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Article

Construction of Trans-4-hydroxy-L-proline-producing Escherichia coli and Optimization of Fermentation Conditions

by
Xinchao Yang
1,†,
Xinyu Li
2,†,
Yuanxiu Wang
1,
Yuehui Liu
1,
Fang Wang
1,
Naxin Sun
1 and
Chunjiang Ye
1,*
1
School of Biological Science and Technology, University of Jinan, Jinan 250022, China
2
Shandong Baicuisheng Special Medical Purpose Formula Food Co., Ltd., Jinan 250019, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2025, 11(2), 54; https://doi.org/10.3390/fermentation11020054
Submission received: 21 December 2024 / Revised: 22 January 2025 / Accepted: 22 January 2025 / Published: 24 January 2025
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
Browse Figures
Versions Notes

Abstract

:
In this study, we combined the citric acid cycle with the biosynthesis pathways of L-proline and L-hydroxyproline to construct a strain that produces L-hydroxyproline directly from glucose and other raw materials, without the addition of L-proline and α-ketoglutarate. The results showed that the level of L-hydroxyproline production was 550 mg/L. Through the optimization of one-way and orthogonal experiments, the optimal shake flask fermentation conditions were obtained, at which time the production of L-hydroxyproline reached 1800 mg/L, which was 3.3-fold higher. The glutamate permease gene GltS was added to the recombinant plasmid pRSFDuet1-p4h-proBA, and the recombinant plasmid obtained was transformed into E. coli T7E by Gibson seamless cloning to obtain the recombinant strain T7E/pRSFDuet1-p4h-GltS-proBA. Finally, by the addition of 30 mmol/L of sodium glutamate, the recombinant strain achieved a yield of L-hydroxyproline of 2150 mg/L, which was about 1.2-fold higher than the yield of L-hydroxyproline without the addition of sodium glutamate.

1. Introduction

Hydroxyproline (Hyp), a valuable chiral amino acid [1,2,3], is present in gelatin, collagen and cell wall proteins, and is also an important component of the secondary metabolites of certain microorganisms [4,5,6]. L-hydroxyproline is a characteristic amino acid of collagen [7,8], which not only maintains the three-dimensional structure of collagen but also inhibits the occurrence of protein agglutination and fibrosis [9,10]. Due to the unique physicochemical properties and structure of L-hydroxyproline, it has been widely used in medicine, material chemistry, food and feed, cosmetology, medical diagnoses, etc., and has broad market prospects.
The traditional method for the production of L-hydroxyproline is animal tissue extraction, which is a very complex process with the disadvantages of low recovery, high cost and serious environmental pollution [11,12]. With the rising cost of raw materials and people’s attention to environmental issues, this traditional extraction method has been gradually eliminated, and microbial fermentation methods have become a promising method due to their economic advantages and environmentally friendly features [13,14]. In the future, this method will completely replace the traditional method of producing L-hydroxyproline.
In this study, by combining the citric acid cycle with the proline and L-hydroxyproline biosynthesis pathways, we constructed a biosynthesis pathway for the direct production of L-hydroxyproline from glucose and other raw materials, without the addition of L-proline and α-ketoglutarate. The innovation in this manuscript is to reduce the synthetic cost of L-hydroxyproline. p4h is the gene of synthetic proline-4-hydroxylase (p4h), and proline-4-hydroxylase is a key factor in converting L-prolitic acid to L-hydroxyls. In E. coli, glutamic acid kinase encoded by proB and the glutamate hemalxidase coded by proA jointly participate in the regulation of L-prolitic acid synthesis metabolism. Laboratory-preserved bacterial fluids were used as templates to obtain the required target genes p4h and proBA, and different E. coli were selected as host bacteria to construct recombinant strains for efficient L-hydroxyproline production.
Shake flask fermentation was optimized by one-way and orthogonal experiments to improve the yield of L-hydroxyproline. The α-ketoglutarate metabolic pathway in the citric acid cycle also affects the biosynthesis of L-hydroxyproline, in which α-ketoglutarate acts as a co-substrate with L-proline, which reduces the production of L-hydroxyproline if it is in insufficient supply. L-hydroxyproline’s precursor is L-proline, which is preceded by glutamic acid, and glutamic acid’s precursor is α-ketoglutarate. The biotransformation process of proline to L-hydroxyproline requires the participation of α-ketoglutarate, part of which, in turn, is required for the production of glutamate, and glutamate and α-ketoglutarate are capable of interconverting each other intracellularly [15,16]. By introducing the GltS glutamate permease gene, the ability of cells to take up glutamate was enhanced, and the amount of glutamate or monosodium glutamate added to the fermentation broth was explored in order to satisfy the supply of glutamate and α-ketoglutarate, which would in turn increase the yield of L-hydroxyproline.

