Establishment of a highly efficient conjugation protocol for Streptomyces kanamyceticus ATCC12853

Abstract Kanamycin B as the secondary metabolite of wild‐type Streptomyces kanamyceticus (S. kanamyceticus) ATCC12853 is often used for the synthesis of dibekacin and arbekacin. To construct the strain has the ability for kanamycin B production; the pSET152 derivatives from Escherichia coli ET12567 were introduced to S. kanamyceticus by intergeneric conjugal transfer. In this study, we established a reliable genetic manipulation system for S. kanamyceticus. The key factors of conjugal transfer were evaluated, including donor‐to‐recipient ratio, heat‐shock, and the overlaying time of antibiotics. When spores were used as recipient, the optimal conjugation frequency was up to 6.7 × 10−6. And mycelia were used as an alternative recipient for conjugation instead of spores; the most suitable donor‐to‐recipient ratio is 1:1 (107:107). After incubated for only 10–12 hr and overlaid with antibiotics subsequently, the conjugation frequency can reach to 6.2 × 10−5 which is sufficient for gene knockout and other genetic operation. Based on the optimized conjugal transfer condition, kanJ was knocked out successfully. The kanamycin B yield of kanJ‐disruption strain can reach to 543.18 ± 42 mg/L while the kanamycin B yield of wild‐type strain was only 46.57 ± 12 mg/L. The current work helps improve the content of kanamycin B in the fermentation broth of S. kanamyceticus effectively to ensure the supply for the synthesis of several critical semisynthetic antibiotics.

5%-10% . The inadequate supply of KB seriously confines the output and leads to the high price of these crucial antibiotics.
To solve the problem of KB in short supply, it is urgent and significant to construct engineered strain with the high-yield ability of KB.
Many efforts have been made to make S. kanamyceticus produce more KB relatively. Thapa, Oh, Lee, and Liou (2007) reported the heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in Streptomyces venezuelae YJ003, but the isolated compound from the transformant was verified to be KA rather than KB.  demonstrated that genes aprD3, aprQ, and aprD4 were disrupted together in Streptomyces tenebrarius H6, which blocked apramycin and oxyapramycin biosynthesis and increased the production of carbamoyl KB. Subsequently, the disruption of tacA made the mutants produce KB instead of carbamoyl KB.
The work demonstrated that the subtle genetic engineering could significantly improve the expected production and eliminate the undesired one. In our study, we hope to change the distribution of the fermentation products in S. kanamyceticus and make KB become the main product. Studies of kanamycin biosynthetic pathways provide information and direction for strain improvement. Park's team discovered that there are two parallel pathways to synthesize KA and KB, in which the first glycosyltransferase kanF accepts both UDP-Glc and UDP-GlcNAc as cosubstrates but preferentially transfers the former (Nepal, Oh, & Sohng, 2009;Park, Park, Nepal, & Han, 2011). However, in 2012, Sucipto reported that kanJ and kanK are responsible for the transformation from KB to KA, that is, KB would be the direct biosynthetic precursors of KA (Sucipto, Kudo, & Eguchi, 2012). These findings lay the solid foundation for the construction of high KB-producing mutants from wild-type S. kanamyceticus.
In this study, we knocked out kanJ and found that the knockout of kanJ led to the accumulation of KB. The KB yield of the kanJ-disruption strain was 10.7-fold higher than that of the original strain. Recently, Gao reported a similar research and got similar conclusions (Gao, Wu, Sun, Ni, & Xia, 2017). Compared with Gao's work, we established a reliable genetic manipulation system for S. kanamyceticus by optimization of conjugation conditions including donor-to-recipient ratio, heat-shock, and overlaying time of antibiotics. Mycelia are used as recipient rather than traditional spores and there are no same reports in S. kanamyceticus. In our experiments, we found that the conjugation frequency of mycelium was higher than that of spores. The highest conjugation frequency of mycelium was 7.9-fold higher than that of spores.

| Microorganisms, plasmids, media, and culture conditions
The microorganisms and plasmids used in this study are listed in Table 1. The Mannitol-Soy-agar (MS) medium and seed medium were used for conjugal transfer and selection of S. kanamyceticus mutant.
Genomic DNA was isolated from wild-type S. kanamyceticus, which was also used as the host. Both S. kanamyceticus and its mutants were cultivated in 250-ml shaken flasks, with seed medium contained 15 g soluble starch, 4.0 g yeast extract, 0.5 g K 2 HPO 4 , and 0.5 g MgSO 4 in 1.0 L tap water. After incubation at 28°C for 30 hr, the fermentation medium was inoculated with 3 ml (10% [vol/vol]) seed culture and incubated for 7 days. The fermentation culture medium contained 35 g soyal bean, 30 g maltose, 25 g soluble starch, 8 g Sodium nitrate, and 0.1 g ZnSO 4 in 1.0 L tap water. Appropriate antibiotics were added to the media when needed at the following concentrations: apramycin, 35-50 μg/ml; nalidixic acid, 50 μg/ml; kanamycin, 25 μg/ml, and chloramphenicol, 25-50 μg/ml.

