【作者】 傅维琦；

【导师】 岑沛霖； 林建平；

【作者基本信息】 浙江大学 ， 生物化工， 2009， 博士

【摘要】 5-氨基乙酰丙酸（5-aminolevulinic acid,ALA）是生物合成叶绿素、血红素、卟啉和维生素B₁₂等四吡咯化合物的前体物质。ALA除作为一种环境相容性及选择性很高的新型光动力学农药外,还在临床上广泛用作光化疗剂和诊断剂。代谢工程技术在ALA的开发中显示出很大的前景,但此前获得的工程菌产量与诱变菌株相比还比较低,底物的转化率不高,并且工艺尚不成熟,离产业化尚有距离。本工作的目的是运用代谢工程原理和技术,构建高效率产ALA的重组大肠杆菌,并优化工程菌的发酵条件和过程控制策略,提高ALA的生产水平,降低ALA的生产成本,为该产品的工业化生产及其在农业和医学领域的大规模应用奠定坚实的基础。本文首先克隆了类球红细菌的hemA基因,构建了重组质粒pET28a（+）-Rs.hemA。然后通过Red同源重组技术敲除大肠杆菌MG1655的琥珀酸脱氢酶基因（sdhAB）,获得了MG1655（sdhAB^-）。大肠杆菌MG1655和MG1655（sdhAB^-）经过溶源化处理后,构建了工程菌MG1655（DE3）/pET28a（+）-Rs.hemA和MG1655（sdhAB^-）（DE3）/pET28a（+）-Rs.hemA用于ALA的生产。考虑到培养基中葡萄糖、甘氨酸和琥珀酸的浓度对于ALA的合成具有重要影响,通过响应面设计优化了培养基中这些成分的浓度并考察了其交互作用。工程菌经过摇瓶试验优化,发现sdhAB缺陷型菌株具有更好的ALA合成潜力。采用工程菌MG1655（sdhAB^-）（DE3）/pET28a-Rs.hemA在151发酵罐上进行批次发酵,ALA的积累量达到2.1 g/l,相对琥珀酸的摩尔转化率达40.1%。采用pKD46介导的Red同源重组技术,成功将嗜热栖热菌的ALA脱水酶基因敲入到大肠杆菌BW25113中替换了大肠杆菌自身的ALA脱水酶基因,获得了ALA脱水酶基因替换菌株。考虑到表达的ALA合成酶在大肠杆菌BW25113和MG1655等菌株中的活力比较低,提高ALA合成酶的活力成为进一步研究的首选目标。重组类球红细菌ALA合成酶基因在大肠杆菌BL21（DE3）和Rosetta（DE3）中进行了表达优化,其中Rosetta（DE3）为稀有密码子优化型菌株。对ALA合成酶表达条件和培养基条件进行了优化,结果表明,相比于宿主BL21（DE3）,Rosetta（DE3）表达的ALA合成酶活力更高,ALA产量也更高。在51发酵罐上进行了ALA合成的实验,以工程菌Rosetta（DE3）/pET28af+)-Rs.hemA为出发菌株,ALA积累量最高达3.8 g/l（29 mM）。对工程菌E.coli Rosetta（DE3）/pET28a（+）-Rs.hemA合成ALA进行了系统研究。在批次发酵中,确定了葡萄糖和甘氨酸的添加能够有效促进ALA的合成。对于前体流加策略进行了研究,确定了合适的前体配比,ALA的产量最高达4.1g/l。考虑到流加发酵过程的pH控制对于细胞生长和ALA合成有重要影响,设计了新型的两阶段pH反馈控制的流加策略,发酵前6h的pH值控制在5.9随后利用前体流加控制pH值在6.2,ALA积累量最高达6.6g/l（50 mM）。为了增强来源于放射形土壤杆菌（A.radiobacter zju-0121）的ALA合成酶基因的表达,根据此前的研究结果,选择大肠杆菌Rosetta（DE3）作为宿主构建了高效表达的重组菌。对重组菌细胞破碎液中的ALA合成酶进行了蛋白电泳分析和活力分析,结果发现,宿主Rosetta（DE3）表达的ALA合成酶活力比此前BL21（DE3）表达的酶活力提高了20%。采用优化的培养条件,在151发酵罐上进行了流加发酵研究,ALA的产量可达6.5g/l。采用亲和层析和凝胶过滤层析纯化获得了重组ALA合成酶。研究了ALA合成酶的基本特性,并通过比较研究筛选得到ALA脱水酶的抑制剂D-葡萄糖和D-木糖。将D-木糖用于151发酵罐进行ALA的合成,积累量最高达7.3g/l（56 mM）。更多还原

