Over-expression of deacylase gene in Streptomyces hosts
Enzymatic modification is more efficient than chemical modification, but deacylase was rate-limiting in the antibiotics production process, as the bioconversion efficiency of deacylases in the original strain is low. Streptomyces strains are suitable heterologous hosts for producing enzymes, particularly GC-rich deacylases, for industrial applications.25,26 To investigate the effect of deacylase gene, pDS701 plasmid consist of deacylase gene was introduced into six Streptomyces species (S. lividans TK24, S. coelicolor, S. albus, S. avermitilis K139, S. ambofaciens 2283 and S. griseus), yielding recombinant strains SLI701, SCO701, SAL701, SAV701, SAM701 and SGR701.13 There were no apparent phenotypic differences between Streptomyces and its recombinant strains. Genotypes of six recombinant strains were verified by polymerase chain reaction (PCR). The 3.3-Kb DNA fragments were amplified. Sequencing and alignment confirmed that all six segments had 100% identity with the deacylase gene of recombinant plasmid pDS701 (Fig. 2). As negative controls, PCR products could not be amplified from S. albus host and wild-type A. utahensis.
Deacylation of different recombinant Streptomyces strains
Six recombinant Streptomyces strains and A. utahensis NRRL 12052 were inoculated, the mycelia were collected to determine the bioconversion efficiency. The reaction mixture containing 2 g/L polymyxin B1 in 0.1 M Tris buffer pH8.0, was incubated at 30℃, 5 h and then analysed by high-performance liquid chromatography (HPLC). All seven samples showed the new peak, which almost having the same retention time with the standard cyclic heptapeptide moiety of polymyxin B1 (4.56 min, Fig. 3b), and a peak of remaining polymyxin B1 substrate (31.10 min, Fig. 3a). The overlapped peak also obtained by injecting the mixture of SAL701 sample and standard cyclic heptapeptide polymyxin B1. So we preliminary verified that all six recombinant species and wild-type strain could deacylate polymyxin B1, with different activity dependent on the host strains. As shown in Table 1, five of the six recombinant strains showed similar bioconversion efficiencies, and although SAV701 showed a lower conversion efficiency (Fig. 3g), it was still higher than that of the wild-type strain (34.0% compared with 23.3%). SAL701 (Fig. 3f) showed the highest efficiency with the highest percentage of bioconversion rate (65.2%). No polymyxin B1 or its cyclic heptapeptide moiety was detected in the whole-cells of all seven species examined. Therefore, the S. albus were considered as the preferred host for enzymatic deacylation. SAL701 strain has many advantages such as easy-cultivating, rapid-growing and high bioconversion efficiency, so it could be applied to bioconversion of polymyxin B1 in a short time.
Table 1 Bioconversion rate by wild-type and different recombinant Streptomyces strains
Strains
|
Description
|
Bioconversion rate (%)
|
A. utahensis NRRL 12052
|
wild-type
|
23.3 ± 3.2
|
SLI701
|
pDS701 integrated into S. lividansTK24
|
58.1 ± 2.1
|
SCO701
|
pDS701 integrated into S. coelicolor
|
60.7 ± 0.4
|
SAL701
|
pDS701 integrated into S. albus
|
65.2 ± 4.5
|
SAV701
|
pDS701 integrated into S. avermitilis K139
|
34.0 ± 3.9
|
SAM701
|
pDS701 integrated into S. ambofaciens 2283
|
59.8 ± 0.9
|
SGR701
|
pDS701 integrated into S. griseus
|
52.3 ± 1.0
|
S. albus
|
Streptomyces without plasmid as negative control
|
0
|
Qualitative analysis of polymyxin B1 and its cyclic heptapeptide
Polymyxin B1 and its cyclic heptapeptide moiety were extracted from the reaction mixture, and their identities were confirmed. The structure of the polymyxin B1 cyclic heptapeptide moiety was verified by using electrospray ionization-mass spectrometry (ESI-MS). The parent ion was observed at m/z 784.4441 [M+Na]+ (calculated 784.4440), and its mass spectrum was consistent with the molecular formula C35H59N11O8 (Fig. 4a). Polymyxin B1 showed parent ions at m/z 602.3822 [M+2H]2+ (calculated 602.3822) and m/z 1203.7566 [M+H]+ (calculated 1203.7572), and its mass spectrum was consistent with the molecular formula C56H98N16O13 (Fig. 4b).7 Impure preparations, polymyxin B containing polymyxin B1, were also successfully deacylated but at a slower rate.