| Construction of the plasmids
On the basis of pSET152, the plasmid pSQ202 was used to knockout the kanamycin biosynthetic gene (kanJ). A chloramphenicol resistance gene (Cmr) was required because no proper selection marker was available in pSQ202. First, the gene was amplified from pSA74 using the Cmr forward (F) and Cmr reverse (R) primers that had been inserted with SacI and XbaI, respectively. Subsequently, to amplify a 1,020-bp fragment containing the upstream sequence of kanJ (kanJ-U) (GenBank ID: AJ628422.2), we used kanJ-U F and kanJ-U R primers, which had been inserted with HindIII and SacI, respectively. To amplify a 1,073-bp fragment containing the downstream sequence of kanJ (kanJ-D) (GenBank ID: AJ628422.2), we used kanJ-D F and kanJ-D R primers, which had been inserted with XbaI and EcoRI, respectively. All three PCR fragments were digested with their corresponding enzymes and inserted to the HindIII/EcoRI cloning sites of pSQ202 generating plasmid pSQ202-J.

| Establishment and optimization of a conjugal transfer system
The intergeneric conjugation between E. coli and S. kanamyceticus was carried out as described previously by Mazodier, Petter, and Thompson (1989) with some modifications. The culture of the donor E. coli ET12567 (pSQ202, pUZ8002) containing conjugative plasmid was grown with the appropriate antibiotics to an optical density at 600 nm (OD600) of 0.4-0.6. To remove the antibiotics, the cells were collected and washed twice with Luria-Bertani broth (LB) and then suspended in 1 ml LB. The S. kanamyceticus spores without heat treatment were washed twice and suspended in 2× YT broth at a concentration of 10 9 per ml. Subsequently, the S. kanamyceticus spores were heated at 45-60°C for 10 min, incubated at 37°C for 2-3 hr and served as recipient. Donor and recipient cells were mixed and spread on MS plates and grown for 14-20 hr at 28°C. The effect of MgCl 2 at 10-40 mM on conjugal efficiency was also evaluated.
For conjugation with mycelia, S. kanamyceticus was incubated in seed medium for 48 hr at 28°C. Mycelia were collected and mixed with exponential donor cells, spread on the seed agar plates, and grown for 8-16 hr at 28°C. After incubation, the plates were covered with 1 ml water containing nalidixic acid (50 μg/ml) and apramycin (35 μg/ ml) or chloramphenicol (50 μg/ml) as required and incubated at 28°C for 3-5 days until the exconjugants appeared.
The frequency of pSQ202 transfer was calculated based on the number of exconjugants on a selective plate divided by the number of recipient cells on a nonselective plate (Liu, Lin, Zhang, & Bian, 2007). The average frequency of three independent experiments was calculated.

| Confirmation of the exconjugants by PCR
The exconjugants genomic DNA was extracted and then was confirmed by two PCR primers. The first pair of primers, Cmr F and Cmr R, was designed to prove that Cmr replaced kanJ; the expected PCR product was 898 bp. The second pair of primers, kanJ F and kanJ R, was used to verify whether the kanJ gene was present in the exconjugants. The amplification product was subjected to sequence analysis, and the result was compared with the sequence in GenBank.

| Antibiotic isolation and analysis
After the wild-type strain and kanJ mutant were cultured in fermentation medium at 28°C for 7 days, the culture broth was collected.
Then, the pH of the culture broth was adjusted to 2 with H 2 SO 4 , and the acidified broth was stirred for 30 min and then centrifuged (1,680 g; 15 min). The supernatant was subsequently readjusted to pH 7 using NaOH and then re-centrifuged (1,680 g; 15 min). The supernatant of the culture broth was prepared for bioassays and further separation and purification. Then further purification and product analysis were referred to a novel method that Qiao's laboratory has developed previously for the direct determination of  (Zhang, He, Zhang, & Liu, 2015). The isolated compound was also restored by dissolving the dried precipitates in water, and then, it was analyzed by electrospray ionization-mass spectrometry (ESI-MS).

| Effect of the concentration of MgCl 2 on E. coli and S. kanamyceticus conjugation
MgCl 2 is commonly added to conjugation medium to improve the frequency of conjugation ( MgCl 2 appeared to be the optimal concentration for conjugation of S. kanamyceticus.

| Effect of heat-shock on E. coli and S. kanamyceticus conjugation
The heat-shock of the S. kanamyceticus spores was assessed in a temperature range between 45 to 60°C in order to determine the optimal temperature for conjugation. After heat-shock, Streptomyces spores were precultured for 2-3 hr at 37°C to shorten the germination time.
Different temperatures were selected to evaluate the effect of heat treatment on the conjugation frequency. As shown in Table 3, the rising temperature affiliated to the conjugation efficiency, and the maximum appeared at 55°C. However, the conjugation frequency rapidly decreased to 0.7 × 10 −7 when the temperature was 60°C.
Based on these results, 55°C was chosen as the best heat-shock condition for the spores and used for all subsequent experiments.

