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Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum.

Siebert D, Wendisch VF - Biotechnol Biofuels (2015)

Bottom Line: To construct a 1-propanol producer, the operon ppdABC from Klebsiella oxytoca encoding diol dehydratase was expressed in the improved 1,2-propanediol producing strain ending up with 12 mM 1-propanol and up to 60 mM unconverted 1,2-propanediol.Production of 1,2-propanediol by C. glutamicum was improved by metabolic engineering targeting endogenous enzymes.Furthermore, to the best of our knowledge, production of 1-propanol by recombinant C. glutamicum was demonstrated for the first time.

View Article: PubMed Central - PubMed

Affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.

ABSTRACT

Background: Production of the versatile bulk chemical 1,2-propanediol and the potential biofuel 1-propanol is still dependent on petroleum, but some approaches to establish bio-based production from renewable feed stocks and to avoid toxic intermediates have been described. The biotechnological workhorse Corynebacterium glutamicum has also been shown to be able to overproduce 1,2-propanediol by metabolic engineering. Additionally, C. glutamicum has previously been engineered for production of the biofuels ethanol and isobutanol but not for 1-propanol.

Results: In this study, the improved production of 1,2-propanediol by C. glutamicum is presented. The product yield of a C. glutamicum strain expressing the heterologous genes gldA and mgsA from Escherichia coli that encode methylglyoxal synthase gene and glycerol dehydrogenase, respectively, was improved by additional expression of alcohol dehydrogenase gene yqhD from E. coli leading to a yield of 0.131 mol/mol glucose. Deletion of the endogenous genes hdpA and ldh encoding dihydroxyacetone phosphate phosphatase and lactate dehydrogenase, respectively, prevented formation of glycerol and lactate as by-products and improved the yield to 0.343 mol/mol glucose. To construct a 1-propanol producer, the operon ppdABC from Klebsiella oxytoca encoding diol dehydratase was expressed in the improved 1,2-propanediol producing strain ending up with 12 mM 1-propanol and up to 60 mM unconverted 1,2-propanediol. Thus, B12-dependent diol dehydratase activity may be limiting 1-propanol production.

Conclusions: Production of 1,2-propanediol by C. glutamicum was improved by metabolic engineering targeting endogenous enzymes. Furthermore, to the best of our knowledge, production of 1-propanol by recombinant C. glutamicum was demonstrated for the first time.

No MeSH data available.


Related in: MedlinePlus

Influence of endogenous DHAP phosphatase HdpA on 1,2-propanediol production by recombinant C. glutamicum strains. Batch cultivation of C. glutamicum WT(pEKEx3-mgsA-yqhD-gldA) (circles) and ΔhdpA(pEKEx3-mgsA-yqhD-gldA) (triangles) were performed, and a optical density at 600 nm (solid symbols) and glucose concentration (open symbols), b 1,2-propanediol (solid symbols) and acetol (open symbols) concentrations, and c glycerol (solid symbols) and DHA (open symbols) concentrations are shown. Means and standard errors of three independent cultivations are shown
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Fig3: Influence of endogenous DHAP phosphatase HdpA on 1,2-propanediol production by recombinant C. glutamicum strains. Batch cultivation of C. glutamicum WT(pEKEx3-mgsA-yqhD-gldA) (circles) and ΔhdpA(pEKEx3-mgsA-yqhD-gldA) (triangles) were performed, and a optical density at 600 nm (solid symbols) and glucose concentration (open symbols), b 1,2-propanediol (solid symbols) and acetol (open symbols) concentrations, and c glycerol (solid symbols) and DHA (open symbols) concentrations are shown. Means and standard errors of three independent cultivations are shown

