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Enhancement of 3-hydroxypropionic acid production from glycerol by using a metabolic toggle switch.

Tsuruno K, Honjo H, Hanai T - Microb. Cell Fact. (2015)

Bottom Line: The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions.The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux.This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Bioinformatics, Graduate School of Systems Life Sciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. ktsuruno@brs.kyushu-u.ac.jp.

ABSTRACT

Background: 3-hydroxypropionic acid (3-HP) is an important platform for the production of C3 chemicals, including acrylic acid, methyl acrylate, and acrylamide. Microbial production of 3-HP is mainly due to glycerol metabolism. In this study, in order to improve microbial 3-HP production, we applied a metabolic toggle switch for controlling the glycerol metabolism to redirect the excess metabolic flux of central metabolic pathway toward an exogenous 3-HP producing pathway in Escherichia coli.

Results: The metabolic toggle switch enables conditional repression of the expression of a target gene during the fermentation. We individually performed conditional repression of glpK, tpiA, and gapA, which are involved in glycerol metabolism. The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions. However, gapA conditional repression contributed to improve 3-HP production (titer, 54.2 ± 1.5 mM; yield, 32.1 ± 1.3 %) compared with that for the wild type strain. Additional deletion of endogenous yqhD, which is responsible for the production of a major byproduct, 1,3-propandiol, further increased 3-HP production (titer, 67.3 ± 2.1 mM; yield, 51.5 ± 3.2 %). The titer and yield were 80 and 94 % higher than those of the wild type strain, respectively. The obtained 3-HP yield from glycerol is comparable with the highest yield ever reported for microbial 3-HP production using glycerol as a sole carbon source. The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux.

Conclusion: The conditional repression of gapA by using the metabolic toggle switch combined with deletion of endogeneous yqhD increased 3-HP production approximately twofold from glycerol. This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.

No MeSH data available.


Related in: MedlinePlus

3-HP production by an E. coli strain harboring the synthetic pathway (TA2463). a Growth curve (OD600). b Time course of glycerol consumption. c Time course of 3-HP production. d Time course of 1,3-PDO production. Error bars represent the standard deviation (n = 3)
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Fig3: 3-HP production by an E. coli strain harboring the synthetic pathway (TA2463). a Growth curve (OD600). b Time course of glycerol consumption. c Time course of 3-HP production. d Time course of 1,3-PDO production. Error bars represent the standard deviation (n = 3)

Mentions: TA2463 produced 37.7 ± 1.6 mM 3-HP and the OD600 and glycerol consumption at 48 h were 4.58 ± 0.18, 163.9 ± 1.8 mM, respectively (Fig. 3a–c). The growth rate and 3-HP production decreased after 24 h, although glycerol remained in the medium. This was also observed in a previous study, in which 200 mM glycerol was used as a substrate for 3-HP production in batch culture [20]. The 3-HP yield from glycerol was 26.5 ± 1.1 % (mol/mol). The 3-HP production and yield observed was slightly lower than that previously reported in a batch culture study using the same enzymes for 3-HP production and similar culture conditions, including the use of a shake flask [21]. This could be due to the different protein expression systems used, which resulted in different protein expression levels. A significant amount of 1,3-propanediol (1,3-PDO) (12.8 ± 1.8 mM) (Fig. 3d), which is converted from 3-HPA in a reaction catalyzed by endogenous NADPH-dependent aldehyde reductase encoded by yqhD, was produced as the main byproduct, which is in agreement with previous studies [32–34]. Acetate has been shown to be a major byproduct of 3-HP production from glycerol in other studies [20–23]. In contrast, only small amounts of acetate (maximum of 2.18 ± 0.86 mM) accumulated at 24 h. Acetate was eventually removed, perhaps as a result of reassimilation in our experimental conditions. This observation corresponds to several reports performing fermentation of E. coli under aerobic conditions using minimal medium with glycerol as the sole carbon source [35–37]. Other metabolites such as lactate were not detected during fermentation. IPTG addition at various time points (3, 6, and 9 h) did not significantly affect growth, glycerol consumption, or 3-HP and 1,3-PDO production (data not shown).Fig. 3


Enhancement of 3-hydroxypropionic acid production from glycerol by using a metabolic toggle switch.

