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. 2021 Apr 21;11(5):230. doi: 10.1007/s13205-021-02733-8

Downregulating of hemB via synthetic antisense RNAs for improving 5-aminolevulinic acid production in Escherichia coli

Fanglan Ge 1, Dongmei Wen 1, Yao Ren 1, Guiying Chen 1, Bing He 1, Xiaokun Li 1, Wei Li 1,2,✉
PMCID: PMC8060372  PMID: 33968574

Abstract

Aminolevulinic acid (ALA), a type of natural non-protein amino acid, is a key precursor for the biosynthesis of heme, and it has been broadly applied in medicine, agriculture. Several strategies have been applied to enhance ALA synthesis in bacteria. In the present study, we employed synthetic antisense RNAs (asRNAs) of hemB (encodes ALA dehydratase) to weaken metabolic flux of ALA to porphobilinogen (PBG), and investigated their effect on ALA accumulation. For this purpose, we designed and constructed vectors pET28a-hemA-asRNA and pRSFDuet-hemA-asRNA to simultaneously express 5-ALA synthase (ALAS, encoded by hemA) and PTasRNAs (2 inverted repeat DNA sequences sandwiched with the antisense sequence of hemB), selecting the region ranging from − 57 nt upstream to + 139 nt downstream of the start codon of hemB as a target. The qRT-PCR analysis showed that the mRNA levels of hemB were decreased above 50% of the control levels, suggesting that the anti-hemB asRNA was functioning appropriately. ALA accumulation in the hemB weakened strains were 17.6% higher than that obtained using the control strains while accumulating less PBG. These results indicated that asRNAs can be used as a tool for regulating ALA accumulation in E. coli.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02733-8.

Keywords: Synthetic antisense RNAs, 5-aminolevulinic acid, 5-aminolevulinic acid dehydratase (ALAD), Metabolic regulation

Introduction

A type of natural non-protein amino acid that occurs in living organisms, 5-aminolevulinic acid (ALA), is a common precursor involved in the biosynthesis of tetrapyrroles such as heme, phycobilins, chlorophyll, and cyanocobalamin (i.e., vitamin B12) (Peng et al. 1997; Kang et al. 2012; Hamza and Dailey 2012), which are involved in various important biological processes such as enzymatic reactions, light-harvesting and electron transport (Mobius et al. 2010). In fact, two unrelated pathways have been identified for ALA biosynthesis in some organisms, including (1) the C5 pathway, plants, algae and many bacteria such as Escherichia coli and archaea (Schon et al. 1986; Sasaki et al. 2002). The C5 pathway, which converts glutamate to ALA, employs three reactions catalyzed by the following three enzymes: glutamyl-tRNA synthetase (encoded by gltX), a NADPH-dependent glutamyl-tRNA reductase (encoded by hemA), and a glutamate-1-semialdehyde aminotransferase (encoded by hemL) (Kang et al. 2017). (2) The other pathway is the C4 pathway that occurs in purple non-sulfur bacteria, yeast, birds, and mammals (Liu et al. 2014; Li et al. 2014; Kang et al. 2012) and involved the formation of ALA from succinyl-CoA and glycine via a single decarboxylating condensation reaction catalyzed by ALA synthase (ALAS) (Warnick et al. 1971). After the formation of ALA, two molecules of ALA were polymerized into a porphobilinogen (PBG) molecule by ALA dehydratase (Ding et al. 2017; Mills-Davies et al. 2017).

Until date, several studies have focused on the overexpression of ALAS-encoding genes from different species in recombinant E. coli and Corynebacterium glutamicum to produce ALA via the C4 pathway, and the production level was significantly increased after optimization of the ALAS expression and cultivation process (Choi et al. 2004; Li et al. 2014; Yang et al. 2016; Ding et al. 2017), which included the addition of ALAS inhibitors, adjusting the precursors and isopropyl-β-d-thiogalactopyranoside (IPTG) concentrations, and codon optimization. Metabolic engineering of C. glutamicum and E. coli have also been conducted to produce ALA through the C5 pathway (Kang et al. 2011; Liu et al. 2014; Zhang et al. 2013). The co-overexpression of glutamyl-tRNA reductase (encoded by hemA) and glutamate-1-semialdehyde aminotransferase (encoded by hemL) in C. glutamicum and E. coli were found to be beneficial for enhancing 5-ALA accumulation (Kang et al. 2011; Zhang et al. 2015, 2017). Furthermore, the redistribution carbon flux in the upstream pathway using different promoter for driving biosynthetic genes and shutting down the downstream flux were also tested in the recent studies. Yang et al. (2016) reported that the increase in the pool of succinyl-CoA can improve ALA production via the C4 pathway by deleting sucCD (encoding succinyl-CoA synthetase), while Noh et al. (2017) blocked the TCA cycle to promote the ALA production via the C5 pathway by deleting sucA.

