Metabolic engineering Escherichia coli for high-level production of pseudouridine

doi:10.21203/rs.3.rs-3007742/v1

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Metabolic engineering Escherichia coli for high-level production of pseudouridine

https://doi.org/10.21203/rs.3.rs-3007742/v1

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Background

Pseudouridine-incorporated mRNA vaccines can enhance protein expression and reduce immunogenicity, leading to a large demand for pseudouridine to be used in the production of mRNA drugs. The production of pseudouridine by chemical or semienzymatic synthesis is limited by low yields and high costs, and the production of pseudouridine via the fermentation of genetically engineered strains is an attractive prospect.

Results

In the present study, a cell factory for the efficient synthesis of pseudouridine was established. First, the pyrimidine precursor biosynthesis pathway in Escherichia coli was systematically modified to increase metabolite flux to pseudouridine biosynthesis. Next, two crucial genes (pseudouridine 5’-phosphate glycosidase gene (psuG) and phosphatase gene) were screened. An artificial pseudouridine biosynthesis pathway with high efficiency was constructed. The accumulation of pseudouridine was further increased by the deletion of pseudouridine catabolism-related genes. Ultimately, the pseudouridine titre in a 5-L bioreactor reached 7.5 g/L.

Conclusion

In this study, a cell factory producing a high yield of pseudouridine was successfully constructed and showed the potential for use in industrial production.

Escherichia coli

Pseudouridine

Precursor

Metabolic engineering

Pseudouridine (Ψ) was isolated from yeast more than 50 years ago [1] and as 5-ribosyl uracil was called the fifth nucleoside to be identified. It is the most abundant natural C-nucleoside and has been found in tRNA, small nuclear RNA, nucleolar RNA, and even in coding regions of mRNA [2]. Pseudouridine exhibits important biological functions, impacts various aspects of RNA biology, and confers distinct structural and functional properties [3]. Pseudouridine modifications can increase the stability of mRNA [4], enhance protein expression and reduce immunogenicity [5, 6] and played critical roles in increasing the efficiency of mRNA vaccines against COVID-19 [7]. Moreover, pseudouridine has been used as a biomarker of kidney disease and several kinds of cancer in disease monitoring and therapy [8, 9, 10].

COVID-19 mRNA vaccines has led to high demand for pseudouridine. Until 19 April 2023, 2,328 clinical trial registrations were shown in the Clinical Trials Database [11] when the keyword “mRNA” was entered as the search term, which suggested that there will be a large demand for pseudouridine in the future. To date, there were three approaches to produce pseudouridine. The first approach involves total chemical synthesis. Several synthetic routes have successfully developed, but multiple steps are required; moreover, the yields are low, and the costs are high [12, 13, 14]. The second approach involves natural biosynthesis. In nature, pseudouridine is synthesized by pseudouridine synthase-catalysed posttranslational isomerization of RNA uridine [15, 16, 17]. Due to the low pseudouridine content in RNA [18], large-scale production of pseudouridine by hydrolysing RNA is unfeasible. The third approach involves enzymatic synthesis. The first report enzymatic synthesis of pseudouridine was published in 2008. Uracil and ribose 5’-phosphate were incubated with pseudouridine-5’-phosphate glycosidase (ΨMP glycosidase) and alkaline phosphatase to prepare pseudouridine sample [19]. A semienzymatic synthesis approach was developed for the synthesis of pseudouridine [20]. In this approach, the substrate ribose 5’-phosphate was hydrolysed from adenosine 5′-monophosphate. Because of the high costs of adenosine 5′-monophosphate led to the enzymatic process unconducive to industrial production.

In recent years, E. coli was widely used to construct cell factories for natural product biosynthesis because of many advantages including fast growth, high efficient genetic manipulation methods and high cell density culture [21]. To overcome the problems of pseudouridine production, several strategies were used to engineer E. coli for efficient production of pseudouridine in this study (Fig. 1). The optimal strain produced 7.5g/L pseudouridine in a 5 L bioreactor by fed-batch fermentation.