2. Materials and Methods

2.1. Strains and Plasmids

In Table 1, the strains and plasmids used for testing are listed. All of the strains and plasmids were obtained from our laboratory.
The composition of the culture medium and antibiotics used in the experiment is as follows: LB medium—aspirin 10 g/L, yeast extract 5 g/L and sodium chloride 10 g/L, adjusted to pH 7.0–7.2; antibiotics: 50 g/L kanamycin.

2.2. Construction of Recombinant Plasmids

2.2.1. Construction of Recombinant Plasmids pRSFDuet-1-p4h-proBA and pET28b-p4h-proBA

Primers were designed according to the target gene sequences p4h, proBA and pRSFDuet-1 plasmid mapping (Supplementary Information). Recombinant plasmids were constructed by using laboratory-preserved bacterial fluids as templates for the p4h and proBA genes, and the pRSFDuet-1 or pET28b plasmids as vectors, and the constructed recombinant plasmids were introduced into the cloned host cells to screen for positive clones.

2.2.2. Enzymatic Validation of Recombinant Plasmids pRSFDuet-1-p4h-proBA and pET28b-p4h-proBA

Picked up well-grown single colonies and inoculated them in 5 mL of LB medium containing kanamycin to incubate overnight at 37 °C with shaking at 200 r/min.
Extracted the reorganized plasmid to verify the enzyme cutting of the plasmid used to limit nucleic acids containing exogenous genes.

2.2.3. Induction Culture of Recombinant E. coli and Protein Expression

Translated the correct reorganized plasmid to receptive E. coli and scratched the medium containing kanamycin. Picked up well-grown single colonies and inoculated in 5 mL of LB medium containing kanamycin to incubate overnight at 37 °C with shaking at 200 r/min. Transferred to the fermentation medium containing kanamycin at a 3% inoculum volume, incubated at 37 °C with 200 r/min shaking for 2~3 h, OD600 to 0.5~0.8, added IPTG at a final concentration of 0.5 mmol/L, and incubated for 12 h. Took 1 mL each of the uninduced and induced bacterial fluids, and analyzed it by SDS-PAGE to confirm the recombinant plasmid induced expression; SDS-PAGE analysis was performed to confirm the expression of the recombinant plasmid.

2.3. Determination of L-hydroxyproline

2.3.1. Qualitative Determination of L-hydroxyproline by Ninhydrin Thin-Layer Chromatography

Proline was determined by ninhydrin thin-layer chromatography [17,18].
Preparation of chromatographic solution: the colorant and chromatographic solution were combined into one, i.e., ninhydrin was added directly to the chromatographic solution. The composition of the chromatographic solution was n-butanol:95% ethanol:glacial acetic acid:water = 4:1:1:1, with 0.1% ninhydrin.

2.3.2. Determination of L-hydroxyproline in Fermentation Broth by High Performance Liquid Chromatography

Mobile phase: 0.1% aqueous phosphoric acid:acetonitrile = 1:1 (V/V); column: Agilent HC-C18, 4.66 × 250 mm, 5 μm; flow rate: 1.0 mL/min; detector: VWD; wavelength: 254 nm; injection volume: 5 μL.
Solution preparation:
(1)
For the 0.1% aqueous phosphoric acid solution, we added 1 mL of phosphoric acid to a 1000 mL volumetric flask, then added water and shook well.
(2)
For the 1 mol/L sodium hydroxide solution, we weighed 4.0 g of sodium hydroxide in a 100 mL volumetric flask, dissolved it in water and shook well.
(3)
For the standard/sample solution, we accurately weighed 50 mg of standard L-hydroxyproline in a 50 mL volumetric flask (i.e., 1 g/L); it was then dissolved in an appropriate amount of purified water before adding 2 mL of 50% p-nitrobenzyl chloroformate in dichloromethane solution. The solution was shaken vigorously for 15 min, 3 mL of 1 mol/L NaOH was added, it was shaken vigorously again for 10 min, and then purified water was added to fix the volume before setting it aside [19,20,21].