Mentions: Typically, glycerol is hardly secreted by C. glutamicum WT, although two enzymes involved in glycerol formation have been found, namely gpp-encoded glycerol-3-phosphatase [38] and butA-encoded (S,S)-butanediol dehydrogenase [39]. In the experiments described above, glycerol was produced by the recombinant strains WT(pEKEx3-mgsA-gldA) and WT(pEKEx3-mgsA-yqhD-gldA) but nearly not by the parent strain WT(pEKEx3). This indicated that the heterologous enzymes present in these recombinants may be involved in glycerol formation. Since it is known that the gldA-encoded glycerol dehydratase from E. coli accepts also dihydroxyacetone, acetol, and methylglyoxal as substrates [40] (Fig. 1), it was tested if dihydroxyacetone formation can be prevented. Secretion of dihydroxyacetone by C. glutamicum WT occurs under certain conditions, e.g., acidic conditions [41], and was observed for WT(pEKEx3) under the conditions of 1,2-propanediol production described above. Two enzymes may be involved in DHA production, namely DHAP phosphatase encoded by hdpA [42] and a predicted kinase related to dihydroxyacetone kinases encoded by cg1497 [43]. To test if these enzymes are relevant for glycerol formation from DHA by the 1,2-propanediol-producing strain WT(pEKEx3-mgsA-yqhD-gldA), both genes were deleted by homologous recombination individually and in combination. The resulting strains C. glutamicum Δcg1497(pEKEx3-mgsA-yqhD-gldA), ΔhdpA(pEKEx3-mgsA-yqhD-gldA), and Δcg1497ΔhdpA(pEKEx3-mgsA-yqhD-gldA) were grown as described above for WT(pEKEx3-mgsA-yqhD-gldA). The deletion of the gene cg1497 had no impact on the 1,2-propanediol formation (data not shown). Upon deletion of hdpA, 1,2-propanediol production increased by about 90 % (Fig. 3b), while the double deletion mutant showed no further increase (data not shown). After 51 h of cultivation, C. glutamicum ΔhdpA(pEKEx3-mgsA-yqhD-gldA) accumulated 46 ± 4 mM 1,2-propanediol, which corresponds to a product yield of 0.249 mol/mol. C. glutamicum WT(pEKEx3-mgsA-yqhD-gldA) and ΔhdpA(pEKEx3-mgsA-yqhD-gldA) grew with comparable growth rates, utilized glucose comparably fast (Fig. 3a), and accumulated comparable concentrations (5 and 7 mM, respectively). However, glycerol was not a significant by-product (<5 mM) of the hdpA deletion strain, while the parental strain accumulated more than 40 mM glycerol (Fig. 3c). Thus, preventing DHA formation from DHAP by deletion of hdpA prevented subsequent formation of glycerol from DHA and improved 1,2-propanediol production.Fig. 3


Metabolic pathway engineering for production of 1,2-propanediol and 1-propanol by Corynebacterium glutamicum.

Siebert D, Wendisch VF - Biotechnol Biofuels (2015)

Influence of endogenous DHAP phosphatase HdpA on 1,2-propanediol production by recombinant C. glutamicum strains. Batch cultivation of C. glutamicum WT(pEKEx3-mgsA-yqhD-gldA) (circles) and ΔhdpA(pEKEx3-mgsA-yqhD-gldA) (triangles) were performed, and a optical density at 600 nm (solid symbols) and glucose concentration (open symbols), b 1,2-propanediol (solid symbols) and acetol (open symbols) concentrations, and c glycerol (solid symbols) and DHA (open symbols) concentrations are shown. Means and standard errors of three independent cultivations are shown
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4478622&req=5

