A single metabolite production by Escherichia coli BW25113 and its pflA.cra mutant cultivated under microaerobic conditions using glycerol or glucose as a carbon source

Abstract

Abundant, low prices and a highly reduced nature make glycerol to be an ideal feedstock for the production of reduced biochemicals and biofuels. Escherichia coli has been paid much attention as the platform of microbial cell factories due to its high growth rate (giving higher metabolite production rate) and the capability of utilizing a wide range of carbon sources. However, one of the drawbacks of using E. coli as a platform is its mixed metabolite formation under anaerobic conditions. In the present study, it was shown that ethanol could be exclusively produced from glycerol by the wild type E. coli, while d-lactic acid could be exclusively produced from glucose by pflA.cra mutant, where the glucose uptake rate could be increased by this mutant as compared to the wild type strain. It was also shown that the growth rate is significantly reduced in pflA.cra mutant for the case of using glycerol as a carbon source due to redox imbalance. The metabolic regulation mechanisms behind the fermentation characteristic were clarified to some extent.

Keywords: ATP, adenosine triphosphate; AcCoA, acetyl-coenzyme A; Biofuel; DCW, dry cell weight; DHA, dihydroxyacetone; Escherichia coli; Ethanol production; GAPDH, -glyceraldehyde-3-phosphate dehydrogenase; GDH, glutamate dehydrogenase; Glucose; Glycerol; KH2PO4, potassium dihydrogen phosphate; KOH, potassium hydroxide; LB, Luria Bertani; LDH, lactate dehydrogenase; M9, type of minimal media; MgSO4, magnesium sulfate; NAD+, nicotinamide adenine dinucleotide; NADH, reduced form of nicotinamide adenine dinucleotide; Na2HPO4, sodium phosphate; NaCl, sodium chloride; NaOH, sodium hydroxide; OAA, oxaloacetic Acid; OD, optical density; PEP, phosphoenol pyruvate; PEP, phosphoenolpyruvic acid; PTS, phospho-transferase system; PYR, pyruvate; Pfl, pyruvate formatelyase; TCA, tri-carboxylic acid; UV, ultra violet; cAMP, cyclic adenosine monophosphate; cAMP-Crp, cAMP receptor protein; pflA.cra mutant.

Figure 1

Metabolic pathways of E. coli cultivated under microaerobic condition using glucose and glycerol.

Figure 2

Comparison of ethanol formation in wild type E. coli (a), and pflA.cra mutant (b) cultivated under microaerobic conditions using glucose and glycerol as a carbon source.

Figure 3

Comparison of the cell growth of the wild type E. coli and its pflA.cra mutant cultivated under microaerobic conditions using glucose and glycerol.

Figure 4

Comparison of the pH changes during the batch culture of E. coli BW25113 and its pflA.cra mutant containing glucose (a) and glycerol (b) as carbon sources.

Figure 5

The glucose consumption and the cell growth of the wild type and its pflA.cra mutant cultivated using glucose as a carbon source under micro aerobic condition.

Figure 6

Overall regulation mechanism of pflA.cra mutant.

Figure 7

Chromatogram of ethanol produced at 18 h using glucose (a and c) and 20 h using glycerol (b and d) as carbon source during batch culture by E. coli BW25113 and its pflA.cra mutant.