Enhancement Of ε-poly-L-lysine Production Through Carbon Source Supply Strategy Optimization And Fermentation Process Regulation By Streptomyces Sp. M-Z18

ε-poly-L-lysine （ε-PL）, consists of 25³5 L-lysine residues with linkages betweenα-carboxyl groups andε-amino groups, is a homopolymer produced by microbial nonribosomal peptide synthetases （NRPSs）.ε-PL shows strong antimicrobial activity against a wide spectrum of microorganisms （including bacteria and fungi）, water soluble, thermalstability, wide used range of pH and safety. It is mainly used as a food preservative in several countries, especially in Japan. At present,ε-PL has been formed billions of yen in Japan market. Therefore, it is important to develop an efficientε-PL fermentation strategy for its industrial manufacture in China.In this dissertation, a highε-PL producing strain, Streptomyces sp. M-Z18 was used as a model to demonstrate the effect of glycerol and mixed with glucose as carbon sources onε-PL production by process optimization and regulation. Meanwhile, the differences between glycerol and glucose onε-PL production were investigated. Based on the well understanding of mechanisms in theε-PL formation from precursor L-lysine, whole-cell biotransformation method forε-PL production directly from L-lysine was established. The main results were described as follows:（1） In order to improveε-PL production of Streptomyces sp. M-Z18, the effects of nutritional conditions （carbon sources, nitrogen sources, phosphate salts and metal ions） onε-PL production and cell growth were investigated in shaking flask. The results of one-time-one-factor showed that glycerol, beef extract, （NH₄）2SO₄, KH₂PO₄, K2HPO₄, MgSO₄·7H₂O and FeSO₄·7H₂O were the optimal nutritions forε-PL production and cell growth; Plackett-Burman design was determined glycerol, （NH₄）2SO₄ and K2HPO₄ were the key nutritions forε-PL production. The optimized conditions were determined by using response surface methodology as follows: 60 g/L glycerol,5 g/L （NH₄）2SO₄,10 g/L beef extract,4 g/L KH₂PO₄,0.8 g/L MgSO₄·7H₂O,0.05 g/L FeSO₄·7H₂O. Under the optimized fermentation conditions,ε-PL production and DCW were achieved at 2.27 g/L and 7.75 g/L, respectively, in shake-flask fermentation. Furthermore, the batch fermentation results showed that the production ofε-PL was yielded 3.5 g/L after 96 h in 5 L fermenter under pH uncontrolled strategy, it was enhanced 3-fold than M3G medium.（2） Based on the effect of pH onε-PL production, this dissertation developed a novel two-stage pH control strategy under the direction by the highest specificε-PL formation rate. By applying this strategy, the maximalε-PL concentration and productivity had reached at 9.13 g/L and 4.76 g/L/day, respectively, it is higher by 16.6% and 52.1% than the optimal one-stage pH control process （pH3.5）. Combined with glycerol and （NH₄）2SO₄ feeding strategy, fed-batch fermentation was performed. After 173 h fermentation, theε-PL concentration, productivity and yield reached at 30.11 g/L, 4.18 g/L/day and 13.2%, respectively. Furthermore, due to the complementary advantages of glucose and glycerol forε-PL fermentation, the effect of glucose-glycerol mixed carbon sources onε-PL fermentation were investigated. The results of experiment showed that glycerol and glucose simultaneously consumed by Streptomyces sp. M-Z18 for cell growth andε-PL synthesis. In addition, glycerol-glucose fermentation could significantly reduce the fermentation time and improve theε-PL productivity much. When the ratio of glycerol to glucose at 30/30 （w/w）, the batch fermentation time was shorten than single carbon source fermentation by 25.4% （glucose） and 32.8% （glycerol）. Finally, fed-batch fermentation with glucose and glycerol as a mixed carbon source （30/30,w/w） achieved maximumε-PL concentration, productivity and yield of 35.14 g/L, 4.85 g/L/d and 12.1%, respectively.（3） When glycerol and glucose were used as carbon sources forε-PL production, it is found that glycerol and glucose make significant differences onε-PL synthesis. To explain these differences detailed, key enzymes activities, metabolic flux analysis （MFA） and reduction degree of carbon sources were investigated. Results from the key enzymes evaluation showed that phosphoenolpyruvate carboxylase activity in the glucose medium was higher than glycerol, however, the activities of aspartate kinase andε-PL synthase in glycerol was superior than glucose. MFA showed that glycerol as carbon source was reduced the flux of amino acids synthesis （except L-lysine） and cell growth, increased the flux of pentose phosphate pathway, TCA cycle anaplerotic reaction, aspartic acid family amino acid biosynthesis and L-lysine pathway. However, the flux of TCA cycle remained unchanged compared with glucose as carbon source. It was indicated that glycerol as carbon source improved the flux of target metabolic and reduced the by-product. The effect of different reduction degrees of carbon sources （gluconic acid, glucose and sorbitol） on cell growth andε-PL synthesis showed that reduction degree played an important role inε-PL production. Based on the above experimental results, the possible mechanisms on glycerol superior than glucose as carbon source forε-PL production were proposed as follows:â‘ glycerol as the polyol molecules replaced water for supporting the spatial structure of the enzymes and thereby improved the aspartate kinase andε-PL synthase activities. Finally, it had increased the flux ofε-PL synthesis pathway and enhancement ofε-PL production;â‘¡glycerol and L-lysine have the same degree of reduction, so it could reduce the metabolic by-products generated to keep redox balance and improve the yield ofε-PL;â‘¢glycerol have more reduction degree than glucose, so it could provide more ATP forε-PL synthesis.（4） To investigate the effects of precursor L-lysine addition concentration onε-PL synthesis, two-stage culture method was performed and found that low concentrations of L-lysine could significantly promote the production ofε-PL. To reveal the relationship between L-lysine and enhancement ofε-PL production, isotope labeling method （L-（U-13C） lysine） and nuclear magnetic resonance （NMR） were used and found that 40% L-lysine as a whole directly involved in theε-PL synthesis. Moreover, this ratio is not improved when external L-lysine concentration is increased. When the effects of L-lysine, glycerol, pH and cell membrane permeability on Streptomyces sp. M-Z18 whole-cell biotransformation process, the system of whole-cell conversion of L-lysine toε-PL were established. Based on the above experimental results,ε-PL synthesis in the system derived from two ways:â‘ conversion of exogenous L-lysine;â‘¡conversion of glycerol to endogenous L-lysine.（5） In order to develop of fed-batch whole-cell biotransformation system in 5 L fermentor, the effect of pH on the process of whole-cell biotransformation was investigated and the highestε-PL production by 15 g/L was achieved at the optimal culture conditions. We have investigated the effect of the ways of added L-lysine onε-PL production and found that addition 1 g/L L-lysine at the late of fermentation was benefit for coupled fermentation with biotransformation. Based on the two-stage pH control strategy and glycerol-glucose mixed carbon source fermentation strategy, we developed two types of coupled fermentation with biotransformation strategies forε-PL production and achievedε-PL production of 33.76 g/L and 37.6 g/L, respectively.