## 3 Results  
### Screening Hybrid Strains by Improving the Mutant rate of Genome Shuffling  
After several rounds of antibiotic screening, further enhanced production of secondary metabolites was hard to obtain [20]. Genome shuffling could assemble the genetic diversity from different strains into a selected mutant by extensive homologous recombination. Thus, the beneficial traits from the starting strains with the desired phenotype could be inherited. In this study, the selected mutants SD10, SD88, SD99, and SD143 were used as the parental strains and the colonies were cultured on antibiotic resistance plates (Table 1). Three successive rounds of genome shuffling were applied and the candidate high producers were screened out by comparing their toyocamycin production. As shown in the Table 1, the toyocamycin production and drug tolerance in _S. diastochronogenes_ 1628 were increased gradually after each round of genome shuffling, and the production of toyocamycin was related to increased drug-screening concentration. For the first round of genome shuffling, 2 strains (R1-31, R1-41) were screened out from 65 clones with the highest toyocamycin production. The production of toyocamycin increased by 6.4-fold and 6.2-fold compared to the wild type _S. diastatchromogenes_ 1628, and reached 443.9 mg/L and 434.4 mg/L, respectively. Afterwards, the second round of genome shuffling was carried out by screening the drug-resistant mutants with a higher drug concentration (Str 250 mg/L, Rif 200 mg/L, Par 35 mg/L). R2-8 were obtained with 558.5 mg/L toyocamycin production, and were 9.3-fold that of the wild type strain. The percentage of positive mutants in the second round of genome shuffling was 13.6% (Table 2). After the third round of genome shuffling, R3-33 were obtained with a higher drug resistance (Str 275 mg/L, Rif 275 mg/L, and Par 40 mg/L), and the production of toyocamycin reached 644.5 mg/L, which was 10.8-fold that of the wild-type production. The toyocamycin production of R1-31, R1-41, R2-8, and R3-33 were increased significantly, compared with the wild-type by one-way analysis of variance (\(p<0.01\)). At the same time, the positive mutant rate decreased (Table 2) among the colonies appearing on the agar plates with the increases in drug concentration. There were several genes commonly mutant during the ribosome engineering, including _rpsL_, _rsmG_, and _rpoB_, but not all mutations occured in these three genes [20; 28]. In order to identify the change of genome occurring, we sequenced and analyzed the _rpsL_, _rmsG_, and _rpoB_ genes between the mutants and these parental strains. As shown in Table 3, for the first-round shuffling, R1-31 had a deficiency at the 40 bp of _rmsG_, which was obtained \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Strains** &
\begin{tabular}{c} **Frequency (\%) of Screening (Positive** \\ **Mutant/Total Colonies)** \\ \end{tabular} & **Toyocamycin Production (mg/L)** & **Increased Rate (\%) a** \\ \hline R1-31 & 10.7 (7/65) & 443.9 & 57.9 \\ R1-41 & 10.7 (7/65) & 434.4 & 54.4 \\ R2-8 & 13.6 (6/44) & 558.5 & 25.8 \\ R3-33 & 7.5 (3/40) & 644.5 & 15.4 \\ \hline \hline \end{tabular}  
* the increased rate was calculated by compassion with front-round highest production.  
\end{table} 
Table 2: The screening and toyocamycin production of drug-resistant mutants. from the strain SD143. R-41 had a mutation at Cys59 Ter, which could affect the activity of RsmG. While in the second round of shuffling, R2-8 retained the mutation Cys59 Ter of R1-41. The third round of shuffling also maintained the mutation Cys59 Ter in the _rmsG_ of R3-33. Interestingly, a new mutation Gly122 Val was introduced in the _rpsL_ of R3-33, which may occur in the screening using higher streptomycin. There was no mutation in the _rpoB_ gene, but all the mutants showed higher rifampicin-resistance during the three rounds of shuffling. This phenomenon may imply the presence of other unknown mutations in the genome which were responsible for the rifampn tolerance.