18 | UV and NTG mutagenesis | pH shock | 32.2 | [24] |
S. albulus R6 | ARTP mutagenesis, antibiotic screening | pH shock | 70.3 | [25] |
S. albulus GS114 | Streptomycin resistance screening | Dynamic pH control | 60.2 | [26] |
Metabolic engineering modifications | ||||
S. albulus CR1-ask | Ask gene targeted mutation | Constant pH 4.0 | 15.0 | [17] |
S. albulus PD-2 | Overexpression of the vgb | Two-stage pH control | 34.2 | [18] |
S. albulus PD-1 | Overexpression of the amtB | Two-stage pH control | 35.7 | [27] |
S. albulus PD-5 | Knockout of plsI overexpression of plsII | Two-stage pH control | 23.6 | [28] |
S. albulus NBRC14147 | Overexpression of ttm and nys | Two-stage pH control | 3.5 | [29] |
S. albulus Q-PL2 | Overexpression of pls | Constant pH 4.0 | 20.1 | [4] |
S. albulus PL05 | Overexpression of pap and ppk2Bcg | Constant pH 4.0 | 59.25 | [30] |
Strain or Plasmid | Description | Source or Reference |
---|---|---|
Strains | ||
S.albulus | ||
M-Z18 | Parent strain, ε-poly-L-lysine low-production strain | [24] |
WG-608 | Parent strain, ε-poly-L-lysine high-production strain | [36] |
OE-ppc | WG608 carrying pIB139-ppc | This study |
OE-zwf | WG608 carrying pIB139-zwf | This study |
OE-dapA | WG608 carrying pIB139-dapA | This study |
OE-lysA | WG608 carrying pIB139-lysA | This study |
OE-pyc | WG608 carrying pIB139-pyc | This study |
OE-pls | WG608 carrying pIB139-pls | This study |
Control-pIB139 | WG608 carrying pIB139 | This study |
OE-ppc-pyc-pls | WG608 carrying pIB139-ppc-pyc-pls | This study |
E. coli | ||
DH5α | General cloning host | Invitrogen |
ET12567 | Donor strain for conjugation between E. coli and Streptomyces | Invitrogen |
Plasmids | ||
pIB39 | Integrative vector based on ϕC31 integrase | [37] |
pIB139-ppc | ppc cloned in pIB139 | This study |
pIB139-zwf | zwf cloned in pIB139 | This study |
pIB139-dapA | dapA cloned in pIB139 | This study |
pIB139-lysA | lysA cloned in pIB139 | This study |
pIB139-pyc | pyc cloned in pIB139 | This study |
pIB139-pls | pls cloned in pIB139 | This study |
pIB139-ppc-pyc-pls | ppc, pls and pyc cloned in pIB139 | This study |
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Zhang, H.; Yang, H.; Zhang, C.; Zhu, D.; Wang, L.; Zhang, J.; Chen, X. Identification and Combinatorial Overexpression of Key Genes for Enhancing ε-Poly-L-lysine Biosynthesis in Streptomyces albulus. Fermentation 2024, 10, 65. https://doi.org/10.3390/fermentation10010065
Zhang H, Yang H, Zhang C, Zhu D, Wang L, Zhang J, Chen X. Identification and Combinatorial Overexpression of Key Genes for Enhancing ε-Poly-L-lysine Biosynthesis in Streptomyces albulus. Fermentation. 2024; 10(1):65. https://doi.org/10.3390/fermentation10010065
Chicago/Turabian StyleZhang, Hongjian, Hao Yang, Chongyang Zhang, Daojun Zhu, Liang Wang, Jianhua Zhang, and Xusheng Chen. 2024. "Identification and Combinatorial Overexpression of Key Genes for Enhancing ε-Poly-L-lysine Biosynthesis in Streptomyces albulus" Fermentation 10, no. 1: 65. https://doi.org/10.3390/fermentation10010065