-----------------------------Fig. 1----------------------------

Enhanced biosynthesis of the pseudouridine precursor in E. coli

The starting substrates and the enzymes used for pseudouridine enzymatic synthesis were existed in E. coli MG1665 [22, 23]. Nevertheless, no pseudouridine was detected by HPLC in the shake flask fermentation broth of E. coli MG1655 (Fig. 2). When phoA and EcpsuG (from MG1665) were overexpressed in MG1665, the strain MG1655(phoAEcpsuG) can produce 29.5 mg/L pseudouridine after flask fermentation (Fig. 2, Additional file: Fig. S1/S2/S3), which suggested that phosphatase and ΨMP glycosidase played a crucial role in pseudouridine biosynthesis.

To increase pseudouridine production, the pseudouridine precursors biosynthesis pathway was engineered to increase metabolite flux to pseudouridine biosynthesis (Fig. 1). thrA and argF were successively knocked out to block the branching pathway [22, 24]. Subsequently the negative regulator pepA [25, 26]was deleted to enhance pyrimidine biosynthesis, which yielded strain ZM3. After that, plasmid pZH2 was transformed into ZM3, yielding strain ZM3(phoAEcpsuG). The pseudouridine yield of ZM3(phoAEcpsuG) increased to 102.7 mg/L (Fig. 2). It was more than threefold higher than that of MG1655(phoAEcpsuG) (Fig. 2), which illustrated that increasing precursors biosynthesis was effective in improving pseudouridine production.

-----------------------------Fig. 2----------------------------

Identification of phosphatase gene for efficient pseudouridine production

The ΨMP synthesis reaction involving ΨMP glycosidase was favoured for pseudouridine production and was promoted by ΨMP dephosphorylation. Therefore, ΨMP glycosidase and phosphatase genes were the focus of this study. First, phosphatase genes were screened to determine the most suitable genes for pseudouridine production. SDT1, YKL033W-A and PHM8 from Saccharomyces cerevisiae have been reported to show dephosphorylation activity of ΨMP [27]. pumD was involved in pseudouridine biosynthesis and was predicted to be a phosphatase gene [28], so pumD was selected to test whether it has ΨMP dephosphorylation activity. Then, the codons of the four abovementioned genes, namely, SDT1, YKL033W-A, PHM8 and pumD, were optimized and overexpressed with EcpsuG in ZM3, which yielded strains ZM3(SDT1EcpsuG), ZM3(YKL033W-AEcpsuG), ZM3(PHM8EcpsuG) and ZM3(pumDEcpsuG), respectively. All four genes led to improved pseudouridine yields after fermentation in shake flasks that were approximately threefold higher than that produced with phoA (Fig. 3A). Among these genes, pumD was the most efficient for pseudouridine production, and the yield of ZM3(pumDEcpsuG) was the highest, reaching 395.7 mg/L (Fig. 3A).

-----------------------------Fig. 3----------------------------

SDS-PAGE (Fig. 3B) analysis of the supernatants showed that all the phosphatases tested were correctly expressed, although there were some variations in expression levels. Enzymatic assays revealed that PhoA had the lowest activity among the five phosphatase (Table 1), which was consistent with fermentation results. PumD had the highest activity and substrate affinity (Table 1). Therefore, pumD was chosen for subsequent experiments.

Table 1

Kinetic parameters* of phosphatase
Enzyme	Substrate	K_m(mM)	k_cat (S ^− 1)	k_cat/K_m (mM ^− 1S ^− 1)
PhoA	ΨMP	86 ± 24.7	3 ± 0.4	0.035
SDT1	ΨMP	22.8 ± 3.9	7 ± 1.2	0.307
YKL033W-A	ΨMP	24.7 ± 4.6	6 ± 1.5	0.243
PHM8	ΨMP	29.5 ± 3.5	9 ± 1.7	0.305
PumD	ΨMP	9.8 ± 1.7	10 ± 1.4	1.02

*Data are presented as mean ± s.d. (n = 3).

