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High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway.

Fang MY, Zhang C, Yang S, Cui JY, Jiang PX, Lou K, Wachi M, Xing XH - Microb. Cell Fact. (2015)

Bottom Line: Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway.In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition.Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. fangmingyue88@gmail.com.

ABSTRACT

Background: As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process.

Results: Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpEfbr /trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED+pVio was realized with a titer of 0.6±0.01 g L(-1) in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition.

Conclusion: Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.

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Related in: MedlinePlus

Production of tryptophan in flask culture with M9-YE medium (10 g L−1glucose) at 20°C for 48 h (n=3).
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Fig2: Production of tryptophan in flask culture with M9-YE medium (10 g L−1glucose) at 20°C for 48 h (n=3).

Mentions: The multivariate module for various tryptophan-producing strains were generated by combining the overexpression of trpEfbr (feedback inhibition removed by point mutation Met293Thr and Ser40Leu) and trpD under T7 promoter, which are reported to be the key genes to enhance tryptophan synthesis [23] and combinatorial knockout of trpR, tnaA, and pheA, generating eight different BL21(DE3) strains, B1–B8, possibly having different capabilities of producing tryptophan (Table 1). The eight engineered strains were subsequently cultivated in flasks with optimized culture medium (Additional file 1: Table S2, Table S3 and Table S4) for tryptophan accumulation, as shown in Figure 2. B6/pED and B8/pED showed the highest tryptophan concentration (around 0.18 g L−1), while B2/pED and B3/pED were the second highest tryptophan-producing strains, and B1/pED were the lowest tryptophan producers.Table 1


High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway.

Fang MY, Zhang C, Yang S, Cui JY, Jiang PX, Lou K, Wachi M, Xing XH - Microb. Cell Fact. (2015)

Production of tryptophan in flask culture with M9-YE medium (10 g L−1glucose) at 20°C for 48 h (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4306242&req=5

Fig2: Production of tryptophan in flask culture with M9-YE medium (10 g L−1glucose) at 20°C for 48 h (n=3).
Mentions: The multivariate module for various tryptophan-producing strains were generated by combining the overexpression of trpEfbr (feedback inhibition removed by point mutation Met293Thr and Ser40Leu) and trpD under T7 promoter, which are reported to be the key genes to enhance tryptophan synthesis [23] and combinatorial knockout of trpR, tnaA, and pheA, generating eight different BL21(DE3) strains, B1–B8, possibly having different capabilities of producing tryptophan (Table 1). The eight engineered strains were subsequently cultivated in flasks with optimized culture medium (Additional file 1: Table S2, Table S3 and Table S4) for tryptophan accumulation, as shown in Figure 2. B6/pED and B8/pED showed the highest tryptophan concentration (around 0.18 g L−1), while B2/pED and B3/pED were the second highest tryptophan-producing strains, and B1/pED were the lowest tryptophan producers.Table 1

Bottom Line: Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway.In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition.Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. fangmingyue88@gmail.com.

ABSTRACT

Background: As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process.

Results: Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpEfbr /trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED+pVio was realized with a titer of 0.6±0.01 g L(-1) in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition.

Conclusion: Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.

Show MeSH
Related in: MedlinePlus