Fig3: Influence of endogenous DHAP phosphatase HdpA on 1,2-propanediol production by recombinant C. glutamicum strains. Batch cultivation of C. glutamicum WT(pEKEx3-mgsA-yqhD-gldA) (circles) and ΔhdpA(pEKEx3-mgsA-yqhD-gldA) (triangles) were performed, and a optical density at 600 nm (solid symbols) and glucose concentration (open symbols), b 1,2-propanediol (solid symbols) and acetol (open symbols) concentrations, and c glycerol (solid symbols) and DHA (open symbols) concentrations are shown. Means and standard errors of three independent cultivations are shown
Mentions: Typically, glycerol is hardly secreted by C. glutamicum WT, although two enzymes involved in glycerol formation have been found, namely gpp-encoded glycerol-3-phosphatase [38] and butA-encoded (S,S)-butanediol dehydrogenase [39]. In the experiments described above, glycerol was produced by the recombinant strains WT(pEKEx3-mgsA-gldA) and WT(pEKEx3-mgsA-yqhD-gldA) but nearly not by the parent strain WT(pEKEx3). This indicated that the heterologous enzymes present in these recombinants may be involved in glycerol formation. Since it is known that the gldA-encoded glycerol dehydratase from E. coli accepts also dihydroxyacetone, acetol, and methylglyoxal as substrates [40] (Fig. 1), it was tested if dihydroxyacetone formation can be prevented. Secretion of dihydroxyacetone by C. glutamicum WT occurs under certain conditions, e.g., acidic conditions [41], and was observed for WT(pEKEx3) under the conditions of 1,2-propanediol production described above. Two enzymes may be involved in DHA production, namely DHAP phosphatase encoded by hdpA [42] and a predicted kinase related to dihydroxyacetone kinases encoded by cg1497 [43]. To test if these enzymes are relevant for glycerol formation from DHA by the 1,2-propanediol-producing strain WT(pEKEx3-mgsA-yqhD-gldA), both genes were deleted by homologous recombination individually and in combination. The resulting strains C. glutamicum Δcg1497(pEKEx3-mgsA-yqhD-gldA), ΔhdpA(pEKEx3-mgsA-yqhD-gldA), and Δcg1497ΔhdpA(pEKEx3-mgsA-yqhD-gldA) were grown as described above for WT(pEKEx3-mgsA-yqhD-gldA). The deletion of the gene cg1497 had no impact on the 1,2-propanediol formation (data not shown). Upon deletion of hdpA, 1,2-propanediol production increased by about 90 % (Fig. 3b), while the double deletion mutant showed no further increase (data not shown). After 51 h of cultivation, C. glutamicum ΔhdpA(pEKEx3-mgsA-yqhD-gldA) accumulated 46 ± 4 mM 1,2-propanediol, which corresponds to a product yield of 0.249 mol/mol. C. glutamicum WT(pEKEx3-mgsA-yqhD-gldA) and ΔhdpA(pEKEx3-mgsA-yqhD-gldA) grew with comparable growth rates, utilized glucose comparably fast (Fig. 3a), and accumulated comparable concentrations (5 and 7 mM, respectively). However, glycerol was not a significant by-product (<5 mM) of the hdpA deletion strain, while the parental strain accumulated more than 40 mM glycerol (Fig. 3c). Thus, preventing DHA formation from DHAP by deletion of hdpA prevented subsequent formation of glycerol from DHA and improved 1,2-propanediol production.Fig. 3

Bottom Line: To construct a 1-propanol producer, the operon ppdABC from Klebsiella oxytoca encoding diol dehydratase was expressed in the improved 1,2-propanediol producing strain ending up with 12 mM 1-propanol and up to 60 mM unconverted 1,2-propanediol.Production of 1,2-propanediol by C. glutamicum was improved by metabolic engineering targeting endogenous enzymes.Furthermore, to the best of our knowledge, production of 1-propanol by recombinant C. glutamicum was demonstrated for the first time.

View Article: PubMed Central - PubMed

Affiliation: Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.

ABSTRACT

Background: Production of the versatile bulk chemical 1,2-propanediol and the potential biofuel 1-propanol is still dependent on petroleum, but some approaches to establish bio-based production from renewable feed stocks and to avoid toxic intermediates have been described. The biotechnological workhorse Corynebacterium glutamicum has also been shown to be able to overproduce 1,2-propanediol by metabolic engineering. Additionally, C. glutamicum has previously been engineered for production of the biofuels ethanol and isobutanol but not for 1-propanol.

Results: In this study, the improved production of 1,2-propanediol by C. glutamicum is presented. The product yield of a C. glutamicum strain expressing the heterologous genes gldA and mgsA from Escherichia coli that encode methylglyoxal synthase gene and glycerol dehydrogenase, respectively, was improved by additional expression of alcohol dehydrogenase gene yqhD from E. coli leading to a yield of 0.131 mol/mol glucose. Deletion of the endogenous genes hdpA and ldh encoding dihydroxyacetone phosphate phosphatase and lactate dehydrogenase, respectively, prevented formation of glycerol and lactate as by-products and improved the yield to 0.343 mol/mol glucose. To construct a 1-propanol producer, the operon ppdABC from Klebsiella oxytoca encoding diol dehydratase was expressed in the improved 1,2-propanediol producing strain ending up with 12 mM 1-propanol and up to 60 mM unconverted 1,2-propanediol. Thus, B12-dependent diol dehydratase activity may be limiting 1-propanol production.

Conclusions: Production of 1,2-propanediol by C. glutamicum was improved by metabolic engineering targeting endogenous enzymes. Furthermore, to the best of our knowledge, production of 1-propanol by recombinant C. glutamicum was demonstrated for the first time.

No MeSH data available.


Related in: MedlinePlus