It is worth noting that all the four aforementioned phosphatases belong to the haloacid dehalogenase protein family [27, 28], which suggests that haloacid dehalogenases are potential sources for screening more efficient phosphatases for use in pseudouridine production. Haloacid dehalogenase family phosphatases detoxify ΨTP and ΨMP in Saccharomyces cerevisiae and humans [27], and genome analysis revealed that Group I C-glycosynthases usually coexist with a haloacid dehalogenase family phosphatase gene located in the vicinity of the C-glycosynthase gene [29], which may support the speculation that haloacid dehalogenases are candidates for screening more efficient phosphatases against ΨMP.

Identification of ΨMP glycosidase gene for efficient pseudouridine production

psuG is widely presented in the genomes of prokaryotes [19]. In this study, EcpsuG and four other psuG genes from different sources were tested. KspsuG (from Klebsiella spallanzanii), RspsuG (from Rhizobium sp. CF142), SspsuG (from Saccharopolyspora spinosa) and SppsuG (from Streptomyces platensis) came from different bacteria belonged to different genera and had different genetic distance to EcpsuG (Fig. 4A). The plasmids carrying the four aforementioned codon-optimized psuG genes co-overexpressed with pumD were transformed into ZM3, yielding strains ZM3(pumDKspsuG), ZM3(pumDRspsuG), ZM3(pumDSspsuG), and ZM3(pumDSppsuG). Compared with that of ZM3(pumDEcpsuG), the titre of pseudouridine from ZM3(pumDRspsuG) was increased by 28% and reached 506.6 mg/L, which was the highest in all the aforementioned strains (Fig. 4B). Pseudouridine were detected in the fermentation broth of Streptomyces platensis [30], but SppsuG was not efficient for pseudouridine production, and the yield of ZM3(pumDSppsuG) was only 78.2 mg/L (Fig. 4B).

-----------------------------Fig. 4----------------------------

SDS-PAGE (Fig. 4C) analysis of the supernatants showed that all the PsuG tested were correctly expressed. Enzymatic assays revealed that RspsuG had the most activity and substrate affinity than the others, and it was 6-fold more efficient and showed 1.57-fold more substrate affinity than EcpsuG (Table 2), which was consistent with fermentation results.

Table 2

Kinetic parameters* of ΨMP glycosidase
Enzyme	Substrate	K_m(µM)	k_cat (S ^− 1)	k_cat/K_m (mM ^− 1S ^− 1)
EcpsuG	Uracil	223 ± 61	5.5 ± 0.52	24.7
KspsuG	Uracil	378 ± 72	2.5 ± 0.31	6.6
RspsuG	Uracil	127 ± 18	18.6 ± 2.1	146.4
SspsuG	Uracil	836 ± 185	1.2 ± 0.09	1.4
SppsuG	Uracil	766 ± 162	1.5 ± 0.17	1.9

*Data are presented as mean ± s.d. (n = 3).

Fine-tuning of gene expression

Various factors, such as plasmid copy number, promoter strength, gene copy number and order of genes, can affect gene expression level [31, 32]. To further improve the yield of pseudouridine, five recombinant E. coli strains were constructed according to different genetic strategies (Fig. 5). There were significant differences in pseudouridine yield under different genetic strategies. ZM3(pZH23) exhibited the highest yield, reached to 592 mg/L (Fig. 5). The only difference between ZM3(pZH23) and ZM3(pumDEcpsuG) was the cloning order of pumD and RspsuG, however, the yield of ZM3(pumDEcpsuG) was lower than that of ZM3(pZH23). When the T7 promoter of pZH23 was replaced by trc promoter, the yield of ZM3(pZH20) decreased. When pumD and RspsuG were cloned in pCDFDuet-1 under individual T7 promoter, although the copy number increased, the yield of ZM3(pZH23) was the lowest (Fig. 5).