2.4. Determination of Proline-4-Hydroxylase P4H Enzymatic Activity

The fermentation broth was centrifuged at 12,000 r/min at 4 °C for 2 min, the supernatant was removed to recover the organisms, and 500 μL of enzyme reaction buffer (240 mmol/L of morpholine ethanesulfonic acid with a pH of 6.5, 20 mmol/L of L-proline, 40 mmol/L of α-ketoglutaric acid, 4 mmol/L of ferrous sulfate, 8 mmol/L of L-ascorbic acid) was added to the weighed centrifuge tube. The cells were added to a weighed centrifuge tube, resuspended with enzyme reaction buffer, and incubated at 30 °C with 200 r/min for 15 min before being transferred to a boiling water bath at 100 °C for heating, terminating the enzyme reaction for about 2 min to determine the concentration of L-hydroxyproline in the reaction solution. In this study, the amount of enzyme required to generate 1 nmol of L-hydroxyproline in 1 min was defined as one unit of enzyme activity. Whole-cell activity was defined as the enzyme activity per milligram of wet bacteriophage per unit volume of fermentation broth in U/mg of wet cell weight.

2.5. Fermentation Conditions for Recombinant E. coli

The fermentation conditions for recombinant E. coli were as follows: pH 6.5, 37 °C, 200 r/min, 2–3 h, 2% inoculation ratio (v/v). The bacterial solution OD600 was 0.5~0.8, IPTG at a final concentration of 0.4 mmol/L was added for induction, and the temperature was lowered to 30 °C for further cultivation to draw the fermentation curve of recombinant E. coli.

2.6. Optimization of Fermentation Conditions

Initial shake flask fermentation medium: 10 g/L of glucose, 15 g/L of tryptone, 3 g/L of dipotassium hydrogen phosphate, 1 g/L of magnesium sulfate, 0.015 g/L of calcium chloride, 0.8 g/L of ferrous sulfate and 2 mmol/L of L-ascorbic acid. An initial pH of 7.0 was loaded at 30 mL/300 mL per flask to explore the effect of fermentation temperature, pH and IPTG concentration on the production of L-hydroxyproline.
An orthogonal experiment was done to optimize the fermentation medium (Table 2). The fermentation pH was adjusted to 6.5, the inoculum was transferred to the 30 mL/300 mL fermentation shake flask medium at 2% (v/v), the fermentation medium was incubated for 2~3 h at 37 °C with 200 r/min oscillation, the temperature was lowered to 30 °C when the OD600 was reduced to 0.5~0.8, and the final concentration of IPTG was added to the induction before allowing the induction to continue for 24 h. The fermentation broth was centrifuged to obtain the supernatant. The concentration of L-hydroxyproline was determined using the chloramine-T oxidation method.

3. Results and Discussion

3.1. Transformation of Recombinant Plasmids and Enzymatic Verification of Positivity

The recombinant plasmids pRSFDuet-1-p4h-proBA and pET28b-p4h-proBA were extracted and transformed into E. coli BL21 (DE3). As shown in Figure 1a,b, the transformed recombinant strains grew on a solid LB medium containing kanamycin, proving that the recombinant plasmids were successfully transformed.
The recombinant plasmids pRSFDuet-1-p4h-proBA and pET28b-p4h-proBA were subjected to enzyme digestion verification. The results of recombinant plasmid pRSFDuet-1-p4h-proBA enzyme digestion verification electrophoresis are shown in Figure 1c, and the results of recombinant plasmid pET28b-p4h-proBA enzyme digestion verification electrophoresis are shown in Figure 1d. The sizes of these fragments were all in accordance with the original ones, indicating that the recombinant plasmid was constructed successfully. Moreover, the recombinant plasmid pRSFDuet-1-p4h-proBA and recombinant plasmid pET28b-p4h-proBA, which were verified to be correct by enzymatic digestion, were both correctly sequenced by DNA sequencing.

3.2. Recombinant E. coli Fermentation Results

Six different groups of recombinant E. coli were cultured and the enzyme activity of wet cell proline-4-hydroxylase was determined; the results are shown in Table 3. The combination with the highest wet cell P4H enzyme activity of the six different groups of recombinant E. coli was T7E/pRSFDuet-1-p4h-proB-proA.
Since the type of host bacteria affects the stability of recombinant plasmid expression, it is important to screen for suitable host bacteria. Taking different types of recombinant E. coli as the horizontal coordinate and the yield of L-hydroxyproline in the fermentation broth as the vertical coordinate, the results are shown in Figure 2, and the relatively optimal combinations are BL21/pET28b-p4h-proB-proA and T7E/pRSFDuet-1-p4h-proB-proA. Recombinant E. coli T7E/pRSFDuet-1-p4h-proB-proA with a yield of 550 mg/L combined with the enzyme activity of P4H in the wet cells of different recombinant E. coli was selected for the next step of fermentation optimization.

