Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli
© Yi et al.; licensee BioMed Central Ltd. 2014
Received: 7 February 2014
Accepted: 15 May 2014
Published: 19 May 2014
Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production.
In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate.
The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt.
KeywordsTrans-4-hydroxy-L-proline Recombinant Corynebacterium glutamicum Recombinant Escherichia coli Proline 4-hydroxylases
Hydroxyproline (Hyp) is a specific amino acid component of collagen. The amount of Hyps varies from 80 to 100 residues per 1000 residues in mammalian collagen, which can be used to estimate collagen content and act as an important indicator to collagen quality . There are five naturally occurring Hyps, including three diastereomers of 4-hydroxyproline and two diastereomers of 3-hydroxyproline. Among them, trans-4-hydroxy-L-proline is the most abundant component in the constitution of collagen and can enhance the procollagen synthesis. Its derivative N-acetyl trans-4-hydroxyproline (oxaceprol) is an atypical inhibitor of inflammation and useful for the treatment of diseases affecting the connective tissues such as osteoarthritis . Trans-Hyp has been widely used in medicine, biochemistry, food, cosmetic and other aspects of industry . Additionally, trans-Hyp has also been found in the composition of some secondary metabolites such as actinomycins and echinocandins .
Trans-Hyp is manufactured industrially most by acid hydrolysis of mammalian collagen because of its rich amount in the collagen. However, it obviously results in many environmental issues and brings great difficulties into the down stream processing . There are several identified pathways of hydroxyproline biosynthesis. In animal tissue, 4-hydroxyproline is catalyzed by prolyl 4-hydroxylase, which takes peptidyl proline as substrate rather than free proline . 4-hydroxy-2-oxoglutaric acid can be enzymatically transformed to hydroxyproline . Some bacteria or fungi have been found to form hydroxyproline via fermentation directly . Although the titer of product is low, these findings show the possibility of utilizing biological processes to produce trans-Hyp.
The proline 4-hydroxylases (P4Hs) have been identified from several microbial strains, which can catalyze the hydroxylation of L-proline at the 4-position to produce trans-Hyp in the presence of 2-oxoglutarate, oxygen and ferrous ion [9–11]. P4Hs have an optimum pH range of 6.0 to 7.5 and temperature range of 30°C to 40°C. Its activity is inhibited by metal ions such as Zn2+ and Cu2+. Lawrence et al. have studied the effect of co-substrates on the hydroxylation of L-proline by P4H and pointed that 2-oxoglutarate was essential for proline hydroxylation since the replacement of 2-oxoglutarate with 2-oxopentanoate, 2-oxoadipate, pyruvate or 2-oxomalonate (all at 0.5 mM) led to no detectable hydroxylation of L-proline . Although 2-oxoglutarate as the oxygen donator is required for hydroxylation of L-proline to 4-hydroxy-L-proline in vitro, it is unnecessary to add extra 2-oxoglutarate in vivo in the production of 4-hydroxy-L-proline by recombinant strains. 2-oxoglutarate is a key metabolic intermediate in the tricarboxylic acid cycle (TCA cycle) in Escherichia coli strain, which can result in the formation of hydroxyproline from glucose and proline directly . Shibasaki et al. have analyzed the possible metabolic pathways of 2-oxoglutatate. They concluded that 2-oxoglutatate can be supplied either through the action of proline dehydrogenase (PutA) from L-proline or through the action of isocitric dehydrogenase (Icd) from glucose. The addition of L-proline to a glucose-containing minimal medium had a positive effect on both the proline 4-hydroxylase activity and production level . But the availability of intracellular proline may still be limited because the biosynthesis of proline in wild type E. coli is strictly regulated to very low level. Thus, the precursor and co-factor in the microbial production of hydroxyproline need to be considered simultaneously.