【Abstract】 5-Aminolevulinic acid（ALA） is generally known as an essential intermediate in the biosynthesis of tetrapyrroles such as heme,porphyrin,chlorophyll,and vitamin B₁₂.Recently,ALA has received wide attention as a selective and biodegradable herbicide or insecticide in agriculture.Also ALA as photosensitizer for photodynamic therapy（PDT） and diagnosis is of potential use for cancers,oral errucous hyperplasia and so on.ALA can be produced by fermentation with mutants of photosynthetic bacteria and recombinant organisms.Among them,metabolic engineering technology offers a new choice to increase the expression of ALA and to enhance the productivity of ALA,and will be a promising biotechnology for ALA mass production in the near future.But ALA production with engineered strains is still costly due to the relatively low ALA accumulation and yield.Also the fermentation technique should be improved further for ALA mass production and potential application.The aim of this work is to construct highly efficient recombinant Escherichia coli and develop new and effective approaches for fermentation process to enhance the ALA productivity,decrease production cost and become a promising biotechnology for ALA industrial production and application in agricultural and medical field via metabolic engineering and process design.Firstly,the ALA synthase（ALAS） gene（hemA） was cloned from the genome of Rhodobacter sphaeroides and recombinant plasmid pET28a（+）-Rs.hemA was constructed.Then,succinate dehydratase gene（sdhAB） in chromosome of E.coli MG1655 was knockout to obtain engineered strain MG1655（sdhAB^-） by using the Red recombinantion system with homologous PCR products.And E.coli MG1655 and MG1655（sdhAB^-） were lysogenized and recombinants E.coli MG1655（sdhAB^-） （DE3）/pEY28a（+）-Rs.hemA and MG1655（DE3）/pET28a（+）-Rs.hemA were prepared for ALA production..Since initial glucose,glycine and succinate were observed to be the key factors in the medium,the main interactions among them for ALA production were studied by response surface analysis.Based on the results from shake flask experiments,E.coli MG1655（sdhAB^-）（DE3）/pET28a（+）-Rs.hemA was a better ALA producer.ALA production was achieved to 2.1 g/l,and molar yield（ALA/succinate） was improved to 40.1%under the appropriate conditions in 15-1 fermenter.ALA dehydratase（ALAD） gene（hemB） of E.coli BW25113 was successfully replaced by Thermus thermophilus HB8’s via pKD46 mediated Red recombination technology.A metabolic engineering strain with low ALAD activity was obtained.But considering ALAS activity was still low in E.coli BW25113 and MG1655,it’s a preference to optimize ALAS.Then ALAS gene containing several codons rarely used by E.coli was optimized in two strains of E.coli:BL21（DE3） and Rosetta（DE3） which is a rare codon optimizer strain.The effects of initial concentration of isopropyl-β-D-thiogalactopyranoside（IPTG）,induction time,and temperature on enzyme activity were studied and compared for two strains.The results indicated that the ALAS expressed by Rosetta（DE3）/pET-28a（+）-Rs.hemA was higher than that by BL21（DE3）/pET-28a（+）-Rs.hemA.ALAS activity was generally higher with Rosetta（DE3） than with BL21（DE3）,so was ALA biosynthesis.Based on the optimal culture system using Rosetta（DE3）,the yield of ALA achieved 3.8 g/l（29 mM） under the appropriate conditions in 5-1 fermenter.ALA production with recombinant E.coli Rosetta（DE3）/pET28a（+）-Rs.hemA was studied in detail.In batch fermentation,the addition of glucose and glycine was effective to improve ALA production.Then the fed-batch fermentation was conducted with continuous feeding of precursors.When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l,respectively,in the feeding,the ALA yield reached 4.1 g/l.But the molar yield（ALA/glycine） was decreased in the fed-batch fermentation compared to batch fermentation.And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production.A two-stage pH value controlling strategy was suggested,in which,the pH value in the first 6 h was regulated at pH 5.9,after then at pH 6.2,and the ALA yield was as high as 6.6 g/l via fed-batch fermentation.To enhance the expression of ALAS gene from Agrobacterium radiobacter zju-0121,E.coli Rosetta（DE3） was picked out as the host to construct an efficient recombinant strain according to the above results.Cell extracts of the recombinant E. coli were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis （SDS-PAGE） under the appropriate conditions.The results indicated that the activity of ALAS expressed in Rosetta（DE3）/pET-28a（+）-Ar.hemA was about 20%higher than that in E.coli BL21（DE3）.Based on an optimal fed-batch culture system described in our previous work,up to 6.5 g/l（50 mM） ALA was produced in a 15-1 fermenter.Then ALAS from E.coli Rosetta（DE3）/pET-28a（+）-Ar.hemA was purified by affinity purification on Ni-NTA agarose and gel filtration chromatography on Sephadex G-25 Medium resin.The recombinant ALAS was characterized,and D-glucose and D-xylose were selected as effective inhibitor for ALAD from E.coli Rosetta（DE3）.Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture with Rosetta（DE3）/pET28a-Ar.hemA,the yield of ALA achieved was 7.3 g/l（56 mM） under the appropriate conditions in the fermenter.更多还原