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.