Corynebacterium glutamicum is one of the most important industrial microorganisms and widely used in amino acids, vitamins and nucleic acids production . Leuchtenberger et al. has summarized the commercial application of C. glutamicum to the fermentative production of amino acids . Lee et al. has reported a novel glutamate and proline producing method through the utilization of phenol in C. glutamicum. Masaaki Wachi reported a strategy for optimizing the industrial production of amino acids by reinforcing the export systems of C. glutamicum. The metabolic pathways of amino acids are sophisticated and controlled tightly in C. glutamicum. But C. glutamicum as the platform of amino acid production has been studied in details and there are lots of molecular tools used for its genetic modifications, which contribute to C. glutamicum as one of the most popular host systems [18, 19]. Additionally, Ikeda et al.  and Kalinowski et al.  have completed genome sequencing of C. glutamicum ATCC13032, which made C. glutamicum into a new era of system biology. To overproduce amino acids by C. glutamicum, not only the modification of biosynthetic pathway and regulation mechanism but also the transportation of amino acids plays significant roles in the final yield of a particular amino acid [22, 23].
Results and discussion
Construction of trans-Hyp producing recombinant strains
There are several genes being speculated as putative L-proline 4-hydroxylase gene in the database, including genes in Pseudomonas stutzeri , Janthinobacterium sp., Bordetella bronchiseptica RB50, Bradyrhizobium japonicum, Achromobacter xylosoxidans C54 and Dactylosporangium. sp. Using PCR, we cloned and obtained the putative genes of P4H from P. stutzer and B. bronchiseptica RB50, named p4hP and p4hB. They were ligated to the corresponding plasmids after digestion and converted to C. glutamicum and E. coli, respectively. The length of p4hP was 918 bps while p4hB was 924 bps. These sequences were 100% identical to the reported genes in NCBI. The gene of trans-P4H from Dactylosporangium sp. (p4hD) had been expressed in E. coli successfully and can transform L-proline with good enzymatic properties [11–13, 24–26]. The length of p4hD was 816 bps encoding a 272-amino-acid polypeptide with the molecular weight of 29,715 daltons [11, 26]. In this study, p4hD was applied with some modifications on the nuclear bases. The original gene sequence of p4hD was analyzed (http://www.kazusa.or.jp/codon/) and the results showed there were some rare codons for both C. glutamicum and E. coli. It has been reported that rare codons are strongly associated with low level of protein expression . Codon optimization for heterologous protein expression has often been shown to drastically increase protein expression . Thus, the rare codons of p4hD gene were substituted for those used with high frequency in C. glutamicum and the GC content was adjusted from 73% to 61% through synonymous conversion, which was close to that of C glutamicum. The modified gene of p4hD was synthesized according to the above modifications (Additional file 1).
Comparison of P4H activities
Comparison of trans -P4Hs activities and trans -Hyp production by different recombinant C. glutamicum and E. coli strains
Specific activities (U/mg · wet cell weight)
C. glutamicum ATCC13032/pEC-XK99E- p4hD
37.4 ± 1.4
0.072 ± 0.001
5.5 ± 0.7
C. glutamicum ATCC13032/pEC-XK99E- p4hP
20.7 ± 1.1
0.106 ± 0.002
7.3 ± 0.5
C. glutamicum ATCC13032/pEC-XK99E- p4hB
40.7 ± 0.8
0.079 ± 0.016
5.4 ± 0.03
C. glutamicum ATCC15940/pEC-XK99E- p4hD
12.9 ± 0.5
0.103 ± 0.001
14.0 ± 0.2
C. glutamicum ATCC21355/pEC-XK99E- p4hD
35.9 ± 0.1
0.087 ± 0.005
6.6 ± 0.2
C. glutamicum ATCC21157/pEC-XK99E- p4hD
12.3 ± 0.9
0.112 ± 0.004
13.3 ± 0.1
C. glutamicum 49-1/pEC-XK99E- p4hD
12.4 ± 0.6
0.113 ± 0.001
13.8 ± 0.5
E. coli BL21/pET-28a -p4hD
60.4 ± 1.8
0.470 ± 0.028
6.5 ± 0.2
E. coli BL21/pET-28a -p4hP
22.2 ± 0.5
0.126 ± 0.007
7.3 ± 0.05
E. coli BL21/pET-28a -p4hB
50.0 ± 2.2
0.115 ± 0.006
6.9 ± 0.1