Effect of reaction buffer on bioconversion of polymyxin B1 by SAL701
The buffer of reaction mixture strongly affects the bioconversion rate. Hydrolysis of polymyxin B1 catalyzed by SAL701 was evaluated in different 0.1 M buffer pH range of 3.0-10.0 at 30 °C for 5 h. As shown in Table 2, the strain displayed the highest activity at pH 8.0 in Tris buffer (65.2%), which was selected as the standard buffer for deacylation. The same pH value (pH 7.5 or 8.0) in three different buffers were compared, and found that Tris buffer (60.8% and 65.2%) was more suitable for reaction than phosphate buffer (60.5% and 62.9%) and disodium hydrogen phosphate-citrate buffer (53.2% and 52.3%).
Table 2 Effect of reaction buffer
pH
|
Bioconversion rate (%)
|
disodium hydrogen phosphate-citrate buffer
|
phosphate buffer
|
Tris buffer
|
3.0
|
21.2 ± 1.2
|
|
|
3.5
|
19.3 ± 3.4
|
-
|
-
|
4.0
|
23.6 ± 0.9
|
-
|
-
|
4.5
|
25.9 ± 4.6
|
-
|
-
|
5.0
|
26.9 ± 3.0
|
|
|
5.5
|
33.3 ± 2.7
|
31.9 ± 2.2
|
-
|
6.0
|
42.8 ± 3.5
|
41.3 ± 4.0
|
-
|
6.5
|
41.4 ± 2.2
|
50.9 ± 3.1
|
-
|
7.0
|
42.9 ± 4.1
|
55.6 ± 3.7
|
-
|
7.5
|
53.2 ± 0.8
|
60.5 ± 0.8
|
60.8 ± 3.0
|
8.0
|
52.3 ± 1.2
|
62.9 ± 0.9
|
65.2 ± 4.0
|
8.5
|
|
|
64.7 ± 1.2
|
9.0
|
-
|
-
|
61.5 ± 1.9
|
9.5
|
-
|
-
|
62.2 ± 2.1
|
10.0
|
|
|
60.3 ± 2.0
|
Also, the activities were increased when the ionic strength of buffer was increased (0.05-0.3 M phosphate buffer and Tris buffer). The bioconversion efficiency increased from 56.3% to 70.1% in Tris buffer and from 52.8% to 61.2% in phosphate buffer (Table 3).
Table 3 Effect of ionic and ionic strength
Buffer
|
Ionic
|
Bioconversion rate (%)
|
0.05 M
|
0.1 M
|
0.2 M
|
0.3 M
|
phosphate buffer
|
-
|
52.8 ± 1.7
|
55.6 ± 3.9
|
59.0 ± 2.2
|
61.2 ± 1.6
|
Tris buffer
|
-
|
56.3 ± 4.2
|
65.2 ± 1.1
|
70.1 ± 2.9
|
69.2 ± 1.0
|
Tris buffer
|
K+
|
-
|
-
|
73.9 ± 3.0
|
-
|
Tris buffer
|
Na+
|
-
|
-
|
72.1 ± 1.5
|
-
|
Tris buffer
|
Mg2+
|
-
|
-
|
74.7 ± 2.1
|
-
|
Tris buffer
|
Cu2+
|
-
|
-
|
69.2 ± 2.0
|
-
|
Tris buffer
|
Fe2+
|
-
|
-
|
72.7 ± 1.3
|
-
|
Deacylase activity did not require metal ions as previously studied. But the inclusion of an ion (0.2 M K+, Na+, Mg2+ or Fe2+), enhanced the deacylase activity (73.9%, 72.1%, 74.7% and 72.7% respectively, compared to 70.1% for enzyme alone, Table 3). Addition of Cu2+ did not increase the activity. Therefore, 0.2 M Tris buffer pH 8.0 with 0.2 M Mg2+ was selected as the standard condition for the deacylation reaction.
Effect of reaction temperature and time on bioconversion of polymyxin B1 by SAL701
The enzymatic activity varies over a temperature range of 25-70°C. Maximum deacylase activity was achieved at 40°C under the optimized reaction buffer (0.2 M Tris buffer, pH 8.0 with 0.2 M Mg2+), the conversion rate of polymyxin B1 was 76.9% (Fig. 5).
The ability of SAL701 to deacylate polymyxin B1 was examined during 96 h. The time course was determined under the optimized buffer conditions. As shown in Fig. 6, the deacylated product occurred at the earliest time tested (5 h), and the bioconversion efficiency was 74.8%, and then gradually increased to 77.7% at 96 h.
Effect of substrate concentration on bioconversion of polymyxin B1 by SAL701
The optimum concentration of polymyxin B1 in the bioconversion mixture was confirmed using SAL701, SCO701, SAM701 recombinant strains and wild-type A. utahensis NRRL 12052, the relatively highly active recombinant Streptomyces strains. The optimal substrate concentration of SAL701 was increased from 2.0 to 6.0 g/L, under conditions in which the bioconversion efficiency was more than 60%, and SCO701, SAM701 were increased from 2.0 to 4.0 g/L (Table 4).