| Effect of donor-to-recipient ratio on E. coli and S. kanamyceticus conjugation
The ratio of donor-to-recipient cell number was a crucial parameter in the intergeneric conjugation of Streptomyces (Enriquez, Mendes, Anton, & Guerra, 2006). In order to establish an optimal spores recipient number for a given number of E. coli donor, we set up 4 series of matings. Different concentration of S. kanamyceticus spores and mycelia was mixed with E. coli ET12567 (pUZ8002, pSQ202) cells, respectively. The conjugation frequency was calculated and shown in Table 4. For spores recipient, no exconjugant was observed at the donor-to-recipient ratio of 10:1. The number of exconjugant colonies was found to increase with the number of spores recipient. The conjugation frequency increased to 6.7 × 10 −6 when the ratio was up to 1:100. Subsequently, mycelia were used as recipient instead of spores. The highest conjugation frequency was obtained at the ratio of 1:1, which was 7.9 times higher than that optimized spores.
However, few exconjugants were observed when the number of mycelia cells was 10 9 .

| Effect of overlaying time of antibiotics on E. coli and S. kanamyceticus conjugation
Donor and recipient cells were mixed and spread on plates with in- Note. Time of heat-shock is 10 min.
S. kanamyceticus). The antibiotic overlaying time was another factor which may influence the conjugation frequency (Table 5). More exconjugants were obtained with the time prolonged from 16 to 18 hr.
To some extent, premature overlaying weakened the growth ability of mycelia on the culture medium. Nevertheless, when the overlaying time of antibiotics was extended to 22 hr, the exconjugants of a single colony could not be observed on the plate and it induced false-positive result of exconjugants. This suggested that the best overlaying time of antibiotics mix-culture plates was 18-20 hr for using S. kanamyceticus spore as recipient, but only 10-12 hr for S. kanamyceticus mycelia.

| Construction of recombinant plasmids pSQ202-J
To construct the kanJ disruption plasmid, pSQ202 was used to delete the inner HindIII fragment (0.8 kb) of the ΦC31 int gene. The plasmid was constructed as described in materials and methods.
The genetic organization and the restriction endonuclease map are shown in Figure 2.

| Transformation of pSQ202-J into S. kanamyceticus
According to the best conjugal transfer condition mentioned above, E. coli ET12567 (pUZ8002, pSQ202-J) was used as donor to TA B L E 4 The effect of donor-to-recipient ratio on Escherichia coli and Streptomyces kanamyceticus conjugation

| D ISCUSS I ON
There are different restriction-modification systems in Streptomyces due to the diversity of them (Sadeghi, Soltani, Nekouei, & Jouzani, 2014 The results revealed that the donor-to-recipient ratio played a crucial role for affecting conjugation frequency in S. kanamyceticus.
Different number of E. coli donor cells was used to explore the optimal one for a given number of spores. However, there were no obvious differences in the consequences and 10 7 was applied for further experiments. As the recipient cells' number increased, the number of exconjugant colonies increased simultaneously when spores were used in the tested range. For mycelia as recipient, in contrast, the increasing donor cells were not expected to achieve the improvement of conjugation frequency, which was not consistent with the results of spores.
The present work firstly demonstrated that S. kanamyceticus mycelia could be used quite effectively as recipient for intergeneric conjugation instead of spores. In addition, compared to 5-7 days culture time for spores, it was only 2-3 days when mycelia were used as recipient, and no heat-shock operation was required. In particular, the optimal conjugation frequency achieved with S. kanamyceticus mycelia (6.2 × 10 −5 ) was more satisfactory than that obtained with spores (6.7 × 10 −6 ). It suggested that mycelia were more appropriate in S. kanamyceticus conjugation experiments.
Generally, several conjugal transfer conditions were screened, and the optimal one was confirmed to transfer the foreign plasmids into S. kanamyceticus. Kanamycin biosynthetic gene (kanJ) was knocked out through derivatives of pSET152, restraining the transformation of metabolites from KB to KA. The ferment result proved that we successfully made KB to be the main product in the fermentation broth. The highest KB yield of ΔkanJ mutant reached 585.33 mg/L, which was 10.7-fold higher than that of the original strain. Thus, the current work provided a strategy to obtain adequate KB as raw materials for synthesis of dibekacin and arbekacin. Importantly, the possibility to create a new engineered strain, by using the similar genetic manipulation, is particularly significant for the future application of other antibiotics production.

ACK N OWLED G M ENTS
The work was supported by the National Natural Science Foundation of China (21672021, 21572018, 21372024 and 21232005) and by the State Key Laboratory of Natural and Biomimetic Drugs.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R S CO NTR I B UTI O N
Shuman Zhang performed the experiment and edited the final version of the manuscript. Tiansheng Chen performed the experiment.
Jia Jia, Liwen Guo, and Huizheng Zhang analyzed data. Chao Li and Renzhong Qiao contributed reagents and analytical tools. Shuman Zhang and Tiansheng Chen contributed equally.

DATA ACCE SS I B I LIT Y
The data will be available on request from the corresponding authors.