The recombinant cells with expressing of different genes showed different levels of catalytic activities toward L-proline. The activity of trans-P4H expressed by E. coli BL21/ pET28a-p4hD was the highest among all the constructed recombinant strains. The new cloned and expressed genes from P. stutzeri and B. bronchiseptica also showed interested activities. As for different host strains, E. coli represented better than C. glutamicum, which may be related to the performance of corresponding plasmid. Four L-proline producing strains of C. glutamicum were used as expression host strains and the resulted recombinant strains showed different enzymatic activities. The highest specific enzymatic activity among C. glutamicum strains was 40.7 U/mg · wet cell by C. glutamicum ATCC13032/pEC-XK99E-p4hB. However, the specific enzymatic activity of recombinant E. coli/pET28a -p4hD was up to 60.4 U/mg · wet cell. The growth of three recombinant E. coli strains was similar. But there was significant difference among the recombinant C. glutamicum strains. The recombinant C. glutamicum strains with higher specific enzymatic activities grew less than those with lower specific enzymatic activities. Additionally, the enzymatic activity of E. coli BL21 /pET28a -p4hD was similar to that of E. coli W1485/pWFH1 and higher than that of E. coli BL21/pET24-p4h1 of [12, 13]. The p4hD in E. coli W1485/pWFH1 was the original one in Dactylosporangium sp., while p4hD in E. coli BL21/pET24-p4h1 was modified. Although the codon optimization in this study was designed for C. glutamicum, the results indicated that it was also successfully in E. coli.
Trans-Hyp production in flasks
The production of trans-Hyp by different recombinant C. glutamicum and E. coli strains was also shown in Table 1. The yields of trans-Hyp by these recombinant strains depended both on the enzymatic activity of P4H and cell growth. E. coli BL21/ pET28a-p4hD had the highest yield, which was coincided of its specific enzymatic activity. Although the recombinant E. coli strains grew similarly in the production medium, there was significant difference in the production of trans-Hyp which did not keep the same level with the specific enzymatic activities. The productions of trans-Hyp by recombinant C. glutamicum strains were also much less than that of E. coli BL21/pET28a-p4hD. It was due to both the less expression of trans-P4H and less cell growth in C. glutamicum. The L-proline production of four C. glutamicum strains was also less than 1 g/L. There was little difference of trans-Hyp production among the recombinant strains of C. glutamicum with same gene p4hD, despite that some strains had better enzymatic performance and proline production.
Hyp production under different L-proline supplementation
Supplementary addition of L- proline (mM)
In order to further increase the biosynthesis of trans-Hyp by recombinant C. glutamicum and E. coli, alternative approaches should be considered as well. In E. coli, the degradation of proline should be overcome. Although the trans-Hyp production by a putA mutant of E. coli was not improved furthermore, the yield based on the proline utilized was enhanced greatly. In both C. glutamicum and E. coli, the expression of recombinant P4H as one of the oxygenases is involved in the physiological metabolism of host cells including the cofactor, co-substrate and oxygen. Moreover, without a powerful proline synthetic pathway in E. coli, the availability and transportation of substrate will limit the transformation seriously.
In this study, two new and a modified trans-P4Hs were expressed in C. glutamicum and E. coli successfully. Different amount of trans-Hyp were produced by these recombinant strains detected. Although the yield in recombinant C. glutamicum was less than that in recombinant E. coli, C. glutamicum as a native proline producing strain was worthy of further optimizing. This is the first report of producing trans-Hyp by introducing L-proline 4-hydroxylase into C. glutamicum.
Strains and plasmids
Strains and plasmids used in this study
Strains & plasmids
F-, ompT, hsdS(rBB-mB-), gal, dcm (DE3)
His4-tag, T7 promoter, Kanr
E. coli - C. glutamicum shuttle expression vector, Kanr
pET-28a containing the p4h gene from P. stutzeri
pEC-XK99E containing the p4h gene from P. stutzeri
pET-28a containing the p4h gene from B. bronchiseptica
pEC-XK99E containing the p4h gene from B. bronchiseptica
pET-28a containing the p4h gene from Dactylosporangium sp.
pEC-XK99E containing the p4h gene from Dactylosporangium sp.
