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

Abstract

BackgroundPropionic acid is used primarily as a food preservative with smaller applications as a chemical building block for the production of many products including fabrics, cosmetics, drugs, and plastics. Biological production using propionibacteria would be competitive against chemical production through hydrocarboxylation of ethylene if native producers could be engineered to reach near-theoretical yield and good productivity. Unfortunately, engineering propionibacteria has proven very challenging. It has been suggested that activation of the sleeping beauty operon in Escherichia coli is sufficient to achieve propionic acid production. Optimising E. coli production should be much easier than engineering propionibacteria if tolerance issues can be addressed.ResultsPropionic acid is produced in E. coli via the sleeping beauty mutase operon under anaerobic conditions in rich medium via amino acid degradation. We observed that the sbm operon enhances amino acids degradation to propionic acid and allows E. coli to degrade isoleucine. However, we show here that the operon lacks an epimerase reaction that enables propionic acid production in minimal medium containing glucose as the sole carbon source. Production from glucose can be restored by engineering the system with a methylmalonyl-CoA epimerase from Propionibacterium acidipropionici (0.23 ± 0.02 mM). 1-Propanol production was also detected from the promiscuous activity of the native alcohol dehydrogenase (AdhE). We also show that aerobic conditions are favourable for propionic acid production. Finally, we increase titre 65 times using a combination of promoter engineering and process optimisation.ConclusionsThe native sbm operon encodes an incomplete pathway. Production of propionic acid from glucose as sole carbon source is possible when the pathway is complemented with a methylmalonyl-CoA epimerase. Although propionic acid via the restored succinate dissimilation pathway is considered a fermentative process, the engineered pathway was shown to be functional under anaerobic and aerobic conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0735-4) contains supplementary material, which is available to authorized users.

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

“…Interestingly, Monk et al () also show an in silico comparison of different E. coli strains for the production of several compounds including 1‐propanol. However, the model uses the enzymes from the sbm operon (Gonzalez‐Garcia, McCubbin, Wille et al, ) to direct succinate to propionyl‐CoA using 19 sets of enzymes that could potentially produce the acyl‐CoA acid. In all cases, the results show no difference between the strains.…”

“…Production of lactate was also decreased, with ethanol and 1‐propanol becoming major electron sinks. The production of 1‐propanol can be expected as a consequence of the promiscuous activity of AdhE (Ammar, Wang, & Yang, ; Gonzalez‐Garcia, McCubbin, Wille et al, ). The mutations detected upstream of the adhE gene would suggest some regulatory modification most likely related to increased expression, explaining the increase in ethanol and 1‐propanol production (Figure ).…”

“…Organic acids, carbohydrates, and alcohol were quantified by ion‐exclusion chromatography using an Agilent 1200 high‐performance liquid chromatography (HPLC) system and an Agilent Hi‐Plex H column (300 × 7.7 mm; PL1170‐6830) with a guard column (SecurityGuard Carbo‐H, Phenomenex PN: AJO‐4490) as previously reported (Gonzalez‐Garcia, McCubbin, Wille et al, ).…”

“…All other media components were obtained from Sigma‐Aldrich Co. (St. Louis, MO). Three different culture media were used: Luria‐Bertani (LB) medium, super optimal broth medium, and a modified M9 mineral medium (Gonzalez‐Garcia, McCubbin, Wille et al, ). Ampicillin (100 µg/ml) and/or kanamycin (50 µg/ml) were supplemented when required.…”

“…Protein extracts were stored at –80°C before trypsin digestion. Trypsin digestion and protein identification were performed as previously reported (Gonzalez‐Garcia, McCubbin, Wille et al, ). Protein Pilot 4.2 software (Applied Biosystems, Foster City, CA) was used to identify all proteins.…”