3.3. Growth Curve of Recombinant E. coli T7E/pRSFDuet-1-p4h-proB-proA

The age of the seed solution had a great influence on fermentation, so the selection of seed solution at the right growth stage helped to increase the L-hydroxyproline yield. The seed solution of recombinant E. coli T7E/pET28b-p4h-proBA was cultured in order to obtain enough strains with good growth conditions for fermentation. In this experiment, incubation time was the horizontal coordinate and OD600 was the vertical coordinate, and the growth curve of recombinant E. coli T7E/pET28b-p4h-proBA was shown in Figure 3:
As can be seen in Figure 3, recombinant E. coli T7E/pET28b-p4h-proBA began to enter the logarithmic growth phase after 4 h of culturing in the LB medium, when it was accessed, and ended the logarithmic growth phase to enter the stable growth phase at about 12 h. Generally, seed sap in the middle-to-late stage of the logarithmic growth phase is selected for fermentation, i.e., seed sap at a seeding age of 10–12 h was selected for transfer to the fermentation medium. In the subsequent shake flask fermentation experiments, the incubation time of the seed liquid was 10~12 h. In the subsequent fermentation experiments, the seed liquid was transferred to the fermentation medium.

3.4. Optimization of Fermentation Medium Components

Based on initial fermentation conditions (Section 2.6), recombinant E. coli T7E/pRSFDuet-1-p4h-proBA was selected for the one-way optimization of the fermentation medium components, including glucose, tryptone, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate and L-ascorbic acid. The use of glucose as the main carbon source for the fermentation of recombinant E. coli T7E/pRSFDuet-1-p4h-proBA allowed the bacterial cells to utilize the carbon source more quickly. If the concentration of glucose in the medium is too high, then overflow of the citric acid cycle can occur, leading to excessive accumulation of acetic acid [22,23,24] and, ultimately, affecting the growth and metabolism of E. coli. Therefore, the glucose concentration must be controlled at a more reasonable level. In addition, the ratio of carbon source, nitrogen source and inorganic salt must also be regulated during the fermentation process [25,26,27]; otherwise, it will affect the growth and protein expression of the recombinant strain, which will ultimately lead to a lower yield of L-hydroxyproline.
As shown in Figure 4a, the highest accumulation of L-hydroxyproline was observed at a glucose concentration of 14 g/L. It can be seen that a too-low or too-high concentration of glucose and other carbon sources was not favorable to the growth and reproduction of bacterial cells and the production of target products [28]. During the fermentation of L-hydroxyproline, when the concentration of glucose was too high, metabolic by-products such as acetic acid were produced [29], which were unfavorable to the accumulation of the target product. As can be seen in Figure 4b, the highest accumulation of L-hydroxyproline was observed when the concentration of tryptone was 21 g/L. The composition of tryptone is complex and contains a large number of small molecule peptides in its constituents, which can be effectively and rapidly utilized by cells. In Figure 4c, it can be seen that increasing the concentration of dipotassium hydrogen phosphate within a certain concentration range was favorable to increasing the yield of L-hydroxyproline. When the concentration of dipotassium hydrogen phosphate exceeded 3 g/L, the yield of L-hydroxyproline showed a rapid decrease. This may be due to the fact that the concentration of dipotassium phosphate was too high, which led to an increase in the osmotic pressure of the solution and inhibited the growth of the recombinant strain, resulting in a decrease in the yield of L-hydroxyproline.
Magnesium ions are activators of many enzymes in the metabolic pathways of organisms, and a certain concentration of magnesium sulfate can effectively promote the growth of bacteria and the expression of recombinant proteins [30]. As shown in Figure 4d, although the highest L-hydroxyproline production was observed at a magnesium sulfate concentration of 2.0 g/L, the difference in L-hydroxyproline production from that at a concentration of 1.5 g/L was not significant. Free L-proline was catalyzed by proline-4-hydroxylase to produce L-hydroxyproline, and a ferrous ion was required as a cofactor to exert the hydroxylation activity of hydroxylase during the reaction. Ferrous ions in the medium can inhibit microbial growth because they alter the permeability of the cell membrane, leading to intracellular nutrient loss, which affects the growth of the organism. In addition, ferrous ions affected the synthesis of a variety of metabolites, such as amino acids, within the cell. As shown in Figure 4e, the accumulation of L-hydroxyproline increased with the increase in ferrous ion concentration within a certain concentration range, and the highest accumulation of L-hydroxyproline was observed when the concentration of ferrous sulfate reached 1.2 g/L. L-ascorbic acid has strong antioxidant abilities, and the addition of appropriate concentration of L-ascorbic acid reduced the oxidation of ferrous ions while playing the role of ferrous ions. Klein C et al. studied the effects of ferrous ions and L-ascorbic acid on proline hydroxylase and found that the presence of appropriate amounts of them helped to enhance the activity of proline hydroxylase [31]. This study also found that adding the appropriate concentration of ferrous sulfate and L-ascorbic acid can improve the production of hydroxyproline. As shown in Figure 4f, the highest L-hydroxyproline yield was obtained when L-ascorbic acid was added to the fermentation broth at a final concentration of 2 mmol/L, and the yield of L-hydroxyproline tended to stabilize and slightly decrease when the concentration of L-ascorbic acid was increased.