As a cofactor of several enzymes, Heme is involved in the electron transport chain and is considered essential for cell survival (Mobius et al. 2010). In the heme biosynthesis pathway, 5-ALA dehydratase (ALAD), encoded by hemB, catalyzes the condensation of 2 ALA molecules into PBG, which plays an important role in the heme pathway (Jaffe et al. 1995). Thus, the inactivation of ALA dehydratase (ALAD) is impractical. Since the past 20 years, some attempts have been made to weaken the expression of this essential downstream gene to decrease the consumption of ALA (Yu et al 2015; Zhang et al. 2019). Researches have revealed that HemB inhibitors such as levulinic acid (Sasaki et al. 1987), D-xylose and D-glucose can effectively promote ALA accumulation (Lee et al. 2003). Recently, Zhang et al. (2018) performed ribosome-binding site (RBS) replacement to downregulate the expression of hemB. In addition, Zhang et al. (2019) replaced the promoter ALA dehydratase with fliCp to weaken the ALA catabolism, while Su et al. (2019) fine-tuned the expression of HemB with CRISPR interference (CRISPRi), and improved the ALA accumulation.

Antisense RNAs (asRNAs) are a class of single-stranded RNA molecules that are capable of pairing complementarily with their target mRNA and inhibiting the gene expression (Nakashima et al. 2006). They are particularly useful for silencing such essential genes whose deletion is usually deemed lethal to the host cell. A few researches applied asRNA for metabolic engineering in bacteria (Sun et al. 2018; Yang et al. 2015; Wu et al. 2014; Na et al. 2013). In this study, we employed synthetic asRNAs with a loop-stem structure to conditionally downregulate the expression of HemB. The transcriptional levels of hemB mRNA, the concentration of ALA and its downstream metabolite, and PBG of the hemB-weakened strains were investigated. In the present study, we have introduced an effective strategy to enhance the ALA yield.

Materials and methods

Bacterial strains, plasmids, and growth conditions

The bacterial strains, plasmids, and primers used in this study are listed under Table 1. Rhodobacter capsulatus was cultivated in Van Niel’s yeast agar medium (composed of 10 g/L yeast extract, 1.0 g/L K2PO4, 0.5 g/L MgSO4, pH adjusted to 7.0–7.2) under anaerobic conditions in the light of tungsten filament at 30 °C. For molecular manipulation, E. coli strains harboring plasmids were routinely grown in Luria–Bertani (LB) medium supplemented with 2.5 µg/mL kanamycin as when required based on the harboring vectors. For ALA fermentation, shake-flask culturing was performed for E. coli strain under 200 rpm at 37 °C in 250 mL Erlenmeyer flasks containing 50 mL fermentation medium (composed of tryptone 10 g/L, yeast extract 5 g/L, glucose 2 g/L, glycine 2 g/L and succinic acid 10 g/L). The growth medium was supplemented with 25.0 µg/mL kanamycin as required. To induce the gene expression, when OD600 reached 0.6, 0.1 mM IPTG was added to the culture medium to induce the gene expression. The incubation temperature was reduced to 28 °C, while the culture pH was maintained at 6.4 with addition of 4.0 M NaOH. Bacterial cell growth was determined by measuring the optical density at 600 nm (OD600) using a UV–visible spectrophotometer (Shimadzu UVmini-1240, Kyoto, Japan).

Table 1.