Table 4 Effect of substrate concentration
polymyxin B1 concentration (g/L)
|
Bioconversion rate (%)
|
SAL701
|
SCO701
|
SAM701
|
A. utahensis NRRL 12052
|
1.0
|
78.4 ± 3.0
|
74.8 ± 2.5
|
75.1 ± 2.1
|
39.4 ± 2.2
|
2.0
|
73.9 ± 1.7
|
72.4 ± 1.7
|
72.3 ± 2.8
|
31.8 ± 1.8
|
3.0
|
70.2 ± 1.3
|
69.4 ± 1.5
|
68.9 ± 0.6
|
26.4 ± 3.0
|
4.0
|
70.7 ± 1.3
|
66.4 ± 2.5
|
64.3 ± 2.8
|
22.5 ± 0.3
|
6.0
|
61.2 ± 0.8
|
58.7 ± 3.1
|
56.1 ± 0.5
|
21.0 ± 0.7
|
8.0
|
43.7 ± 0.3
|
43.6 ± 3.2
|
50.4 ± 1.5
|
23.0 ± 1.7
|
10.0
|
47.3 ± 1.3
|
36.8 ± 2.7
|
41.4 ± 1.7
|
19.5 ± 1.2
|
12.0
|
33.9 ± 1.6
|
37.9 ± 2.8
|
30.1 ± 3.9
|
21.2 ± 3.9
|
Thermostability of SAL701
The thermostability of the mycelia of recombinant S. albus was evaluatedat different temperatures under the optimized conditions above (Fig. 7). The enzyme showed no decrease inactivity after storage at 4°C and 30°C for at least 48 h. However, the mycelia also maintained 90% of its activity following storage at 50°C for at least 12 h.
Recycling of mycelia of SAL701
The mycelia was used to bioconvert polymyxin B1, the number of times required for the mycelia to be reused was investigated. The mycelia of SAL701 could be recycled at least three times without loss of catalytic activity on the basis of maintaining the bioconversion efficiency at 70.2% under the optimized conditions above (Table 5).
Table 5 Number of times of reutilizing the mycelia
Number of times
|
Bioconversion rate (%)
|
1
|
72.4 ± 0.2
|
2
|
73.5 ± 3.1
|
3
|
70.2 ± 3.0
|
4
|
50.3 ± 3.7
|
5
|
30.9 ± 1.8
|
6
|
23.4 ± 1.6
|
7
|
6.3 ± 1.2
|
8
|
6.9 ± 2.4
|
Substrate spectrum of SAL701
The aforementioned results established the characteristic of SAL701 and necessitated further research about their substrate spectrum. The chemical structure of β-lactam antibiotics also consists a β-lactam ring and a side chain. Different β-lactam substrates were used to determine the bioconversion rate. But no enzymatic deacylation was observed for the β-lactam compounds penicillin G and cephalosporin C.
Hydrolysis of specific amide bonds of SAL701
Enzyme-catalyzed reactions are specific. Amidohydrolase from E. coli N.C.I.B. 8743 is stereospecific for the deacylation of α-amino acids, acylated L-α-amino acids but not D-α-amino acids are hydrolyzed, whereas DL-amino acids are only 50% hydrolyzed.27-29 SAL701 catalyzes hydrolysis of amide bond 1 (Fig. 1), which is the closest amide bond to the cyclic heptapeptide of polymyxin B1, rather than at other amide bonds, suggesting its high specificity for the catalytic site. The specificity of deacylase could avoid the existence of impurities, reduce the difficulty in the final purification process, and improve the economic benefits of the process.
An enzyme’s specificity depends on its structure. As a representative functional deacylase, the sequence similarity to many other deacylases. Although a few deacylase structures have been reported, that of a functional deacylase has not been determined. Homology protein structure models for deacylase was constructed to identify the structural basis for the programming of this distinct specificity (Fig. 8) based on the known crystal structure of the MacQ (PDB ID 5C9I; resolution: 1.80 Å, and identity of 39.1% with deacylase). MacQ derived from Acidovorax sp. strain MR-S7 are comprised of 806 amino acid residues, and exhibits acylase activity against β-lactam antibiotics and N-acylhomoserine lactones (AHLs). Structural comparison with MacQ revealed that deacylase has a similar structure and active site organization, indicating that the key enzyme-co-product interactions among MacQ are well-conserved, deacylase and similar enzymes such as the catalytic site specificity. Ser/Thr/Cys is the necessary residue of catalysis, and initiates amide bond cleavage of various substrate compounds as a nucleophile. Similar to Ser1β, which act as a catalytic residue in MacQ, the docking results indicated that Thr190 in deacylase from A.utahensis NRRL 12052, which located near amide bond 1, can facilitate hydrolysis with high regioselectivity.24,30,31 The relatively close distance of the hydroxyl group to the amide carbon atom of the peptide bond, suggests an important role for the residues in catalysis, as revealed by substrate docking.