3.5. Optimization of Culture Conditions of Fermentation Medium

The effects of the inoculum amount, fermentation temperature, pH and IPTG concentration on the production of L-hydroxyproline were explored separately on the basis of the initial shake flask fermentation medium. The results are shown in Figure 5.
As can be seen in Figure 5a, the content of L-hydroxyproline was highest when the inoculum amount was 2%. If the inoculum amount was too little, the proliferation rate of bacteria would be slowed down and the fermentation time would be prolonged; while, if the inoculum amount was too much, it may cause the strain to enter into the senescence period earlier and produce secondary metabolites, which is also unfavorable to the production of L-hydroxyproline. In Figure 5b, it can be seen that, when the fermentation temperature was 26 °C, the accumulation of L-hydroxyproline was only 226 mg/L. With an increase in fermentation temperature, the acid production increased rapidly, and the highest production was achieved when the fermentation temperature was 30 °C, when the acid production was as high as 992 mg/L, which was four times that of the lowest acid production. As the temperature continued to increase, the L-hydroxyproline production decreased rapidly. The incubation temperature not only affected the correct folding of proteases [14] but also affected the accumulation of L-hydroxyproline. The optimal growth temperature of E. coli was around 37 °C, but the proper temperature was more favorable for the expression of target proteins, and controlling the fermentation temperature at a relatively low level was favorable for improving plasmid stability and enhancing the stability of exogenous proteins [32]. Therefore, 30 °C was more suitable for the fermentation culture of recombinant E. coli T7E/pRSFDuet-1-p4h-proB-proA. Data from Yi et al. also showed higher protein levels and enzyme activity at 30 °C [33].
As shown in Figure 5c, the maximum accumulation of L-hydroxyproline was observed at an initial pH of 6.5, and the accumulation of L-hydroxyproline decreased rapidly when the pH was higher than 6.5. This suggested that high pH may seriously affect the enzyme activity of proline-4-hydroxylase in the bacterial cells, and that acidic or alkaline initial culture conditions were not conducive to the accumulation of L-hydroxyproline. The results of optimizing the concentration of IPTG are shown in Figure 5d: the accumulation of L-hydroxyproline increased with the increase in concentration of IPTG within a certain concentration range, and the highest accumulation of L-hydroxyproline was observed when the concentration of IPTG was 0.5 mmol/L; however, there was not much difference in the product amount when the concentration of IPTG was 0.4 mmol/L. Therefore, an IPTG concentration of 0.4 mmol/L was more appropriate.

3.6. Analysis of Results of Orthogonal Experiments

In this experiment, E. coli T7E/pRSFDuet-1-p4h-proB-proA was used as the starting strain for optimization, inoculated at 2% (v/v) and fermented at a pH of 6.5 at 30 °C with 0.4 mmol/L IPTG for 24 h. On the basis of the previous experiments that used one-factor optimization of the fermentation medium, the appropriate glucose concentration, tryptone concentration, dipotassium hydrogen phosphate concentration, magnesium sulfate concentration, calcium chloride concentration, iron sulfate concentration and L-ascorbic acid concentration were selected, as shown in Table 2, to carry out a seven-factor, three-level orthogonal experiment so as to further optimize the content of each component of the fermentation medium.
According to the results of the orthogonal experiments shown in Table 4, the concentrations of the components in the optimal fermentation medium were: 14 g/L of glucose, 21 g/L of tryptone, 3 g/L of dipotassium hydrogen phosphate, 2 g/L of magnesium sulfate, 0.045 g/L of calcium chloride, 1.2 g/L of ferrous sulfate and 2 mmol/L of L-ascorbic acid.