Strains, plasmids and primers used in this work

Strains or plasmids Relevant property Source
Strains
Rhodobacter capsulatus The source of hemA ATCC 11166
E. coli DH5α Wild type; subcloning host Laboratory stock
E. coli Rosetta(DE3) Expressing host Laboratory stock
E. coli R0 E. coli Rosetta(DE3) harboring pET28a ( +) This study
E. coli R1 E. coli Rosetta(DE3) harboring pET28a ( +)-.hemA This study
E. coli R2 E. coli Rosetta(DE3) harboring pET28a ( +)-hemA-asRNA This study
E. coli R3 E. coli Rosetta(DE3) harboring pRSFDuet-1 This study
E. coli R4 E. coli Rosetta(DE3) harboring pRSFDuet-hemA This study
E. coli R5 E. coli Rosetta(DE3) harboring pRSFDuet-hemA-asRNA This study
E. coli B0 E.coli BL21 harboring pET28a ( +) This study
E. coli B1 E.coli BL21 harboring pET28a ( +)-hemA This study
E. coli B2 E.coli BL21 harboring pRSFDuet-1 This study
E. coli B3 E.coli BL21 harboring pRSFDuet-hemA This study
Plasmids
pET28a ( +) Kanr, T7 promoter Laboratory stock
pET28a( +)-hemA pET28a ( +) carrying hemA from R. capsulatus This study
pET28a( +)-hemA-asRNA pET28a ( +) carrying hemA from R. capsulatus and PTasRNA This study
pRSFDuet-1 Kanr, T7 promoter Novagen
pRSFDuet-hemA pRSFDuet-1 carrying hemA from R. capsulatus This study
pRSFDuet-hemA-asRNA pET28a ( +) carrying hemA from R. capsulatus and PTasRNA This study
Primers
Pf-hemA 5′-CTGCATATGGACTACAATCTCGCGCTCGACAAAG-3’ This study
Pr-hemA 5′- ATAGGATCCAGAATGGCTCAGGCAGAGGCC-3’ This study
Pf-PTasRNA 5′-CTCAAGCTTCTCTGCAGGTCGTAAATCACTG This study
Pr-PTasRNA 5′-TACTCGAGCTAGGAGGAATTAACCATGCAGTGG This study
Pf-16sRNA 5′-GCTCGTGTTGTGAAATGTT-3’ This study
Pr-16sRNA 5′-GCTCGTGTTGTGAAATGTT-3’ This study
Pf2-PTasRNA 5′-CAAAGATCGGCAACACCAGG-3’ This study
Pr2-PTasRNA 5′-TTAATCCAACGCCCTCGTCG-3’ This study
Pf-hemB 5′-CAGGTACAGGCGATTCGTCA-3’ This study
Pr-hemB 5′-TCACGACGGTTCATTGGGTT-3’ This study
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The design of the asRNA

The DNA sequence of PTasRNA synthesized by BGI (Beijing, China), was cloned into the pUC57 vector; the detail DNA sequence of this synthesized asRNA has been added to the supplemental material (Fig. S1). A paired-termini (PT) designed to stabilize asRNA cassettes contained 2 inverted repeat DNA sequences (each PT contained 38 bp) sandwiched with the antisense sequence of hemB (Fig. 1). High GC content in the PT is beneficial to strengthen the pairing and to withstand RNase degradation of RNA. The flank of the antisense sequence of hemB (196 bp) was designed with 2 restriction endonuclease sites NcoI and BamH I to facilitate the displacement of different asRNAs sequences.

Fig. 1.

Fig. 1

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Schematic of the constructed plasmids for simultaneously expressing PTasRNA and hemA. a The predicted secondary structure of the PTasRNA. Ptrc trc promoter, lacO lac operator, PT 38-bp paired-terminal sequence. b A schematic of the pRSFD-hemA-asRNA. hemA from R. capsulatus was inserted into pRSFDuet, resulting into expressing plasmids pRSFDuet-hemA. The artificially synthesized anti-hemB PTasRNA sequence, driven by the trc promoter, was inserted into the pRSFDuet-hemA, obtained the expression vector pRSFDuet-hemA-asRNA. c The schematic of the pET28a-hemA-asRNA. hemA from R. capsulatus was inserted into pET28a, resulting into expressing plasmids pET28a-hemA. The anti-hemB PTasRNA sequence was inserted into the pET28a-hemA, obtained the expression vectors pET28a-hemA-asRNA

Construction of plasmid simultaneously co-expressing hemA and anti-hemB asRNA

hemA (Genbank Id: X53864) was obtained by PCR amplification using the total DNA of R. capsulatus as a template. PCR products with NdeI and BamHI restriction sites at the proximal ends were inserted into pET28a (+) and pRSFDuet digested with the same enzymes, resulting in the formation of plasmid pET28a-hemA and pRSFDuet-hemA. The sequences of the constructed plasmid were confirmed by DNA sequencing.

