Strain engineering for high‐level 5‐aminolevulinic acid production in Escherichia coli

Abstract

Herein, we report the development of a microbial bioprocess for high-level production of 5-aminolevulinic acid (5-ALA), a valuable non-proteinogenic amino acid with multiple applications in medical, agricultural, and food industries, using Escherichia coli as a cell factory. We first implemented the Shemin (i.e., C4) pathway for heterologous 5-ALA biosynthesis in E. coli. To reduce, but not to abolish, the carbon flux toward essential tetrapyrrole/porphyrin biosynthesis, we applied clustered regularly interspersed short palindromic repeats interference (CRISPRi) to repress hemB expression, leading to extracellular 5-ALA accumulation. We then applied metabolic engineering strategies to direct more dissimilated carbon flux toward the key precursor of succinyl-CoA for enhanced 5-ALA biosynthesis. Using these engineered E. coli strains for bioreactor cultivation, we successfully demonstrated high-level 5-ALA biosynthesis from glycerol (~30 g L −1) under both microaerobic and aerobic conditions, achieving up to 5.95 g L −1 (36.9% of the theoretical maximum yield) and 6.93 g L −1 (50.9% of the theoretical maximum yield) 5-ALA, respectively. This study represents one of the most effective bio-based production of 5-ALA from a structurally unrelated carbon to date, highlighting the importance of integrated strain engineering and bioprocessing strategies to enhance bio-based production.

Strain engineering and bioprocessing strategies for biobased production of porphobilinogen in Escherichia coli

“…The Shemin/C4 pathway was implemented in E. coli via heterologous expression of hemA from R. sphaeroides in BW∆ ldhA (Miscevic et al 2021 ). The resulting control strain, DMH, was cultivated under aerobic conditions in a batch bioreactor with ~ 30 g L −1 of glycerol as the carbon source.…”

“…Genetic implementation of the Shemin/C4 pathway in BW∆ ldhA was previously described (Miscevic et al 2021 ). Heterologous expression of the hemA gene cloned in the pK184 vector was under the control of the P lac promoter.…”

Modular control of multiple pathways of Corynebacterium glutamicum for 5-aminolevulinic acid production

“…Recently, great efforts had been made to construct recombinant E. coli and C. glutamicum by expressing an exogenous 5-ALA synthetase (ALAS) gene in order to improve 5-ALA production via the C4 pathway (Li et al 2014 ; Yang et al 2016 ; Ding et al 2017 ; Chen et al 2020 ; Yu et al 2020 ; Miscevic et al 2021 ). So far, the production of 5-ALA has been significantly improved through fermentation process optimization, pathway engineering or tolerance engineering.…”

“…For example, it was reported that HemB inhibitors such as levulinic acid (Sasaki et al 1987 ), D-xylose (Lin et al 2009 ) and D-glucose (Lee et al 2003 ) could effectively promote ALA accumulation and more recently, Zhang et al ( 2019 ) replaced the promoter of ALA dehydratase with fliCp to weaken ALA catabolism. In a different type of approach, Su et al ( 2019 ) fine-tuned the expression of hemB with CRISPR interference (CRISPRi) to improve ALA accumulation, while Miscevic et al ( 2021 ) repressed hemB expression by applying CRISPRi to target various sequences in the promoter and ORF regions for achieving the same results. However, to a certain extent, the repression of hemB impaired the growth of cells (Miscevic et al 2021 ).…”

“…In a different type of approach, Su et al ( 2019 ) fine-tuned the expression of hemB with CRISPR interference (CRISPRi) to improve ALA accumulation, while Miscevic et al ( 2021 ) repressed hemB expression by applying CRISPRi to target various sequences in the promoter and ORF regions for achieving the same results. However, to a certain extent, the repression of hemB impaired the growth of cells (Miscevic et al 2021 ). Similarly, the optimization of the C5 pathway not only increased the 5-ALA titer, but also resulted in the accumulation of downstream toxic compounds, such as protoporphyrin IX, which can induce changes in the overall metabolism and regulation of recombinant strains (Zhanget al 2015 ; Miscevic et al 2021 ).…”