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Construction of a novel anaerobic pathway in Escherichia coli for propionate production

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ABSTRACT

Background: Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application.

Results: In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose.

Conclusions: With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Electronic supplementary material: The online version of this article (doi:10.1186/s12896-017-0354-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


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Engineering of an anaerobic propionate fermentation pathway in E. coli. Bold arrows indicate engineered pathway; stars indicate deleted genes; pck* was a mutated form of the pck in the promoter region to increase its expression
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Fig1: Engineering of an anaerobic propionate fermentation pathway in E. coli. Bold arrows indicate engineered pathway; stars indicate deleted genes; pck* was a mutated form of the pck in the promoter region to increase its expression

Mentions: E. coli is a potential propionate producer, which carries a cryptic Sbm operon. This operon is constitutively inactivated under natural conditions, which consists of four genes: sbm, ygfD, ygfG and ygfH. These genes encode enzymes that catalyze conversion of succinate to propionate in a cobalamin-dependent metabolic pathway [18]. The Sbm cycle, as illustrated in Fig. 1, includes: Sbm, a methylmalonyl-CoA mutase, which catalyzes rearrangement of succinyl-CoA to methylmalonyl-CoA; YgfG, a methylmalonyl-CoA decarboxylase, which catalyzes decarboxylation of methylmalonyl-CoA to form propionyl-CoA; and YgfH, a propionyl-CoA: succinate-CoA transferase, which transfers CoA group of the propionyl-CoA product to a molecule of succinate, thus primes another round of succinate to propionate decarboxylation. The function of YgfD is unknown, but experiments showed that it might form a functional complex with Sbm [19]. As illustrated in Fig. 1, the function of the cycle is for consumption of succinate. It is most possible that succinate is no longer a major substrate for these E. coli strains, so that this operon is silenced during the evolution.Fig. 1


Construction of a novel anaerobic pathway in Escherichia coli for propionate production
Engineering of an anaerobic propionate fermentation pathway in E. coli. Bold arrows indicate engineered pathway; stars indicate deleted genes; pck* was a mutated form of the pck in the promoter region to increase its expression
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5391575&req=5

Fig1: Engineering of an anaerobic propionate fermentation pathway in E. coli. Bold arrows indicate engineered pathway; stars indicate deleted genes; pck* was a mutated form of the pck in the promoter region to increase its expression
Mentions: E. coli is a potential propionate producer, which carries a cryptic Sbm operon. This operon is constitutively inactivated under natural conditions, which consists of four genes: sbm, ygfD, ygfG and ygfH. These genes encode enzymes that catalyze conversion of succinate to propionate in a cobalamin-dependent metabolic pathway [18]. The Sbm cycle, as illustrated in Fig. 1, includes: Sbm, a methylmalonyl-CoA mutase, which catalyzes rearrangement of succinyl-CoA to methylmalonyl-CoA; YgfG, a methylmalonyl-CoA decarboxylase, which catalyzes decarboxylation of methylmalonyl-CoA to form propionyl-CoA; and YgfH, a propionyl-CoA: succinate-CoA transferase, which transfers CoA group of the propionyl-CoA product to a molecule of succinate, thus primes another round of succinate to propionate decarboxylation. The function of YgfD is unknown, but experiments showed that it might form a functional complex with Sbm [19]. As illustrated in Fig. 1, the function of the cycle is for consumption of succinate. It is most possible that succinate is no longer a major substrate for these E. coli strains, so that this operon is silenced during the evolution.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application.

Results: In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose.

Conclusions: With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Electronic supplementary material: The online version of this article (doi:10.1186/s12896-017-0354-5) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus