Molecular characterization of the superior lignin peroxidase-producing Streptomyces lavendulae R-St-1 mutants and fusants
Authors: Batayyib Reem S.M. , Al-Twaty Nada H. , El-Hamshary Ola I.M. 
Published: February 2023

Background
The extracellular lignin peroxidase (LiP) secreted by bacterial isolates is the key enzyme in lignin degradation in several species of Streptomyces (actinomycetes). Random mutations were induced for bacterial strains using ultraviolet (UV) and ethyl methanesulfonate (EMS). Moreover, protoplast fusion is an important tool in strain improvement to achieve genetic recombination and developing hybrid bacterial strains. The molecular analysis of mutants and fusants by random amplification of polymorphic DNA (RAPD-PCR) was done.

Objective

Streptomyces lavendulae R-St strain, which produces the highest LiP, was discovered and investigated in a previous study by the authors. It has been deposited in NCBI under the accession number ‘OL697233.1.’ S. lavendulae was used in the present study to produce novel, higher LiP-producing mutants using EMS-mutagenesis and UV light. Most mutant strains that produce LiP fuse their protoplasts. To assess the genetic diversity of isolated S. lavendulae R-St-1 with its mutants and fusants, RAPD-PCR was used.

Materials and methods
Lignin was extracted and purified from black wood liquor. UV and EMS were used for creating super LiP-producing mutants of S. lavendulae R-St. Protoplast fusion between EMS and UV-treated mutants was performed for isolating LiP-productive fusants (s) from S. lavendulae R-St-1 as the original isolate. Fermentation medium (FM) (g/l) was used for lignin-degrading bacterial screening after dilution of the soil samples: K2HPO4, 4.55, KH2PO4, 0.53, MgSO4,0.5, NH4NO3, 0.1, yeast extract, 0.1, lignin (0.1% v/v), agar 15, and the pH should be 7.0. The aforementioned FM medium was supplemented with 50 mg/l of azure B and toluidine dyes and 100 mg/l of tannic acid. FM was used without any supplements and agar for the isolation of lignin-degrading bacteria using lignin (0.1% v/v). The molecular analysis of mutants by RAPD-PCR was applied using different primers, and different separate bands were determined.

Results and discussion

S. lavendulae R-St-1 strain was mutagenized with alkylating EMS (200 mm) and UV. Results showed that from the S. lavendulae R-St-1 (W.T) isolate, two EMS-treated mutants (Rst/60/7E and Rst/40/8E), which showed activities of 8.5 and 7.3 U/ml, respectively, and two UV-treated mutants (Rst/9/2U and Rst/9/6U), which showed activities of 9.4 and 7.8 U/ml, respectively, were the most efficient ligninolytic mutants. Protoplast fusion between two higher LiP-producing mutants (cross 1 and 2) proved to be the most effective, and the two isolated fusants C1/St/5 and C1/St/6 showed activity of 12.8 and 11.8 U/ml, respectively, after protoplast fusion between Rst/9/6U and Rst/60/7E mutants of S. lavendulae R-St-1 (W.T). To determine molecular variability of two EMS mutants, and their recombinant fusants as well as S. lavendulae (W.T) (parental), three random primers were used. RAPD primer (P1) was employed. Fusant C1/St/5 shared the parental isolate with the bands 850 and 300 bp, whereas fusant C1/St/6 had five new unique bands (1470, 750, 650, 520, and 250 bp). The DNA loci of the obtained banding profiles using P1, P2, and P3 primers were 12, 17, and three loci after RAPD assay. A total of 14 unique loci were obtained using the primers P1 and P2.