3.7. Fermentation of Recombinant E. coli

The recombinant E. coli T7E/pET28b-p4h-proBA was cultured according to the optimized fermentation conditions and medium, and the fermentation curves of the recombinant strains were plotted, with the time of fermentation after induction as the horizontal coordinate and the content of L-hydroxyproline and the OD600 of fermentation broth as the vertical coordinates; the results are shown in Figure 6:
The recombinant strain E. coli T7E/pRSFDuet-1-p4h-proBA yielded approximately 1800 mg/L of L-hydroxyproline under optimal fermentation conditions. During the fermentation process, after the optimization of the fermentation medium’s composition and fermentation conditions, we found that the production of recombinant E. coli L-hydroxyproline showed a positive correlation with the value of the bacterial concentration OD600, and as the recombinant strain grew, proline-4-hydroxylase began to be expressed under the induction of IPTG, and the L-hydroxyproline gradually accumulated. The fermentation curve graph shows that the strain kept growing and proliferating from 0 to 20 h, and the OD600 value increased steadily, which laid a good foundation for the accumulation of the product. The bacteria began to enter the stabilization period at 20 h. From 24 h onwards, the growth of L-hydroxyproline accumulation was slow, probably due to the gradual depletion of the glucose concentration in the fermentation broth and the change in the pH of the solution with the accumulation of the product. Therefore, increasing the value of the OD600 of the fermentation broth, supplementing with nutrients, such as a carbon source, and adjusting the solution pH should be effective means with which to further increase L-hydroxyproline production.

3.8. Changes in Enzyme Activity of Recombinant E. coli Wet Cells After Optimization

The enzyme activity of wet cell proline-4-hydroxylase was measured with fermentation time as the horizontal coordinate and the enzyme activity of wet bacteriophage proline-4-hydroxylase as the vertical coordinate, and the results are shown in Figure 7. With the growth of recombinant E. coli, proline-4-hydroxylase was accumulated and expressed under the induction of IPTG, and the enzyme activity of the wet cell reached its highest at about 24 h, probably due to the fact that the pH of the solution changed with the accumulation of fermentation products, which led to a decrease in the wet cell enzyme activity while still maintaining a high enzyme activity at 24~48 h.

3.9. Effect of the Addition of Monosodium Glutamate on the Production of L-hydroxyproline

Glutamate is an intermediate in the L-hydroxyproline biosynthesis pathway, and supplementation with glutamate or monosodium glutamate helps to increase L-hydroxyproline production. Exogenous glutamate uptake was low due to the deficiency of the cellular GltS sodium glutamate transporter protein, and glutamate permease enhanced the uptake of glutamate and monosodium glutamate from the solution environment.
After 24 h of fermentation, the supernatant was centrifuged and the yield of L-hydroxyproline was determined. As shown in Table 5, the yield of L-hydroxyproline increased with the increase in the concentration of monosodium glutamate within a certain range, and the yield of L-hydroxyproline almost ceased to increase when the concentration of the added sodium glutamate exceeded 40 mmol/L. Since there was little difference in the L-hydroxyproline yield between 30 mmol/L and 40 mmol/L, the most suitable sodium glutamate addition was 30 mmol/L, which produced a yield of L-hydroxyproline of 2150 mg/L. The yield of L-hydroxyproline was increased by about 1.2 times compared to the yield without the addition of sodium glutamate.
In this study, the use of recombinant E. coli for L-hydroxyproline production using glucose and other raw materials was examined, and the optimization of L-hydroxyproline production in the shake flask fermentation stage was initially carried out, but the production scale-up process in the fermenter stage should to be investigated in order to optimize it accordingly. In order to obtain more product, the yield of L-hydroxyproline can also be increased by means such as batch replenishment.
Although E. coli is the most commonly used host strain for efficient expression of exogenous proteins, it is not a generally recognized as safe (GRAS)-certified safe strain, and the products obtained by direct fermentation using E. coli may contain substances that cause pyrogenic reactions in the human body, so the products cannot be used directly as additions to food and drug products, etc. Therefore, it is necessary to replace them with certified safe strains that are capable of efficiently expressing the exogenous proteins.
Since the applications of L-hydroxyproline are becoming more and more extensive, the demand for L-hydroxyproline in the market has increased, and the quality requirements for L-hydroxyproline have also increased accordingly. In the future, in-depth research can be carried out on the following aspects: bioinformatics can be used to select enzymes with high efficiency in catalyzing the synthesis of L-hydroxyproline from the gene pool, and enzymes with good stability and high catalytic efficiency can be developed for better use in industrial production; the metabolic pathway of the host bacterium can also be altered through synthetic biology technologies so as to make more precursors flow to the synthetic pathway of L-hydroxyproline, thus improving the quality of L-hydroxyproline and its applications. Thus, the yield and conversion rate of L-hydroxyproline can be improved.