The rrnB T1 terminator (GenBank: U13872.1; the sequences are shown in Fig. S1), synthesized by BGI (Beijing, China), was digested with BamHI/HindIII and inserted into the corresponding site of pET28a-hemA to terminate the hemA transcription. The PTasRNA sequence was digested with SacI/NotI and cloned into the corresponding restriction site of the abovementioned plasmid to obtain the recombination product pRSFDuet-hemA-asRNA (Fig. 1b). hemA was amplified by PCR with the primer pairs Pf-PTasRNA/ Pr-PTasRNA and the plasmid pUC57-PTasRNA as a template, followed by digestion with HindIII and XhoI, and ligation with pET28a-hemA using the same restriction enzyme digestion to construct pET28a–hemA-asRNA (see Fig. 1c).

Analysis of ALA and PBG

The fermentation liquid was centrifuged for 5 min at 12,000 rpm, and the supernatant obtained was diluted to the appropriate concentration. The concentration of ALA in the culture medium was determined as per the method described by Mauzerall and Granick (Mauzerall et al. 1956). Briefly, 1 mL of a suitable sample diluent or the standard solution was mixed with 0.5 mL 1 M sodium acetate (pH 4.6) and 0.25 mL of acetylacetone (2,4-pentanedione) in a cuvette, followed by heating at 100 °C for 15 min. Then, the mixtures were cooled to the room temperature, and the same amount of Ehrlich’s reagent (p-dimethylaminobenzaldehyde in 95% EtOH with HClO4 as catalyst) (Mauzerall et al. 1956) was added into them. After incubation at the room temperature for 30 min, the absorbance was measured at 554 nm. For determination of the concentration of PBG, 1 mL of a suitable sample diluent was mixed with 1 mL of Ehrlich’s reagent. After 30 min of the reaction, the absorbance was measured at 554 nm. The concentrations of ALA and PBG were then deduced from the standard curve prepared separately. All assays were performed in triplicate, and the results were presented as means ± SD (n = 3).

RNA extraction and qPCR

Total RNA extraction from E. coli cells was performed using the RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instruction. Total RNA was treated with DNase I to remove the genomic DNA completely. cDNA was synthesized from the total template RNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). RT-PCR was performed with 50 ng cDNA using the SYBR Premix Ex Taq II (Takara) in 25 µL reaction volume under the following cycling conditions: 95 °C for 30 s; followed by 40 cycles at 95 °C for 5 s; and then at 60 °C for 30 s. Next, 16S rRNA was used for the normalization of the RT-PCR values with the primers Pf-16sRNA and Pr-16sRNA. hemB and PTasRNA were amplified with the primer pairs Pf-hemB/Pr-hemB and Pf2-PTasRNA/Pr2-PTasRNA.

Results

Effect of different expression hosts and expression vector on the ALA yield

Under natural conditions, E. coli strain Rossett and BL21 can produce ALA with extremely low yield approximately 30–70 mg/L through the C-5 pathway, which is influenced by several factors. The 5-ALA synthase (ALAS, encoded by hemA), which catalyzes the one-step condensation of succinyl-CoA and glycine to form ALA (Jaffe et al. 1995). It is a crucial enzyme involved in the C4 pathway. Since E. coli does not possess the C4 pathway, a heterologous ALAS must be introduced in its system. As the host and expression vector can influence the ALA yield, we selected pET28a (a low-copy number in E. coli Rosetta and BL21 strain) and pRSFDuet (a high copy number in E. coli Rosetta and BL21 strain) as expression vectors, respectively. hemA from Rhodobacter capsulatus was inserted into pET28a and pRSFDuet, resulting into expressing plasmids pET28a-hemA and pRSFDuet-hemA, respectively, followed by overexpression in E. coli Rosetta (DE3) and E. coli BL21, respectively. The expression of hemA and the accumulation of ALA were examined in four cell lines using different IPTG concentrations and in the absence of IPTG (data not shown). The results obtained showed that E. coli Rosetta/pET28a-hemA had the highest ALA yield of 529 mg/mL among the four strains, followed by that of E. coli BL21/pET28a-hemA (432 mg/mL) (Fig. 2), and then by that of E. coli Rosetta/pRSFDuet-hemA (389 mg/mL). E. coli BL21/pRSFDuet-hemA exhibited the lowest ALA accumulation with 342 mg/mL concentration, suggesting that E. coli Rosetta (DE3) was the preferred host, and that pET28a was the preferred expression vector for ALA production.

