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

Abstract

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

Engineering Escherichia coli for propionic acid production through the Wood–Werkman cycle

“…Cells were grown in complex media 0.73 Srirangan et al (2013) Propionate production was achieved by expressing the sbm operon and the methylmalonyl-CoA mutase from Methylobacterium extorquens AM1. Cells were grown in complex media 3.42-70.57 Li et al (2017) Propionate production was achieved by expressing the sbm operon and the methylmalonyl-CoA epimerase from Propionibacterium acidipropionici.…”

“…The first one used the native silent "sleeping beauty mutase (sbm)" operon (Haller, Buckel, Rétey, & Gerlt, 2000). The pathway was completed with a methylmalonyl-CoA mutase from Methylobacterium extorquens AM1 and expressed in a succinate overproducing strain to improve production (Li et al, 2017). Recently, we showed that the coexpression of a methylmalonyl-CoA epimerase allowed for propionate production in a chemically defined medium under aerobic and anaerobic conditions (Gonzalez-Garcia, McCubbin, Wille et al, 2017).…”

Cofactor selectivity in methylmalonyl-CoA mutase, a model cobamide-dependent enzyme

“…MCM catalyzes the interconversion of (R)-methylmalonyl-CoA and succinyl-CoA, a bidirectional reaction used in propionate metabolism [12,75,76], catabolism of branched amino acids and odd-chain fatty acids [76,77], polyhydroxybutyrate degradation [78], secondary metabolite biosynthesis [79], and autotrophic carbon dioxide fixation [4,80]. MCM-dependent pathways have been harnessed industrially for the bioproduction of propionate, bioplastics, biofuels, and antibiotics [81][82][83][84][85][86][87][88].…”

“…MCM catalyzes the interconversion of (R)-methylmalonyl-CoA and succinyl-CoA, a bidirectional reaction used in propionate metabolism (Hosotani et al, 1980;Savvi et al, 2008;Vrijbloed et al, 1999), catabolism of branched amino acids and odd-chain fatty acids (Fenton et al, 2014;Savvi et al, 2008), polyhydroxybutyrate degradation (Charles et al, 1997), secondary metabolite biosynthesis (Hunaiti and Kolattukudy, 1984), and autotrophic carbon dioxide fixation (Berg et al, 2007;Han et al, 2012). MCM-dependent pathways have been harnessed industrially for the bioproduction of propionate, bioplastics, biofuels, and antibiotics (Akawi et al, 2015;Aldor et al, 2002;Dayem et al, 2002;Gonzalez-Garcia et al, 2017;Gross et al, 2006;Li et al, 2017;Reeves et al, 2007;Srirangan et al, 2013).…”

Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase

“…Amongst the many products that have been produced in E. coli, PA was first produced in E. coli by Kandasamy et al [ 6 ] using the acrylate pathway. More recently, several investigations have reported PA production using the silent native operon known as the sleeping beauty mutase, sbm [ 18 – 22 ]. The sbm operon encodes four enzymes, three of them allegedly able to complete a succinate dissimilation cycle: methylmalonyl-CoA mutase (ScpA), biotin-independent methylmalonyl-CoA carboxylase (ScpB), and propionyl-CoA:succinate CoA transferase (ScpC) (Fig.…”