4. Conclusions

We successfully constructed recombinant E. coli producing L-hydroxyproline from glucose and other raw materials, without the addition of exogenous L-proline, using laboratory-preserved bacterial fluids as templates for p4h and proBA; pET28b and pRSFDuet-1 as vectors; and E. coli BL21, Rosetta and T7E as hosts. The optimal recombinant strain was E. coli T7E/pRSFDuet-1-p4h-proBA, with an L-hydroxyproline yield of about 550 mg/L. The optimal shake flask fermentation medium and fermentation conditions were obtained through one-way and orthogonal experiments, and the L-hydroxyproline yield was about 1800 mg/L, which was an increase of about 3.3-fold compared with the non-optimized L-hydroxyproline yield. Finally, by adding 30 mmol/L of monosodium glutamate, the yield of L-hydroxyproline was about 2150 mg/L, which was about 1.2-fold higher than the yield of L-hydroxyproline when no monosodium glutamate was added.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11020054/s1, Figure S1. Plasmid mapping of pRSDute-1-p4h-proBA plasmid. Figure S2. Plasmid mapping of pET28b-p4h-proBA plasmid.

Author Contributions

Conceptualization, X.Y.; Methodology, X.L.; Validation, Y.W.; Formal analysis, Y.L.; Investigation, F.W.; Resources, N.S. and C.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the following projects: 1. Shandong Province Key R&D Program (Medical Food Special Program) (2018YYSP028); 2. Shandong postdoctoral innovation project; 3. Doctor’s Fund of the University of Jinan.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declared that they have no conflicts of interest in this work. Author Xinyu Li was employed by the company Shandong Baicuisheng Special Medical Purpose Formula Food Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Fermentation 11 00054 g001
Figure 1. Transformation of recombinant plasmids and enzymatic verification of positivity. (a) Recombinant strain of E. coli BL21/pET28b-p4h-proBA; (b) recombinant strain of E. coli BL21/pRSFDuet-1-p4h-proBA; (c) M: 1 kb DNA ladder marker, lane 1: XhoI single digestion verification, lane 2: NdeI/XhoI double digestion verification, lane 3: NcoI/HindIII double digestion verification; (d) M: 1 kb DNA ladder marker, lane 1: XhoI single digestion verification, 2: NcoI/XhoI double digestion verification.
Figure 1. Transformation of recombinant plasmids and enzymatic verification of positivity. (a) Recombinant strain of E. coli BL21/pET28b-p4h-proBA; (b) recombinant strain of E. coli BL21/pRSFDuet-1-p4h-proBA; (c) M: 1 kb DNA ladder marker, lane 1: XhoI single digestion verification, lane 2: NdeI/XhoI double digestion verification, lane 3: NcoI/HindIII double digestion verification; (d) M: 1 kb DNA ladder marker, lane 1: XhoI single digestion verification, 2: NcoI/XhoI double digestion verification.
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Fermentation 11 00054 g002
Figure 2. Fermentation results of different recombinant E. coli bacteria. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
Figure 2. Fermentation results of different recombinant E. coli bacteria. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
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Fermentation 11 00054 g003
Figure 3. Growth curve of recombinant E. coli T7E/pET28b-p4h-proBA. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
Figure 3. Growth curve of recombinant E. coli T7E/pET28b-p4h-proBA. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
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Fermentation 11 00054 g004
Figure 4. Optimization experiments for each component in the fermentation medium: (a) glucose, (b) tryptone, (c) K2HPO4, (d) MgSO4, (e) FeSO4, (f) L-ascorbic acid. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
Figure 4. Optimization experiments for each component in the fermentation medium: (a) glucose, (b) tryptone, (c) K2HPO4, (d) MgSO4, (e) FeSO4, (f) L-ascorbic acid. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
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Figure 5. Optimization of culture conditions for fermentation media: (a) inoculum concentration, (b) temperature, (c) pH, (d) IPTG. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
Figure 5. Optimization of culture conditions for fermentation media: (a) inoculum concentration, (b) temperature, (c) pH, (d) IPTG. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