Construction of recombinant strains
Primers used in this study for gene cloning and plasmid construction
Sequences (5’ → 3’)
p4hP -pEC-XK99E -A
p4hB -pEC-XK99E -A
Luria broth (LB) medium, tryptone 10 g/L; yeast extract 5 g/L; solium chloride 10 g/L, was used for seed cultivation of E. coli strains. LBG medium containing 1% glucose additionally was used for C. glutamicum seed cultivation.
The medium (MEC) for batch culture of E. coli in shake flasks contained: glucose 10 g/L, glycerol 5 g/L, CO(NH2)2 10 g/L, yeast extract 10 g/L, K2HPO4 1 g/L, NaCl 2 g/L, MgSO4 · 7H20 0.2 g/L, FeSO4 · 7H2O 1 mM, MnSO4 · 4H2O 10 mg/L, ZnSO4 · 7H2O 10 mg/L, VB1 200 ug/L.
The medium (MCG) for batch culture of C. glutamicum in shake flasks contained: glucose 10 g/L; glycerol 5 g/L; CO(NH2)2 10 g/L; corn syrup 15 g/L; K2HPO4 1 g/L; NaCl 2 g/L; MgSO4 · 7H20 0.2 g/L; FeSO4 · 7H2O 1 mM; MnSO4 · 4H2O 10 mg/L; ZnSO4 · 7H2O 10 mg/L; VB1 200 mg/L; ethyl alcohol absolute 1.5%.
The seed culture of E. coli strains was prepared by transferring 1 ml of glycerol stock to 30 ml of LB medium in a 250-ml flask, which was incubated overnight at 37°C and 220 rpm. Then 6% of seed culture was inoculated into 30 ml of MEC medium in a 250 ml flask and incubated at 37°C and 220 rpm for about 36 h. The initial pH of the medium was adjusted to 7.4. Induction (0.5 mM IPTG) was performed when the optical density was around 0.5, and the growth temperature was reduced from initial 37°C to 30°C. The cultivation of C. glutamicum was similar to that of E. coli expect that they were conducted at 30°C in the whole process using MCG medium. Experiments were performed in parallel on the same media without any induction.
The intracellelular trans-P4H activities were measured by the whole-cell reaction procedures. After 8 hours induction in fermentation medium, cells were harvested by centrifugation at 12000× g for 20 min. The harvested cells were resuspended in the reaction mixture as followed. Each reaction mixture contained 80 mM 2-[N- morpholino] ethanesulfonic acid (MES) buffer (pH 6.5), 4 mM L-proline, 8 mM 2 - ketoglutarate, 2 mM FeSO4, 4 mM L-ascorbic acid The final cell concentration was about 100 g wet weight/L. The reaction mixtures were incubated at 35°C for 10 min and then cellular activity was inactivated completely by heat treatment at 100°C and 5 minutes. The amount of trans-4-hydroxy-L-proline in the supernatant of each mixture after centrifugation was determined. The amount of the enzyme which forms 1 nmol of Hyp in one minute was defined as 1 U.
The cell concentration was determined by measuring the optical density of appropriately diluted sample at 600 nm (E. coli, OD600) and 620 nm (C. glutamicum OD620) with a UV-visible spectroscopy system (Xinmao, Shanghai, China). Hyp was oxidized by Chloramine T and analyzed using spectrophotometric determination .
All measurements for growth, trans-Hyp production and trans-P4H activity were performed in triplicate, and the data were averaged and presented as the mean ± standard deviation.
This study was supported by the National High Technology Research and Development Program of China (Grant No’s. 2012AA022104 & 2012AA021205), the Fundamental Research Funds for the Central Universities (Grant No’s. 222201313007 & 222201314048), the National Special Fund for State Key Laboratory of Bioreactor Engineering (No.2060204).
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