Exotic glycerol dehydrogenase expressing Escherichia coli increases yield of 2,3-butanediol

Abstract

Background

The thriving of biodiesel industry has led to produce 10% (v/v) crude glycerol, thus creating an overflow problem. Biofuel production is restricted by Escherichia coli due to its toxicity to bacterial cells. Therefore, a platform chemical and fuel additive 2,3-butanediol (2,3-BD) with low toxicity to microbes could be a promising alternative for biofuel production by recombinant E. coli using glycerol as the sole substrate.

Results

A novel expression system of E. coli was developed to express the dhaD gene encoding glycerol dehydrogenase (GDH) to produce value-added metabolic products through aerobic biotransformation of glycerol. The dhaD gene obtained from Klebsiella pneumoniae SRP2 was expressed in E. coli BL21(DE3)pLysS using an E. coli–K. pneumoniae shuttle vector pJET1.2/blunt consisting of chloramphenicol-resistance gene under the control of the T7lac promotor. RT-PCR analysis and dhaD overexpression confirmed that the 2,3-BD synthesis pathway gene was expressed on RNA and protein levels. Therefore, the recombinant E. coli exhibited a 38.9-fold higher enzyme activity (312.57 units/mg protein), yielding 8.97 g/L 2,3-BD, a 2.4-fold increase with respect to the non-recombinant strain.

Conclusions

The engineered strain E. coli BL21(DE3)pLysS/pJET1.2/blunt-dhaD, carrying the 2,3-BD pathway gene dhaD from our newly isolated Klebsiella pneumoniae SRP2 strain, displayed the best ability to synthesize 2,3-BD from low-cost biomass glycerol. The value of expression of an important glycerol metabolism gene dhaD is the highest ever achieved with an engineered E. coli strain. From these results, the first reported dhaD expression system has paved the way for improvement of 2,3-BD production and is efficient for another heterologous gene expression in E. coli.

Keywords

Coli BL21 / T7lac Promoter / budC Gene / Cloning Vector pJET1 / pLysS Strain

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