-----------------------------Fig. 5----------------------------

Knockout of pseudouridine catabolism-related genes

The involvement of psuK has been identified in pseudouridine metabolism in Escherichia coli UTI89 and is specifically phosphorylated pseudouridine to be pseudouridine 5’-phosphate. psuT was predicted to function as pseudouridine uptake which coorperated with psuK and psuG to metabolite extracellular pseudouridine [19, 33]. Based on their functions, knockout of psuK and psuT was expected to contribute to pseudouridine accumulation. psuK and psuT were successively deleted in ZM3, yielding ZM4 and ZM5, respectively. Plasmid pZH23, which exhibited the most efficient pseudouridine production, was transformed into ZM4 and ZM5, yielding ZM4(pZH23) and ZM5(pZH23), respectively. The deletion of psuK exerted no positive effect on pseudouridine production (Fig. 6). After deleting psuT, the yield of ZM5(pZH23) increased by 8%, reaching 631 mg/L (Fig. 6). The result of fermentation of the psuT mutant suggested that blocking pseudouridine uptake was conducive to the accumulation of pseudouridine.

-----------------------------Fig. 6----------------------------

Fed-batch fermentation of pseudouridine in a 5-L bioreactor

The strain ZM5(pZH23), which exhibited the highest yeild of pseudouridine in shake flasks, was used to explore the production potential of a fed-batch fermentation strategy under the indicated cultivation conditions. As shown in Fig. 7, during the growth stage, the 10 g/L glucose concentration initially used was almost exhausted after 4 h, and glucose was then fed at the appropriate rate. In the initial 8 h, the cells grew exponentially but no product accumulated. IPTG was added to the medium after 8 h of cultivation, and the fermentation temperature was decreased to 30°C which was optimal for the reaction of ΨMP glycosidase synthesis [20]. Then, pseudouridine began to accumulate. The cell concentration reached maximum at 32 h, after that the OD₆₀₀ value began to decrease steadily, but pseudouridine continued to accumulate, exhibiting a cell growth-independent production profile. At 52 h, the final pseudouridine titre reached 7.5 g/L, indicating a productivity rate of 0.14 g/L/h.

-----------------------------Fig. 7----------------------------

In this study, a cell factory producing pseudouridine was successfully constructed. The deletion of argF, thrA and pepA which were involved in pseudouridine precursors biosynthesis could increase pseudouridine production. psuG from Rhizobium sp. CF142 and pumD from Streptomyces sp. ID38640 constituted the most efficient combination for pseudouridine biosynthesis. The deletion of psuT further increased the yield of pseudouridine. Ultimately, the yield of pseudouridine reached 7.5 g/L in a 5-L bioreactor via fed-batch fermentation. To our knowledge, this is the first report about engineered E. coli to produce pseudouridine. This study provides a foundation for the large-scale industrial production of pseudouridine via microbial fermentation.

Bacterial strains and culture conditions

The strains used in this study are listed in Additional file: Table S1. E. coli DH5α was used as the cloning host. E. coli MG1665 was used in genome-editing experiments and as the host strain for pseudouridine production. E. coli cells were cultivated at 37°C in Luria − Bertani (LB) medium for clone construction experiments and seed preparation. Kanamycin (50 mg/L), ampicillin (50 mg/L), and spectinomycin (50 mg/L) were added as needed.

The fermentation medium (20 g of glucose, 10 g of yeast extract, 6 g of KH₂PO₄, 16.4 g of K₂HPO₄, 5 g of (NH₄)₂SO₄, 1.1 g of citric acid, 1 g of MgSO₄, 15 mg of MnSO₄, and 2 mg of V_B1 in 1000 mL water) was used for shake flask fermentation. Isopropyl-β-D- thiogalactopyranoside (IPTG) was added to the medium at a final concentration of 0.1 mM when the OD₆₀₀ reached 0.6, and the temperature was adjusted to 30°C.

The fed-batch fermentation was initiated with 10 g/L glucose. The other components in the medium were the same as those in the flask fermentation medium. After 8 h of culture, IPTG was added to the medium to a final concentration of 0.1 mM, and the temperature was adjusted to 30°C. The pH was maintained at 7.0 via the automated addition of a 25% (v/v) NH₄OH solution. The dissolved oxygen (DO) saturation level was controlled to be greater than 25% by adjusting the aeration and agitation rates. The concentration of glucose was maintained to be less than 5 g/L by feeding 600 g/L glucose at the appropriate rate.

