Study On Breeding And Fermentation Of ?-poly-L-lysine Producing Strains And Preliminary Physiological Analysis Of The Improved Production

?-poly-L-lysine??-PL?is a natural high molecular polymer composed of 25-35 L-lysine monomers by dehydration condensation of?-COOH and?-NH₂.Because of its solubility in water,heat stability,biodegradability,broad spectrum of inhibition,high safety,and good clinical effect,?-PL has been widely used as a food preservative,drug carrier,therapeutic agent,emulsifier and hydrogels in many countries such as Japan,Korea,the United States,and China.In this paper,Streptomyces sp.G67 was used as the starting strain.Firstly,several rounds of compound mutagenesis,genome shuffling combining with ribosome engineering were applied to Streptomyces sp.G67,and single,double and triple-resistance mutants were screened successively,dramatically improving the shake flask production.High-production mechanism was then analyzed from physiological levels.Secondly,a pH shock combined with pH-assised dissolved oxygen process?PS-PAD?strategy was constructed,with the potential for industrial application was established.Feed-batch fermentation was then carried out in 5 L bioreactors under this strategy and the physiological changes of the strains during fermentation process was studied.Finnaly,the?-PL biosynthetic pathway of the two strains were constructed and compared.The specific research contents are as follows:?1?Streptomyces sp.G67 was bred by ultraviolet?UV?mutagenesis,Atmospheric and room temperature plasma?ARTP?mutagenesis and ethyl methanesulfonate?EMS?mutagenesis.The results showed that the mutagenic effect of ARTP and EMS mutagenesis were significantly better than that of UV mutagenesis.Therefore,EMS and ARTP were selected for the compound mutagenesis of G67.The acquisition of streptomycin resistance?Str^r?in the six antibiotics increased most significantly in?-PL production,so Str was chosen as the resistance marker for compound mutagenesis.After 3 rounds of“compound mutagenesis+Str^r”screening,a single-resistance high-production mutant GS-1 was obtained with shake flask production increased from 1.9 g·L^-11 to 2.81 g·L^-1,which was an increase of 47.9%.Amplified fragment length polymorphism?AFLP?analysis indicated that“compound mutagenesis+Str^r”screening rapidly increased the gene polymorphism of high-producting mutants,which lead to the increase of?-PL production.?2?Multiple-resistance mutants breeding was screened out by the method of genome shuffling combining with ribosome engineering.firstly,the novel ARTP mutagenesis was used to construct“Gen^r mutant library”.After three successive rounds of genome shuffling,a Str^rGen^r double resistance mutant GSG-1 was obtained with shake flask production of 3.56g·L^-1,which was 87.3%higher than G67.AFLP analysis indicated that“Genome shuffling+Str^r”can increase the gene polymorphism of high production mutants better than mutagenisis.Secondly,the rifmycin resistance?Rif^r?of double resistance mutant was improved by three successive rounds of ribosome engineering,and a Str^rGen^r Rif^r triple-resistance mutant GSGR-1 was constructed with shake flask production of 4.23 g·L^-1,which was 2.23 times of the original strain G67.Sequencing results of the resistance genes showed that the 856th amino acid?Q856H?of the RNA polymerase?subunit was replaced in GSGR-1,leading to the increase of?-PL production.?3?Firstly,the differences between the mutants and starting strain G67 in cell morphology,culture characteristics,the fermentation levels of?-PL in shake flask and 5 L bioreactor,antibiotic resistance and mutation site were compared.Then,by analysis of the physiological changes of the mutants during synthesis of?-PL,the high?-PL production mechanism was preliminary explained from physiological level.?4?Genome sequencing and functional annotation of starting strain S.albulus M-Z18 and double resistance high-producing strain Streptomyces sp.GSG-1 were carried out.The?-PL metabolic map was constructed by KEGG analysis.Subsequently,the differences of the metabolic pathways of?-PL between M-Z18 and GSG-1 were compared,and the high production mechanism of GSG-1 was speculated:The?-PL biosynthesis pathway of GSG-1 is similar to that of M-Z18.Due to the gene absences of acetyl-CoA synthetase?EC 6.2.1.13?,glucose dehydrogenase?EC 1.1.1.47?,L-Lys 6-transaminase?EC 2.6.1.36?and L-Lys 2,3-aminomutase?EC 5.4.3.2?,the TCA circulation flux of GSG-1 increased,the synthesis of by-products?such as?-D-glucose,alcohol?decreased,and the decomposition pathway of L-Lys weakens.Finally,the L-Lys and ATP concentration in GSG-1 was improved,leading to the enhancement of?-PL production.These results were verified by batch fermentation experiments on 5 L fermentor.?5?PS-PAD strategy?pH shock-pH asisted DO control strategy?was established to solve the problem that respiratory activity and?-PL yield of the mutant strain decreased rapidly in the late stage of fed-batch fermentation,and the fed-batch fermentation level of the mutant strain was greatly improved.The specific steps were:1,pre-culture stage:pH was set at 5.5and maintained for 10 h;2,pH shock phase:pH naturally decreased from 4.0 to 3.3,and maintained for 5 h;3,recovery phase:pH was set to 3.8 and maintained until the end of fermentation;4,Slightly adjusting pH?3.8-4.2?when during feeding period to control the dissolved oxygen between 15%-30%until the end of fermentation.By using this strategy,GSGR-1 has achieved the?-PL production of 82.3 g·L^-1,and this is the highest reported?-PL production as far as we know.To explore the effects of PS-PAD strategy on the synthesis and metabolism of?-PL,the physiological changes of GSGR-1 under PS-PAD strategy and constant pH strategy were compared.It was found that under PS-PAD strategy,cell respiratory activity,respiratory chain activity,and the key enzyme activities of the?-PL metabolic pathway and central metabolic pathway were all enhanced and maintained until the end of fermentation.This proved that the PS-PAD strategy can maintain the increase of the mycelia vitality?-PL production caused by acid pH shock for a long time.These results preliminarily revealed the reason for the enhancement of?-PL production.