Tsuruno K, Honjo H, Hanai T - Microb. Cell Fact. (2015)

3-HP production by an E. coli strain harboring the synthetic pathway (TA2463). a Growth curve (OD600). b Time course of glycerol consumption. c Time course of 3-HP production. d Time course of 1,3-PDO production. Error bars represent the standard deviation (n = 3)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4594890&req=5

Fig3: 3-HP production by an E. coli strain harboring the synthetic pathway (TA2463). a Growth curve (OD600). b Time course of glycerol consumption. c Time course of 3-HP production. d Time course of 1,3-PDO production. Error bars represent the standard deviation (n = 3)
Mentions: TA2463 produced 37.7 ± 1.6 mM 3-HP and the OD600 and glycerol consumption at 48 h were 4.58 ± 0.18, 163.9 ± 1.8 mM, respectively (Fig. 3a–c). The growth rate and 3-HP production decreased after 24 h, although glycerol remained in the medium. This was also observed in a previous study, in which 200 mM glycerol was used as a substrate for 3-HP production in batch culture [20]. The 3-HP yield from glycerol was 26.5 ± 1.1 % (mol/mol). The 3-HP production and yield observed was slightly lower than that previously reported in a batch culture study using the same enzymes for 3-HP production and similar culture conditions, including the use of a shake flask [21]. This could be due to the different protein expression systems used, which resulted in different protein expression levels. A significant amount of 1,3-propanediol (1,3-PDO) (12.8 ± 1.8 mM) (Fig. 3d), which is converted from 3-HPA in a reaction catalyzed by endogenous NADPH-dependent aldehyde reductase encoded by yqhD, was produced as the main byproduct, which is in agreement with previous studies [32–34]. Acetate has been shown to be a major byproduct of 3-HP production from glycerol in other studies [20–23]. In contrast, only small amounts of acetate (maximum of 2.18 ± 0.86 mM) accumulated at 24 h. Acetate was eventually removed, perhaps as a result of reassimilation in our experimental conditions. This observation corresponds to several reports performing fermentation of E. coli under aerobic conditions using minimal medium with glycerol as the sole carbon source [35–37]. Other metabolites such as lactate were not detected during fermentation. IPTG addition at various time points (3, 6, and 9 h) did not significantly affect growth, glycerol consumption, or 3-HP and 1,3-PDO production (data not shown).Fig. 3

Bottom Line: The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions.The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux.This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Bioinformatics, Graduate School of Systems Life Sciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. ktsuruno@brs.kyushu-u.ac.jp.

ABSTRACT

Background: 3-hydroxypropionic acid (3-HP) is an important platform for the production of C3 chemicals, including acrylic acid, methyl acrylate, and acrylamide. Microbial production of 3-HP is mainly due to glycerol metabolism. In this study, in order to improve microbial 3-HP production, we applied a metabolic toggle switch for controlling the glycerol metabolism to redirect the excess metabolic flux of central metabolic pathway toward an exogenous 3-HP producing pathway in Escherichia coli.

Results: The metabolic toggle switch enables conditional repression of the expression of a target gene during the fermentation. We individually performed conditional repression of glpK, tpiA, and gapA, which are involved in glycerol metabolism. The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions. However, gapA conditional repression contributed to improve 3-HP production (titer, 54.2 ± 1.5 mM; yield, 32.1 ± 1.3 %) compared with that for the wild type strain. Additional deletion of endogenous yqhD, which is responsible for the production of a major byproduct, 1,3-propandiol, further increased 3-HP production (titer, 67.3 ± 2.1 mM; yield, 51.5 ± 3.2 %). The titer and yield were 80 and 94 % higher than those of the wild type strain, respectively. The obtained 3-HP yield from glycerol is comparable with the highest yield ever reported for microbial 3-HP production using glycerol as a sole carbon source. The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux.

Conclusion: The conditional repression of gapA by using the metabolic toggle switch combined with deletion of endogeneous yqhD increased 3-HP production approximately twofold from glycerol. This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.

No MeSH data available.


Related in: MedlinePlus