Fig. 2.

Fig. 2

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ALA yield of different recombinant bacteria hosts

Design and characterization of asRNAs targeting hemB

Considering that the disruption of hemB resulted in poor cellular viability (Zhang et al. 2018), we employed synthetic antisense RNAs (asRNAs) to conditionally downregulate the hemB expression and achieve ALA enrichment in E. coli. To increase the stability of asRNAs, we synthesized a DNA fragment carrying a paired termini (PT) and inserted the asRNA targeting hemB between them to generate a stem-loop structure. The anti-hemB PTasRNA sequence, driven by the trc promoter, was inserted into the pRSFDuet-hemA and pET28a-hemA, to obtain the expression vectors pET28a-hemA-asRNA and pRSFDuet-hemA-asRNA, respectively. Subsequently, these expression vectors were introduced into the E. coli Rosetta strain.

The sequence that binds the target mRNA constitutes a crucial portion of an asRNA molecule. Moreover, Crosbyetal and Nakashima et al. (2006), suggested that the target region on mRNA covering the RBS and start codon is an ideal target for asRNA-mediated gene silencing. Therefore, we selected the target region ranging from − 57 nt upstream to + 139 nt downstream of the start codon.

The expression profiles of the anti-hem B PTasRNA in strain Rosetta/ pET28a-hemA-asRNA and strain Rosetta/pRSFDuet-hemA-asRNA were quantitated by fluorescence quantitative real-time RT-PCR (qRT-PCR). Meanwhile, the strain Rosetta/pET28a-hemA and Rosetta/ PRSFDuet-hemA were used as control. As illustrated in Fig. 3, the expression level of the control in the presence of 1 mmol/L IPTG was set to 1; as expected, anti-hem B PTasRNA exhibited much higher expression level in the two experimental groups when compared with the control groups, and the relative difference reached the highest level at 8 h. This result indicated that the PTasRNA vector tightly regulated the expression of PTasRNA, which is expressed at a significantly high level.

Fig. 3.

Fig. 3

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Comparison of the transcriptional levels of PTasRNA mRNA and hemB mRNA of different recombinant E. coli strains. a The level of PTasRNA mRNA in E. coli Rosetta/pET28a-hemA-asRNA. b The level of PTasRNA mRNA in E.coli Rosetta/pRSFDuet-hemA-asRNA. c The level of hemB mRNA in E. coli Rosetta/pET28a-hemA-asRNA. d The level of hemB mRNA in E. coli Rosetta/pRSFDuet-hemA-asRNA

To evaluate whether this asRNA system could work with high performance, the transcriptional levels of hemB mRNA from asRNA-regulated strains were calculated relative to the control. As shown in Fig. 3, > 50% of the downregulation of the hemB expression was observed in the strain with pET28a-hemA-asRNA than that in the control. This observation demonstrated that the expression of asRNA could knockdown the hemB transcriptional level. Moreover, the growth of strains expressing asRNA were slightly affected (as shown in Fig. 4), and the growth lag phase was found to be longer than that of the control strains, probably due to the lower expression level of HemB in the former.

Fig. 4.

Fig. 4

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Cell growth of different recombinant E.coli strains

Effects of hemB downregulation on ALA accumulation

ALAD (encoded by hemB) is the first enzyme involved in the conversion of ALA to porphyrin. To measure the effect of anti-hemB PTasRNA on the accumulation of ALA, Rosetta strain with pET28a-hemA-asRNA, a Rosetta strain with pRSFDuet-hemA-asRNA, and the respective control strains were cultured in the presence of 0.1 mM IPTG, respectively. The yield of ALA and PBG were determined during the fermentation process. As showed in Fig. 5, as compared with that in the control strain, the strain Rosetta/pET28a-hemA-asRNA produced more ALA, but accumulated lesser PBG. After 20 h of incubation, the recombinant strains Rosetta/pET28a-hemA-asRNA and pRSFDuet-hemA-asRNA produced approximately 1231 mg/L and 607 mg/L of ALA, respectively (Fig. 5b), which were 17.6% and 9.5% greater than those obtained with the control strains. Similarly, as shown in Fig. 5, the Rosetta strain with pRSFDuet-hemA-asRNA accumulated more ALA and less PBG than the control strain with pRSFDuet-hemA. Our results demonstrated that targeting hemB asRNA can weaken the metabolic flux of ALA to PBG, and improve the ALA production, which is consistent with the results of a previous study (Yu et al. 2015).