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Figure 6. Fermentation of recombinant Escherichia coli. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
Figure 6. Fermentation of recombinant Escherichia coli. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
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Figure 7. Enzyme activity of recombinant E. coli wet cells. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
Figure 7. Enzyme activity of recombinant E. coli wet cells. Data presented are the average of three independent cultivations, and error bars represent standard deviations.
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Table 1. Strains and plasmids.
Table 1. Strains and plasmids.
Host/PlasmidRelevant Features
E. coli BL21(DE3)F-ompT hsdSB (rB- mB-) gal dcm (DE3)
E. coli DH5αClone host
pWFP 1Ampicillin resistance containing proline-4-hydroxylase gene, Glutamate kinase gene, glutamate dehydrogenase gene
pET28bKanamycin resistance
pRSFDuet-1Kanamycin resistance
Table 2. Factor levels for orthogonal experiments.
Table 2. Factor levels for orthogonal experiments.
NumberFactorsLevel 1Level 2Level 3
AGlucose (g/L)101214
BPeptone (g/L)151821
CDipotassium hydrogenphosphate (g/L)123
DCalcium chloride (g/L)0.0150.030.045
EFerrous sulfate (g/L)0.81.21.6
FMagnesium sulfate (g/L)11.52
GVitamin C (mmol/L)246
Table 3. Enzyme activities of different recombinant E. coli wet cell P4Hs.
Table 3. Enzyme activities of different recombinant E. coli wet cell P4Hs.
NumberDifferent Recombinant
E. coli		Enzyme Activity in U/mg Wet Cell Weight
1BL21/pET28b-p4h-proB-proA929.74
2BL21/pRSFDuet-1-p4h-proB-proA352.49
3T7E/pET28b-p4h-proB-proA140.36
4T7E/pRSFDuet-1-p4h-proB-proA1406.46
5Rosetta/pET28b-p4h-proB-proA250.13
6Rosetta/pRSFDuet-1-p4h-proB-proA385.54
Table 4. Table of the visual analysis results of the orthogonal experiments.
Table 4. Table of the visual analysis results of the orthogonal experiments.
NumberA
Glucose		B
Peptone		C
K2HPO4		D
CaCl2		E
FeSO4		F
MgSO4		G
VC		L-Hydroxyproline (mg/L)
112123331067.88
232312211571.05
322231121191.69
413332131317.48
522323111334.32
63121313819.27
733221311442.28
812212321170.89
92113231959.92
1032131231247.16
1113233211265.98
1231333321293.71
131132122890.59
1421222231142.17
1523113221173.86
1623311331433.37
1733122121306.59
1811111111059.96
K16772.786165.626815.377228.407265.057029.317633.51
K27235.337582.997032.287183.837468.107290.817027.33
K37680.067939.567840.527275.946955.027368.057027.33
k11128.801027.601135.901204.731210.841171.551272.25
k21205.891263.831172.051197.311244.681215.141171.22
k31280.011323.261306.751212.661159.171228.011171.22
R151.21295.66170.8515.3585.5156.46101.03
Optimum level3333231
Optimal combinationA3B3C3D3E2F3G1
Note: Ki is the sum of the experimental indicators of the same factor at the same level; ki is the average value of the experimental indicators of the same factor at the same level; R is the extreme deviation, which indicates the magnitude of change between experimental indicators of the same factor within the factor’s value range.
Table 5. Effect of sodium glutamate on the production of L-hydroxyproline.
Table 5. Effect of sodium glutamate on the production of L-hydroxyproline.
Sodium Glutamate (mM)01020304050
L-hydroxyproline (mg/L)1777.211836.642001.062149.632189.252171.42
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Yang, X.; Li, X.; Wang, Y.; Liu, Y.; Wang, F.; Sun, N.; Ye, C. Construction of Trans-4-hydroxy-L-proline-producing Escherichia coli and Optimization of Fermentation Conditions. Fermentation 2025, 11, 54. https://doi.org/10.3390/fermentation11020054

AMA Style

Yang X, Li X, Wang Y, Liu Y, Wang F, Sun N, Ye C. Construction of Trans-4-hydroxy-L-proline-producing Escherichia coli and Optimization of Fermentation Conditions. Fermentation. 2025; 11(2):54. https://doi.org/10.3390/fermentation11020054

Chicago/Turabian Style

Yang, Xinchao, Xinyu Li, Yuanxiu Wang, Yuehui Liu, Fang Wang, Naxin Sun, and Chunjiang Ye. 2025. "Construction of Trans-4-hydroxy-L-proline-producing Escherichia coli and Optimization of Fermentation Conditions" Fermentation 11, no. 2: 54. https://doi.org/10.3390/fermentation11020054

APA Style

Yang, X., Li, X., Wang, Y., Liu, Y., Wang, F., Sun, N., & Ye, C. (2025). Construction of Trans-4-hydroxy-L-proline-producing Escherichia coli and Optimization of Fermentation Conditions. Fermentation, 11(2), 54. https://doi.org/10.3390/fermentation11020054

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