Construction of recombinant plasmids

The plasmids used in this study are listed in Additional file: Table S1. The primers used for gene cloning are given in Additional file: Table S2. Ezmax Universal CloneMix (Tolobio) was used for plasmid construction. The plasmid pET-30a (+) was used as a cloning vector and for pseudouridine production. phoA DNA fragments originating from E. coli MG1655 were amplified from its genome DNA by PCR and ligated with linearized pET-30a (+) that had been digested by NdeI ,resulting in pZH1. The EcsuG DNA fragments (from E. coli MG1655) were amplified by PCR and ligated to linearized pZH1 that had been digested by EcoRV, resulting in pZH2. The RBS sequence (aaaggaggatatacat) was inserted in front of the psuG start codon. SDT1, YKL033W-A and PHM8 originating from Saccharomyces cerevisiae, and pumD originating from Streptomyces sp. ID38640 was cloned into pET-30a (+) with EcpsuG using a similar method, yielding plasmids pZH3, pZH4, pZH5, and pZH6. KspsuG (from Klebsiella spallanzanii), RspsuG (from Rhizobium sp. CF142), SspsuG (from Saccharopolyspora spinosa) and SppsuG (from Streptomyces platensis) were cloned into pZH6 digested with KpnI and XhoI, yielding plasmids pZH7, pZH8, pZH9, and pZH10,respectively. All heterologous genes were synthesized by GenScript Bio Inc. (Nanjing, China), and the codons of the heterologous genes were optimized based on E. coli with GenSmart codon optimization performed by GenScript Bio. The sequences of the codon-optimized genes are presented in Additional file : Table S3.

Construction of recombinant strains

The CRISPR‒Cas9 system was applied to knock out genes in E. coli following methods described in a previous study [34]. Herein, the process used for the deletion of the pepA gene in E.coli MG1665 is described as an example of the method used. A gRNA plasmid was from plasmid pTargetF was amplified by PCR using primers pepA20F/pepA20R, and the PCR products were transformed into competent E. coli DH5α cells after DpnI digestion. The two homologous arms (approximately 500 bp) from E. coli MG1655 were amplified by PCR using the primers pepA-F1/pepA-R1 and pepA-F2/pepA-R2. The full-length DNA fragment for homologous recombination was obtained by overlapping PCR products using the primers pepA-F1 and pepA-R2. A single colony of MG1665 containing the plasmid pCAS9 was cultivated overnight in LB medium at 30°C. Then, the cultures were transferred to fresh LB medium at a 2% inoculum volume, and arabinose was added to the culture to a final concentration of 10 mM. When the OD₆₀₀ reached 0.6, the cells were collected to prepare electrocompetent cells after washing twice with precooled 10% (v/v) glycerol. The gRNA plasmid and homologous DNA fragment were added into an electroshock cup to be transformed into competent cells through electroporation (1-mm cuvette, 1.8 kV). After electroporation, 1 mL of LB was immediately added, and the cells were allowed to recover at 30°C for 1 h prior to plating on LB agar supplemented with ampicillin and spectinomycin. After 20 h, single transformants were verified by colony PCR using the primers pepVerF and pepVerR. The correct mutant strain was inoculated into 3 mL of LB medium cultured at 30°C, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the medium at a final concentration of 0.1 mM to cure the pTargetF derivative. The colonies cured in the pTarget series were used in the next round of gene knockout. The other mutant strains were constructed with a similar method.

Protein Expression, Purification and SDS-PAGE analysis

BL21(DE3) harboring overexpression plasmids were cultured overnight in LB medium containing 50 mg/L kanamycin at 37°C. Then, the cultures were transferred to 30 ml LB medium containing 50 mg/L kanamycin and grown at 37°C until the OD₆₀₀ reached 0.6–0.8. IPTG was subsequently added to a final concentration of 0.2 mM and incubate for 20 h at 25°C. The culture was centrifuged at 4000 rpm and 4°C for 5 minutes. The resulting pellet was resuspended in lysis buffer (20 mM phosphate buffer pH 8.0, 300 mM NaCl, 10 mM imidazole). The cells were lysed with a sonicator for a total of 10 minutes (3 second pulses and 6 second breaks). After sonication, the lysis solution was centrifuged at 15,000 rpm and 4°C for 20 minutes. The supernatant was purified with Ni-NTA column. The supernatant and loading buffer were boiled for 5 min, then 10 µl mixture was loaded for SDS-PAGE (12% polyacrylamide gel) analysis.