Fig. 5.

Fig. 5

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ALA and PBG accumulation of recombinant E. coli strains. a ALA and PBG yields in Rosetta/pET28a-hemA-asRNA strain. b ALA and PBG accumulation in strain Rosetta/ pRSFDuet-hemA

Discussion

The amino acid 5-ALA is an important intermediate in the biosynthesis of heme, that is essential for cell survival. Decreasing the consumption of ALA by 5-ALA dehydratase (ALAS) t was considered to be an appropriate strategy for accumulating more ALA. Levulinic acid, D-xylose and D-glucose have been reported to be effective inhibitors of HemB, and important for ALA accumulation (Sasaki et al.1987; Lee et al. 2003). Recently, RBS modification, promoter replacement and CRISPR interference were successfully employed to weaken the expression of hemB and increased the accumulation of ALA.

asRNAs was detected in all genomes, but it was predominately exploited in the eukaryotes for gene regulation. For bacteria, the antisense technology is less developed and applied, with only limited studies performed on the metabolic engineering application of asRNAs. Previously, Nakashima et al. designed a sRNA with a paired termini (PT) structure to stabilize the middle functioning asRNA, which could reach a 78% reduction in the acetate kinase activity in E. coli (Nakashima et al. 2006). Later, Yang et al. employed synthetic asRNAs to down-regulate the key genes involved in the fatty acid biosynthesis of E. coli to achieved fivefold malonyl-CoA enrichment (Yang et al. 2015). Recently, Sun et al. (2018) developed a PTasRNA tool in cyanobacteria to downregulate the gene expression by up to 90% in comparison with that in the wild-type (Sun et al. 2018). These successful attempts promoted us to utilize PTasRNA as a tool to control the expression of hemB and increase the accumulation of ALA.

First, we expressed hemA from R. capsulatus with pET28a and pRSFDuet and then overexpressed in E. coli Rosetta (DE3) and E. coli BL21, respectively. Our results revealed that E. coli Rosetta/pET28a-hemA gave the highest ALA yield among the 4 strains, with Rosetta/pET28a-hemA producing more ALA than E. coli Rosetta/pRSFDuet-hemA, which suggested that E. coli Rosetta (DE3) was the preferred host that contained a plasmid (pRARE) constituted from 6 rare-codon tRNAs for the codons CCC. E. coli displays a frequency of approximately 0.6% of the rare codon CCC (Sorensen et al. 1989). There are 6 rare codons (CCC) in hemA (408 codons) obtained from R. capsulatus (nearly 1.47% frequency), which explains that the codon optimizer host strain was profitable for the expression and promotion of the ALA synthesis.

We employed an artificial stem-loop structure to improve the stability of anti-hemB asRNA, and selected the region ranging from − 57 nt upstream to +139 nt downstream of the start codon as a target, followed by the insertion of the PTasRNA into the same expression plasmid of hemA, to obtain pRSFDuet-hemA-asRNA and pET28a-hemA-asRNA. The qRT-PCR analysis revealed that the anti-hemB asRNAs exhibited much higher expression level with abundance and stability, with > 50% downregulation of the hemB expression, which confirmed that the anti-hemB asRNA was functioning appropriately. While the growth of the strains expressing asRNA were only slightly affected. As compared with the control strains, the experimental group strains produced more ALA while accumulating less PBG. Both the strains Rosetta/pET28a-hemA-asRNA and Rosetta/pRSFDuet-hemA-asRNA showed similar results, suggesting that targeting hemB asRNA can weaken the metabolic flux of ALA to PBG, for improving the ALA yield. Based on our cumulative results, we concluded that asRNA is a useful tool to downregulate the activity of HemB, which is an essential enzyme involved in the heme biosynthesis, for enhancing the yield of ALA.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PPTX 78 KB) (77.7KB, pptx)

Acknowledgements

This study was funded by the Sichuan provincial Science & Technology Department (2018JY0106, 2018JY0310).

Compliance with ethical standards

Conflict of interest

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the research paper. The authors declare that they have no conflict of interest in the publication.

Footnotes

Fanglan Ge and Dongmei Wen are equally contributed to this paper.

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