Genome shuffling and ribosome engineering of Streptomyces virginiae for improved virginiamycin production

Medium and culture conditionsSlant and plate medium contained (per liter): 20.0 g soluble starch, 1.0 g KNO3, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g NaCl, 0.01 g FeSO4·7H2O, 15.0 g agar. The pH was adjusted to 7.2-7.4 before autoclaving.Seed and production medium contained (per liter): 7.5 g casitone, 7.5 g yeast extract, 15 g glycerol, 2.5 g NaCl. The pH was adjusted to 6.5 before autoclaving.The R2YE regeneration medium was prepared as described by Wang et al.Both slant and plate cultures were incubated for 4-7 days at 28 °C and relative humidity of 30-60 %. A loopful of spores was transferred into a 500 mL Erlenmeyer flask with 50 mL seed medium. After incubation at 28 °C for 24 h on a rotary shaker at 220 rpm, a 1.5 mL portion of the seed culture was inoculated into 50 mL of production medium in a 500 mL Erlenmeyer flask. Production culture was incubated at 28 °C for 24 h on a rotary shaker at 220 rpm. All the fermentative experiments were performed in triplicate.Protoplast preparation and regenerationSpore suspensions of the starting strains were subsequently inoculated in 50 mL of mycelia medium in 500-mL Erlenmeyer flasks with 0.3 % glycine. After incubation, mycelia were harvested by centrifugation at 6000 rpm for 10 min at 4 °C, washed twice with P buffer, and treated with lysozyme for several quarters at appropriate temperature. Then, lysozyme was removed by washing twice with P buffer, the protoplasts were obtained. The rates of protoplast preparation and regeneration were obtained by determining colony counts using the following formulas:Protoplast preparation ratio (%) = (C-B)/C*100Protoplast regeneration ratio (%) = (A-B)/(C-B)*100Where A is the colony number of protoplasts obtained from the solid regeneration medium (the mycelia with enzyme-treated were spread on the solid regeneration medium); B is the colony number of mycelia with water treatment at 30 °C for 30 min obtained from the solid regeneration medium (the mycelia with enzyme-treated were spread on the solid plate medium); C is the total number of colonies obtained from the solid regeneration medium before hydrolysis of lysozyme.Previous studies on protoplast preparation and regeneration have shown that the concentration of enzyme, time for enzyme treatment and mycelia age are key factors, hence these three main variables were tested for the optimization of protoplast preparation and regeneration conditions to conduct protoplast fusion using a L9 (33) orthogonal test. The experimental conditions and set-up of the design are shown in Table 1. The detailed method was described above.Protoplast inactivation and screening of fusantsThe protoplast suspension was inactivated by heat or UV radiation. For heat inactivation, the protoplasts were held at 65 °C for some time to choose the optimal inactivation condition; for UV inactivation, the protoplast suspensions were placed under a preheated 30-W UV lamp at a vertical distance of 10 cm for a period of time to choose the optimal inactivation condition.The fusants were screened by the high-throughput method in Fig1. The probability of fusant (the frequency of recombinants) was calculated using the following formula:Probability of fusant (%) = [1-(D+E)/(2F)]* 100Where D is the total number of colonies with UV-inactivated protoplasts, E is the total number of colonies with heat-inactivated protoplasts, F is the total number of colonies after fused.Streptomycin resistance-aided genome shufflingGenome shuffling was carried out using the described methods with modifications [24, 25]. Aliquots of protoplasts from each mutant were mixed. One half of the mixture was inactivated with UV radiation for 10 min and the other was held at 65 °C for 20 min. Both of the inactivated protoplasts were mixed in a cell ratio of 1:1, centrifuged at 3000 rpm for 10 min and resuspended in 4 mL stabilizer solution containing 40 % PEG1000 and 25 mM CaCl2. Then, the protoplasts were fused at 30 °C for 5 min. After washing twice with 10 mL of P buffer, the fusants was resuspended in 5 mL P buffer, then transferred to the regeneration plate medium containing 20 μg/mL streptomycin. After incubation at 28 °C for several days, the appeared colonies were isolated to carry out fermentation test in microwell plates. Mutant strains with the maximum VGM activity were roughly screened by the high-thoughput screening method and further by HPLC to determine the levels of VGM to obtain the G1 strains. Five successive rounds of genome shuffling were performed by the same methods, and in the second, third, fourth and fifth rounds of genome shuffling, the concentration of streptomycin in regeneration was increased to 40, 60, 80, and 100 μg/mL, respectively.