Introduction
Increasing concerns over climate change and fossil fuel depletion have provoked a need for sustainable production of chemicals and fuels. One approach of particular attractiveness is the use of biological cell factories, such as microorganisms, as biocatalysts to drive chemical conversions from renewable and clean carbons, so called “bio-based production”. The emergence of novel biotechnological tools for strain engineering, in particular systems biology, synthetic biology, metabolic engineering, and genetic/genomic engineering, has promoted the development of hyper strains for bio-based production (Choi et al. 2019). In addition to the sustainability, bio-based production can have economical potentials over synthetic organic chemistry particularly for the production of complex chemicals in the pharmaceutical and fine chemical industries, such as amino acids, organic acids, and vitamins (Keasling 2010).
5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid existing in most living organisms as a metabolic intermediate toward biosynthesis of essential tetrapyrrole/porphyrin pigment compounds, such as heme (Schlicke et al. 2015) (Fig. 1). In nature, there are two major metabolic routes for 5-ALA biosynthesis, i.e., (1) C4 (also known as Shemin) pathway (mainly existing in mammals, fungi, and purple sulphur bacteria), in which succinyl-CoA and glycine are structurally fused by 5-aminolevulinate synthase (ALAS or HemA) to form 5-ALA (Kang et al. 2004), and (2) C5 pathway (existing in most bacteria, all archaea and plants), in which glutamate is converted to 5-ALA via three enzymatic reactions of ligation, reduction, and transamination catalyzed by glutamyl-tRNA synthase (GluTS), glutamyl-tRNA reductase (GluTR), and glutamate-1-semialdehyde-2,1-aminomutase (GSAM), respectively (Woodard and Dailey 1995).
Practically, 5-ALA has broad applications in many fields, such as medicine (Inoue 2017; Juzeniene et al. 2002), agriculture (Hotta et al. 1997), and food preservation (Li et al. 2016). Hence, technologies for 5-ALA production have been developed. While 5-ALA can be chemically derived from various precursors, such as levulinic acid (MacDonald 1974), tetrahydrofurfurylamine (Kawakami et al. 1991), 5-bromo esters (Ha et al. 1994), andN -furfurylphthalimide (Takeya et al. 1996), these synthetic approaches are deemed uneconomical and the production processes are often complicated for implementation with low yields (Kang et al. 2017). As a result, bio-based production of 5-ALA using either multi-enzyme systems (Meng et al. 2016) or various cell factories has been explored (Sasaki et al. 2002), in particular photosynthetic microorganisms, such asRhodobacter sphaeroides , Rhodopseudomonas palustris , andChlorella sp. (Sasaki et al. 1995), Streptomyces coelicolor(Tran et al. 2019), Corynebacterium glutamicum (Zhang and Ye 2018), as well as genetically tractable Escherichia coli(Ding et al. 2017; Zhang et al. 2015; Zhang et al. 2019).
In this study, we explored strain engineering strategies for high-level 5-ALA production in E. coli . Native biosynthesis of 5-ALA inE. coli is achieved via the C5 pathway, which is adopted in most previous studies for 5-ALA biosynthesis using E. coli as a cell factory (Kang et al. 2011; Li et al. 2014; Zhang et al. 2015). However, this metabolic route is considered mechanistically complex and energetically ineffective, particularly upon high-level 5-ALA biosynthesis, as it requires multiple tightly regulated enzymes (Wang et al. 1999) and utilization of ATP/NADPH as limiting cofactors (Li et al. 1989). On the other hand, for high-level biosynthesis of a target metabolite, it is critical to ensure intracellular abundance of the corresponding precursors, i.e., succinyl-CoA/glycine for the Shemin pathway or glutamate for the C5 pathway in the 5-ALA case. This is normally achieved by proper metabolic direction of the dissimilated carbon flux in key pathways, otherwise artificial supplementation of structurally related carbons, which are often expensive, becomes necessary. Considering the above technical aspects/limitations, we chose to implement the Shemin pathway into E. coli for heterologous 5-ALA biosynthesis with metabolic direction of the dissimilated carbon flux toward succinyl-CoA in the tricarboxylic acid (TCA) cycle. Moreover, without supplementation of structurally related carbons, glycerol was used as the sole carbon source for cultivation of engineered E. coli strains due to its low cost (Ciriminna et al. 2014) and highly reduced nature, generating approximately twice the number of reducing equivalents upon its degradation compared to traditional fermentable sugars (Murarka et al. 2008; Yazdani and Gonzalez 2007).
By initializing the formation of essential porphyrin compounds, 5-ALA is among the most conserved metabolites across all biological kingdoms (Petříčková et al. 2015; Yu et al. 2015). However, 5-ALA normally acts as a metabolic intermediate toward porphyrin biosynthesis with minimal accumulation, limiting its overproduction. Given the imperative physiological role of porphyrin compounds, attempting to accumulate 5-ALA by inactivation of the immediate post-5-ALA conversion catalyzed by 5-aminolevulinate dehydratase (HemB, encoded by hemB ) would abolish porphyrin biosynthesis (Fig. 1) and, therefore, be detrimental to the cells. Hence, instead of gene inactivation, we applied Clustered Regularly Interspersed Short Palindromic Repeats interference (CRISPRi) to repress hemB expression and, therefore, increase 5-ALA accumulation without imposing physiological impacts to the 5-ALA-producing cells. Using various engineered E. coli strains for bioreactor cultivation, we demonstrated high-level 5-ALA biosynthesis under both microaerobic and aerobic conditions with glycerol as a sole carbon source.