Screening, Breeding And Fermentation Researches On ε-poly-L-lysine Producing Strains

ε-poly-L-lysine （ε-PL） is a homo-poly-amino acid where L-lysine monomers are linkedthrough microorganism by peptide bonds between the carboxyl and the epsilon-amino groups.Its polymerization degree is generally ranging from25to35. Because it is water soluble,biodegradable, edible and non-toxic toward humans and environment, ε-PL and its derivativeshave been of interest for a broad range of industrial applications such as food preservatives,emulsifying agent, dietaryagent, biodegradable fibers, highly water absorbable hydrogels,drug carriers, etc. Therefore, the application value commercial production of ε-PL areconsidered highly promising.The work in this paper was done following several basic aspects. First, five species ofε-PL-producing strains were screened from soils with an improved detection method; Thenthe traditional mutagenic method and newly emerging Genome Shuffling approach wereadopted for rapidly breeding these organisms to improve the ε-PL productivity; Subsequently,we made a combination of these shuffled strains through stochastic protoplast fusion andobtained a hybrid with significant improvement for ε-PL production; In the end, ε-PLproductivity in the hybrid was further enhanced by Genome Shuffling, response surfacemethodology and fermentation technique optimization, which achieved the advancedfermentation level in China. The specific studies were as follows:（1） In order to isolate Actinomyces from soils, composite inhibitors consisted of K2Cr2O7（30mg/L）、norfloxacin （3mg/L）、penicillin （2mg/L） and nystatin （80mg/L） were added tomedium to inhibit the growth of bacteria and fungi; The detection method for screeningε-PL-producing strains was improved. The whole agar was removed with grown Actinomycesand was then covered on another agar containing the methylene blue to form a sandwich,which avoided the toxicity problem and this modified method proves effective;42ε-PL-producing strains were obtained from soils with this improved strategy at least includingS. albulus, S. griseofuscus, S. graminearus, S. hygroscopicus and S. padanus. The latter4strains among them were not reported before.（2） Five wild-type ε-PL-producing strains were mutagenized by UV and NTG to obtain theinitial mutant library. UV irradiation was performed by exposing strains to an UV light（power of8W） at a distance of30cm for4min. NTG mutagen concentration was0.5mg/mLwith a treated time of70min; Five substances were choosen as sieves to select mutants:glucose （Glc）, ε-PL, KH₂PO₄, sulfaguanidine （SG） and succinic acid （SA） as sole carbonsource. The threshold concentrations of these substances （except for SA） for wild-type strainswere190g/L、0.06g/L、30g/L、20g/L respectively, and supplemented with LiCl of0.05g/L;After the scale-up breeding and selection, mutants with higher ε-PL production in shake-flaskfermentation test were obtained for four strains （S. albulus, S. graminearus, S. griseofuscus, S.padanus） of three substances （Glc, SG, SA）, from0.38-0.49g/L to0.50-0.72g/L. Thesemutants were then adopted for Genome Shuffling. （3） On premise of obtaining forms of mycelia, conditions of protoplast preparation、regeneration and fusion were optimized as follows:0.5%of lysozyme,30℃of temperature,120min of treated time,30%（W/V）of PEG6000, fusion for10min under37℃; Protoplastswere deactivated by UV irradiation （8W,30cm,60min） and heating （70℃,40min）; Afterthe scale-up Genome Shuffling for four strains （S. albulus, S. graminearus, S. griseofuscus, S.padanus） of three substances （Glc, SG, SA）, the ε-PL production of shuffled strains wasimproved from0.38-0.49g/L to0.75-0.82g/L; Two of the shuffled strains GRF3-4andPAF3-2showed higher ε-PL production in fed-batch fermentation, from7-8g/L to13-15g/L;Compared to wild-type strains, the improvement of ε-PL production in shuffled strains wasdue to the higher enzyme activities （HK, PK, PEPC, AK, CS） in metabolic pathway; Aftermutation and Genome Shuffling,16S rDNA sequence of the strains changed few bases but itcould not change the taxonomic status.（4） Protoplast fusion was carried out between two species of all possible hybridizationsamong shuffled strains: S. albulus （Glc, SA）, S. graminearus （Glc, SG）, S. griseofuscus（GLU,SA）, S. padanus （SG, SA）, S. hygroscopicus （wild-type）, involving10kinds of pairingcombinations but no significant improvements for ε-PL production were observed in thesetwo-spesies hybridizations; However, a hybrid designated Streptomyces sp. FEEL-1wasobtained with ε-PL production of1.1g/L （37%higher than the parents） in shake-flask whenmixed all above strains together and carried out an hybridization among five species; RAPDrevealed that FEEL-1was probably hybridized from S. padanus, S. griseofuscus and S.albulus; The ε-PL production of FEEL-1was obtained as24.5g/L in fed-batch fermentation,which was40-70%higher than those in its parents; Activities of several enzymes in FEEL-1（HK, PK, PEPC, AK, CS） were more active than those in shuffled strains, which was apossible reason for the enhancement of ε-PL production.（5） Genome Shuffling was employed again to further improve ε-PL production ofStreptomyces sp. FEEL-1by AEC as resistance index. A mutant named Streptomyces sp.FEEL-G67was obtained with ε-PL production of1.73g/L in shake-flask and29.82g/L infed-batch fermentation because the key enzyme activity of AK was improved by1.07-foldthan FEEL-1; Plackett-Burman design was adopted to determine that yeast extract, K2HPO₄and MgSO₄were key nutritions for ε-PL production. The optimized medium was achieved byresponse surface methodology as follows （g/L）: glucose50, yeast extract7.5,（NH4）2SO₄5,K2HPO₄·3H₂O2.5, MgSO₄·7H₂O2, ZnSO₄·7H₂O0.04, FeSO₄·7H₂O0.03. Under thiscondition, ε-PL production was improved from1.73g/L to2.32g/L in shake-flask and32.25g/L in fed-batch fermentation; Base on the effect of pH on ε-PL production, we verified thatthe higher pH was beneficial to growth of biomass and lower pH was favorable to synthesis ofε-PL. A two-stage pH control strategy （pH3.8-4.0） was suggested by the highest specificε-PL production rate and ε-PL production was obtained as36.79g/L in fed-batch fermentationunder this process; On this basis, a fed-batch fermentation was carried out by adding yeast extract and the resulting biomass was as high as49.7g/L, meanwhile the ε-PL production wasachieved as41.24g/L, which was the highest yield reported in China.