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

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. © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Open Access *Correspondence: [email protected] 1 Department of Biology, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada Full list of author information is available at the end of the article Background With increasing fossil fuel price and environmental concern, alternative and renewable energy sources have become attractive. Biodiesel, a renewable and promising combustion fuel, is synthesized from vegetable oils and animal fats. However, the biodiesel synthesis process (transesterification) generates 10% crude glycerol as a core by-product which is the cheapest feedstock or negative-value biomass to produce a high-value green product, 2,3-BD (Rahman et al. 2015). 2,3-BD is an important platform chemical, and it is also known as an excellent building block in the synthesis of valuable chiral chemicals (Celinska and Grajek 2009; Zeng and Sabra 2011). Chemical biosynthesis processes for the production of optically pure 2,3-BD are difficult to control, complicated and expensive. Thus, the biotransformation process has been considered as the preferred method for the production of optically pure 2,3-BD (Celinska and Grajek 2009; Ji et al. 2011; Zeng and Sabra 2011). Several engineered strains of microorganisms including Saccharomyces cerevisiae, Enterobacter cloacae, Bacillus licheniformis and E. coli have been used for the production of optically pure 2,3-BD (Yan et al. 2009; Lian et al. 2014; Li et al. 2015; Wang et al. 2012). In the past few years, several microorganisms including cyanobacteria, fungi and bacteria have been proved to produce biofuels and fuel additives using different biomasses (Domínguez de María 2011; Gross 2012; Yan et al. 2009]. E. coli is extensively used as a model organism Page 2 of 11 Rahman et al. Bioresour. Bioprocess. (2018) 5:3 for biofuel production using pentose and hexose sugars from lignocellulosic biomass (Bokinsky et al. 2011). Now, it has been proved that recombinant E. coli can produce numerous biofuels including ethanol, acetone, butanol, α-pinene, isoprenol, isobutanol and fatty alcohols through biosynthetic pathways (Bokinsky et al. 2011; May et al. 2013; Atsumi et al. 2008; Zhang et al. 2013; Yang et al. 2013). Nevertheless, these biofuels are highly toxic to E. coli, and the production of new end products which are less or non-toxic to microbial cells is needed to obtain high product yield (Baez et al. 2011) using lowcost or negative-cost biomass. Thus, less toxic metabolic products such as 2,3-BD, 1,3-propanediol (1,3-PDO) and acetoin can be produced through biotechnological routes (Ji et al. 2011). Moreover, an industrially important platform chemical 2,3-BD could be produced through the oxidative pathway of recombinant E. coli (Xu et al. 2007). A high heating value (27,200 J/g) bulk chemical 2,3-BD could be used as liquid fuel or fuel additive (Xiao et al. 2012); it has very low toxicity to bacterial cells (Oliver et al. 2013). Therefore, 2,3-BD could be a promising alternative for biofuel production through recombinant E. coli strains. Several bacterial species including Klebsiella pneumoniae, K. variicola, K. oxytoca, Serratia marcescens and Enterobacter cloacae have been used to produce 2,3-BD with high yields through optimization of culture conditions or genetic engineering (Celinska and Grajek 2009; Kim et al. 2013), but these strains are pathogenic or opportunistic pathogens and have been categorized under risk group-2 microorganisms, unsuitable for industrial-scale biotransformation (Ji et al. 2011; Kim et al. 2013). The microorganisms that may cause disease in humans and animals but are unlikely to be a serious hazard to laboratory personnel, the community, animals or the environment are called risk group 2. However, the strain E. coli BL21(DE3)pLysS has been known to be non-pathogenic and does not carry any virulence factor or pathogenic mechanism causing infections (Chart et al. 2000). Consequently, E. coli BL21(DE3)pLysS strain could be the best candidate for the safe synthesis of bioproducts (Chart et al. 2000; Zhang et al. 2013). Therefore, in this research work, a preliminary attempt has been made to establish a method for 2,3-BD production efficiently using E. coli BL21(DE3)pLysS. Moreover, in the oxidative pathway of glycerol metabolisms, there are three key enzymes, viz., glycerol dehydrogenase (GDH), α-acetolactate synthase and acetoin reductase which are involved in 2,3-BD biosynthesis (Celinska and Grajek 2009; Zhang et al. 2013). Consequently, GDH is the first enzyme in the oxidative pathway for converting glycerol to dihydroxyacetone(DHA), and then 2,3-BD is produced through pyruvate (Rahman et al. 2015) (Fig. 1). Several works have been reported on the production of 2,3-BD by recombinant E. coli through metabolic engineering of the genes, budB and budC, responsible for α-acetolactate synthase and acetoin reductase enzymes, respectively (Li et al. 2012), but there is no report on the dhaD gene in the same pathway which is responsible for GDH enzyme production. Therefore, it is important to construct an efficient 2,3-BD biosynthesis pathway that includes the related gene cluster to improve 2,3-BD production. In this backdrop, our aim was to construct a novel dhaD expression system in E. coli and its application for 2,3-BD production under completely aerobic condition. In this work, a systematic approach has been taken to construct and optimize 2,3-BD production by an efficient engineered E. coli BL21(DE3)pLysS strain. This research work is the first step for systematic metabolic engineering, which we successfully made as the novel expression system of the dhaD gene, and a high enzyme activity (GDH) was achieved through batch biotransformation process using glycerol as the sole carbon source.