【作者】 陈旭升；

【导师】 毛忠贵；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 ε-聚赖氨酸（ε-poly-L-lysine,ε-PL）是微生物通过非核糖体合成方式催化25³5个L-赖氨酸单体,以α-COOH和ε-NH₂相互缩合的方式而形成的一种同型L-赖氨酸链状聚合物,分子量一般为2500⁴500 Da。由于其具有抑菌性广、水溶性强、热稳定性好、pH值使用范围广以及安全性高等优点,目前主要作为食品防腐剂应用在食品防腐和保鲜领域,已经在日本形成数十亿日元市场。同时,作为一种阳离子型生物聚合物,ε-PL还广泛应用于生物材料和药物载体研究。因此,研究开发ε-PL发酵技术以提高ε-PL发酵水平,实现低成本、高效率ε-PL发酵生产,为我国建立ε-PL产业提供技术支撑。本论文利用一株ε-PL高产菌Streptomyces sp. M-Z18,提出了工业甘油、甘油和葡萄糖混合物、前体L-赖氨酸作为发酵底物的供给策略,通过发酵过程优化与调控技术,显著提高了ε-PL发酵水平;考察了甘油和葡萄糖作碳源引起ε-PL合成的差异;研究了Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL的机制;建立了转化前体L-赖氨酸耦合发酵生产ε-PL的工艺。具体研究内容如下:（1）在确定工业甘油作为发酵碳源的前提下,筛选出相匹配的牛肉浸膏、（NH4）2SO₄、KH₂PO₄、K₂HPO₄、MgSO₄·7H₂O和FeSO₄·7H₂O作为Streptomyces sp. M-Z18合成ε-PL的营养成分;借助Plackett-Burman设计确定了甘油、硫酸铵和K₂HPO₄是影响ε-PL合成的关键营养成分;利用响应面分析构建出最优营养组合为:甘油60 g/L,（NH4）2SO₄ 5 g/L,牛肉浸膏10 g/L,KH₂PO₄ 4 g/L,MgSO₄·7H₂O 0.8 g/L,FeSO₄·7H₂O 0.05 g/L。利用该优化培养基自然发酵生产ε-PL（pH值不控制）,摇瓶ε-PL产量达到2.27 g/L,菌体干重达到7.75 g/L;5 L发酵罐分批发酵ε-PL产量为3.5 g/L,是出发培养基（M3G培养基）的3倍。（2）在研究pH值对ε-PL发酵过程影响的基础上,以最大ε-PL比合成速率为调控目标,建立了一种利用甘油为碳源的两阶段pH值控制策略发酵生产ε-PL工艺。该工艺使得ε-PL分批发酵产量和产率达到9.13 g/L和4.76 g/L/d,较最优单一pH值控制发酵（pH3.5）分别提高16.6%和52.1%。结合甘油和硫酸铵流加技术,ε-PL发酵产量达到30.11 g/L,ε-PL产率为4.18 g/L/d,转化率达到13.2%。根据葡萄糖和甘油发酵生产ε-PL的互补性优点,提出了甘油-葡萄糖双碳源发酵生产ε-PL工艺。研究发现,Streptomyces sp. M-Z18不仅能够同步消耗甘油和葡萄糖用于菌体生长和ε-PL合成,并且显著缩短了发酵时间,提高了ε-PL合成速率。当葡萄糖和甘油混合比例为30/30（w/w）时,发酵速率比任何单一碳源都显著提高:较葡萄糖快25.4%,较甘油快32.8%。甘油-葡萄糖双碳源（30/30,w/w）补料-分批发酵使得ε-PL发酵产量达到35.14 g/L,ε-PL产率为4.85 g/L/d,转化率为12.1%。（3）在考察甘油和葡萄糖对Streptomyces sp. M-Z18合成ε-PL影响时,发现甘油和葡萄糖引起了ε-PL合成的显著差异。通过对两种碳源下ε-PL发酵过程关键酶活性变化考察,发现磷酸烯醇式丙酮酸羧化酶（PEPC）在以葡萄糖为碳源的发酵过程中表现出较高活性,而天门冬氨酸激酶（ASPK）和ε-PL合成酶（Pls）在以甘油为碳源的发酵过程中活性较高;两种碳源下ε-PL发酵过程代谢通量分析表明:以甘油为碳源减小了杂氨基酸合成和菌体合成通量,增大了磷酸戊糖途径、TCA循环回补途径、天门冬氨酸族氨基酸合成途径和L-赖氨酸合成等途径的代谢流量,而TCA循环代谢通量基本保持不变,这说明甘油作碳源能够使得更多的碳代谢流流向ε-PL的合成途径,减少了代谢副产物的生成,提高了产物转化率;考察不同还原度碳源（葡萄糖酸、葡萄糖和山梨醇）对菌体生长和ε-PL合成影响,发现碳源还原度越接近前体L-赖氨酸还原度越有利于ε-PL合成。在上述实验结论的基础上,提出了甘油优于葡萄糖合成ε-PL的可能机制在于:①甘油作为小分子多元醇通过水取代机理稳定了酶的空间构象而提高了ε-PL合成途径中天门冬氨酸激酶和ε-PL合成酶活性,从而增大了ε-PL合成途径代谢通量,提高了ε-PL产量;②甘油与L-赖氨酸还原度相同,实现了底物和产物前体的氧化还原平衡,减少了代谢副产物生成,提高了产物转化率;③甘油比葡萄糖为ε-PL合成提供了更多的能量辅因子ATP。（4）利用两阶段培养方法考察前体L-赖氨酸对ε-PL合成影响时,发现低浓度（1² g/L）L-赖氨酸能够显著促进ε-PL合成。利用同位素标记方法（L-（U-13C）赖氨酸）和核磁共振分析技术（NMR）研究L-赖氨酸转化机制时,发现L-赖氨酸是作为整体直接参与ε-PL合成,且细胞转化前体L-赖氨酸比例为40%左右,该比例不会随着前体L-赖氨酸浓度的增加而提高。在对甘油、pH值、L-赖氨酸和细胞膜通透性等因素的考察基础上,建立了Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL体系并强化了该转化过程。通过对转化过程的研究,发现转化体系合成ε-PL来源于两条途径:①转化外源L-赖氨酸合成ε-PL途径;②转化甘油形成内源L-赖氨酸合成ε-PL途径。（5）在考察pH值对5 L发酵罐规模Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL过程影响的基础上,建立了5 L发酵罐规模补料-分批转化体系,使得ε-PL合成能力达到15 g/L。前体L-赖氨酸添加方式对ε-PL发酵过程影响研究结果表明,在发酵后期添加1 g/L L-赖氨酸有利于实现发酵与前体L-赖氨酸转化的同步进行。结合甘油单一碳源和甘油-葡萄糖双碳源发酵生产ε-PL工艺,初步建立转化前体L-赖氨酸耦合发酵生产ε-PL工艺。两种ε-PL生产工艺分别实现ε-PL发酵产量达到33.76 g/L和37.6 g/L。更多还原

【Abstract】 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:①glycerol 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;②glycerol 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;③glycerol 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:①conversion of exogenous L-lysine;②conversion 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.更多还原