摘要
ε-聚赖氨酸（ε-poly-Lysine，ε-PL）是少数链霉菌将L-赖氨酸单体通过α-COOH与ε-NH_2脱水缩合而成的一种氨基酸同聚物，聚合度为25-35。由于其抑菌谱广、效价高、安全无毒，再配合其水溶性好、作用pH广、热稳定强等特点，ε-PL已被广泛用作食品防腐剂。此外，ε-PL还被用于食疗剂、药物载体、基因芯片、电子材料等领域。因此，其应用价值及市场前景是非常广阔的。
本论文首先从土壤中筛选获得了五种野生型的ε-PL产生菌；其次利用传统诱变方法及新兴的Genome Shuffling技术对这些野生菌株进行育种改造，以提升其ε-PL发酵水平；然后对获得的高产菌株进行了种间随机的原生质体融合，将其优良性状集中于同一个细胞内，进一步提升了ε-PL的产量；最后对种间融合获得的高产杂合子产ε-PL进行了强化，包括Genome Shuffling、培养基优化及发酵工艺优化，最终使其ε-PL产量达到了国内领先水平。具体研究内容如下：
（1）从土壤中筛选放线菌时，为了有效抑制杂菌（细菌、霉菌）的生长，在分离培养基中添加了复合抑制剂：重铬酸钾30mg/L、青霉素2mg/L、氟哌酸3mg/L、制霉菌素80mg/L；对ε-PL产生菌的筛选方法做了改进，利用“双层琼脂法”代替“直接添加美蓝”法，将“菌落生长”与“排斥美蓝显色”分成了两个阶段，能够有效避免美蓝对微生物的毒害，提高了筛选效率；利用该法从土壤中筛选获得了ε-PL产生菌42株，其中至少有白色链霉菌S. albulus、禾粟链霉菌S. graminearus、吸水链霉菌S. hygroscopicus、灰褐链霉菌S. griseofuscus、稠李链霉菌S. padanus等五种菌株，其中后四种菌株在ε-PL产生菌中未见报道。
（2）采用紫外线（UV）与亚硝基胍（NTG）对五种ε-PL产生菌进行诱变。UV诱变剂量为：功率8W，距离30cm照射4min。NTG诱变剂量为0.5mg/mL处理70min；选择了五种物质作为“筛子”：葡萄糖、ε-PL、KH_2PO_4、磺胺胍、琥珀酸为唯一碳源，确定了其（除琥珀酸）对野生菌株的临界浓度分别为：190g/L、0.06g/L、30g/L、20g/L，并复合以0.05g/L的LiCl；开展大批量的诱变及筛选工作，摇瓶发酵筛选得到了其中四种菌株（S. albulus、S. graminearus、S. griseofuscus、S. padanus）三种“筛子”（葡萄糖、磺胺胍、琥珀酸为唯一碳源）的ε-PL产量提高株，由0.38-0.49g/L提高到0.50-0.72g/L，并将其作为后续Genome Shuffling的出发菌株。
（3）在获得“丝状”菌体的前提下，优化了原生质体制备、再生及融合的条件为：0.5%的溶菌酶、酶解温度30℃、酶解时间120min、促融剂PEG6000浓度30%（W/V）、融合过程为37℃保温10min；采取双亲灭活法进行原生质体融合，灭活条件为：紫外线（8W，30cm）照射60min、70℃水浴40min；对四种菌株（S. albulus、S. graminearus、S. griseofuscus、S. padanus）三种“筛子”（葡萄糖、磺胺胍、琥珀酸为唯一碳源）的突变株进行了三轮Genome Shuffling育种，获得了不同菌株不同“筛子”的ε-PL产量提高株，由0.38-0.49g/L提高至0.75-0.82g/L；选取其中的两株融合子GRF3-4、PAF3-2及其野生菌株进行补料分批发酵，融合子在控制pH的扩大培养中ε-PL产量由7-8g/L（野生型）提高到13-15g/L；融合子与野生菌相比，代谢途径中一些关键酶（HK、PK、PEPC、AK、CS）的活性提高，这是导致ε-PL合成能力增强的初步原因；经过诱变及GenomeShuffling育种，菌株的16S rDNA序列会发生极少碱基的变化，但不会引起菌种分类地位的改变。
（4）对Genome Shuffling育种获得的高产融合子S. albulus（葡萄糖、琥珀酸为唯一碳源）、S. graminearus（葡萄糖、磺胺胍）、S. griseofuscus（葡萄糖、琥珀酸为唯一碳源）、S. padanus（磺胺胍、琥珀酸为唯一碳源）、以及S. hygroscopicus的野生型，共包含39株菌在内的五种菌株进行两两的种间原生质体融合育种，在杂合子中没有筛选到ε-PL产量有大幅提高的菌株；将以上所有菌株混合后进行五种菌株的种间随机融合，获得了一株高产杂合子Streptomyces sp. FEEL-1，摇瓶产量为1.1g/L，比亲株提高了37%以上；随机引物扩增多态DNA（RAPD）结果初步表明菌株FEEL-1的亲本为S. albulus、S.griseofuscus与S. padanus；杂合子Streptomyces sp. FEEL-1在控制pH的补料分批发酵中ε-PL产量达到24.5g/L，比其亲本提高了40-70%；胞内酶活（HK、PK、PEPC、AK、CS）的提高是ε-PL产量提高的初步原因，也证明了种间随机融合的有效性。
（5）以S-2-氨乙基-L-半胱氨酸（AEC）作为抗性指标，对杂合子Streptomyces sp. FEEL-1进行了EMS诱变及三轮Genome Shuffling育种，获得了一株Streptomyces sp. FEEL-G67，其天冬氨酸激酶AK的酶活提高了1.07倍，ε-PL摇瓶产量提高至1.73g/L，补料分批发酵产量达到29.82g/L；利用PB-CCD的响应面方法对其发酵培养基（M3G）作了优化，发现对Streptomyces sp. FEEL-G67摇瓶发酵影响显著的三个因素为：酵母粉、K2HPO_4、MgSO_4，且最终优化出的培养基为（g/L）：葡萄糖50，酵母粉7.5，(NH4)2SO_45，K2HPO_4·3H_2O2.5，MgSO_4·7H_2O2，ZnSO_4·7H_2O0.04，FeSO_4·7H_2O0.03。在该条件下，ε-PL摇瓶产量从1.73g/L提高到2.32g/L，补料分批发酵产量达到32.25g/L；基于对pH的发酵工艺优化，证实了高pH有利于菌体生长，而低pH有利于ε-PL的合成。以最大ε-PL比合成速率为目标，提出了两阶段pH控制策略（pH3.8-4.0），在该条件下进行补料分批发酵可使ε-PL达到36.79g/L；在此基础上流加有机氮源（酵母粉），不仅使生物量达到49.7g/L，也进一步使ε-PL产量提升至41.24g/L，这是目前国内ε-PL发酵的最高水平。
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, KH_2PO_4, 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, K2HPO_4and MgSO_4were key nutritions for ε-PL production. The optimized medium was achieved byresponse surface methodology as follows (g/L): glucose50, yeast extract7.5,(NH4)2SO_45,K2HPO_4·3H_2O2.5, MgSO_4·7H_2O2, ZnSO_4·7H_2O0.04, FeSO_4·7H_2O0.03. 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.
引文
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