Enzymatic assays of phosphatase and ΨMP glycosidase

Phosphatase catalytic reaction was performed in 20 mM phosphate buffer (pH 7.0), 10.0 mM MgCl₂, 1.0 µM phosphatase, 0.5-10mM pseudouridine 5’-monophosphate. Reaction were performed in 100 µL scale and incubated at 30°C. ΨMP glycosidase catalytic reaction was performed in 20 mM phosphate buffer (pH 7.0), 100 mM ribose 5-phosphate, 0.5-10mM uracil, 1.0 mM MnCl₂, 1.0 µM ΨMP glycosidase. Reaction were performed in 100 µL scale and incubated at 30°C. The kinetic parameters (K_m and k_cat) were measured with substrate concentrations between 0.5 and 10 mM and calculated according to Michaelis-Menten plot.

Analytical Methods

The concentration of pseudouridine was measured by high-performance liquid chromatography (Agilent 1260 Series) equipped with a TC-C18 column (250 mm ×4.6 mm, Agilent) and monitored at a wavelength of 260 nm. The column was operated at 30°C with a mobile phase consisting of 0.2% ammonium acetate and acetonitrile (95:5, v/v) at a flow rate of 1.0 mL/min.

Ψ: Pseudouridine; ΨMP: pseudouridine-5’-phosphate glycosidase; psuG: Encoding gene of pseudouridine 5’-phosphate glycosidase; KspsuG: psuG gene from Klebsiella spallanzanii; RspsuG: psuG gene from Rhizobium sp. CF142; SspsuG: psuG gene from Saccharopolyspora spinosa; SppsuG: psuG gene from Streptomyces platensis.

Acknowledgements

The authors acknowledge the excellent suggestions provided by prof Yongquan Li and Xuming Mao (Zhejiang University).

Authors' information

Min Zhou

E‑mail: [email protected]

Address: Institute of Biopharmaceuticals, School of Pharmaceutical Sciences, Taizhou University, Taizhou 318000, China

Ruyu Tang

E‑mail: [email protected]

Address: Institute of Biopharmaceuticals, School of Pharmaceutical Sciences, Taizhou University, Taizhou 318000, China

Liyuan Wei

E‑mail: [email protected]

Address: Institute of Biopharmaceuticals, School of Pharmaceutical Sciences, Taizhou University, Taizhou 318000, China

Jidong Wang

E‑mail: [email protected]

Address: Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life Science, Huzhou University, Huzhou 313000, China

Huan Qi

E‑mail: [email protected]

Address: Key Laboratory of Vector Biology and Pathogen Control of Zhejiang Province, College of Life Science, Huzhou University, Huzhou 313000, China

Author contributions

MZ conceived, structured the work, conducted fermentation experiments in bioreactors and wrote the manuscript. RYT constructed plasmids and strains. LYW conducted protein overexpression experiments and fermentation experiments in shake flasks. JDW and HQ performed compound isolation,purification and analysis.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Funding

This work was supported by scientific research start-up funding of Taizhou University (T20200201031).

Ethics approval and consent to participate

Not applicable. The manuscript does not contain data collected from humans

or animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Metabolic engineering Escherichia coli for high-level production of pseudouridine

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Background

Results and Discussion

Identification of phosphatase gene for efficient pseudouridine production

Identification of ΨMP glycosidase gene for efficient pseudouridine production

Fine-tuning of gene expression

Knockout of pseudouridine catabolism-related genes

Fed-batch fermentation of pseudouridine in a 5-L bioreactor

Conclusions

Materials and Methods

Bacterial strains and culture conditions

Construction of recombinant plasmids

Construction of recombinant strains

Protein Expression, Purification and SDS-PAGE analysis

Analytical Methods

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1