發(fā)布時(shí)間：2018-08-12 13:58

【摘要】：檸檬酸作為生產(chǎn)量最大的有機(jī)酸,廣泛應(yīng)用于食品、醫(yī)藥、洗滌劑和化妝品等領(lǐng)域。目前檸檬酸主要通過(guò)黑曲霉進(jìn)行深層有氧發(fā)酵來(lái)生產(chǎn),產(chǎn)量和轉(zhuǎn)化率均已達(dá)到較高水平,但根據(jù)Alvarez-Vasquez的模型,仍然有提高空間,要進(jìn)一步加強(qiáng)檸檬酸的生產(chǎn)需要從基因組和轉(zhuǎn)錄組水平探索黑曲霉高產(chǎn)檸檬酸機(jī)制,以此來(lái)指導(dǎo)代謝調(diào)控。此外,檸檬酸生產(chǎn)菌株經(jīng)過(guò)多輪誘變形成短粗菌絲且細(xì)胞壁增厚,遺傳轉(zhuǎn)化困難且缺少有力的代謝調(diào)控工具,需要研究適用于檸檬酸生產(chǎn)菌株的轉(zhuǎn)化方法和代謝調(diào)控元件。本論文對(duì)黑曲霉檸檬酸工業(yè)生產(chǎn)菌株H915-1建立了遺傳轉(zhuǎn)化方法,并以H915-1及其誘變株為研究對(duì)象,通過(guò)比較基因組學(xué)和轉(zhuǎn)錄組學(xué),探討了黑曲霉高產(chǎn)檸檬酸的機(jī)制,進(jìn)而發(fā)現(xiàn)了低pH誘導(dǎo)的啟動(dòng)子Pgas可以作為動(dòng)態(tài)調(diào)控的基因元件,最后通過(guò)調(diào)整葡萄糖轉(zhuǎn)運(yùn)蛋白的表達(dá)提高了檸檬酸的產(chǎn)量。主要研究結(jié)果如下:(1)對(duì)黑曲霉H915-1的原生質(zhì)體形成條件進(jìn)行優(yōu)化并建立了遺傳轉(zhuǎn)化系統(tǒng)。最優(yōu)酶解液配比為5 mg×m L~(-1)溶壁酶、0.2 U×m L~(-1)幾丁質(zhì)酶和460 U×m L~(-1)葡萄糖醛酸酶;優(yōu)化后的原生質(zhì)體制備條件:滲透壓穩(wěn)定劑為0.7 M KCl,菌體量15 mg,酶解溫度37°C,菌球直徑50μm。采用PEG介導(dǎo)法,利用共轉(zhuǎn)化的方式,可以使2個(gè)表達(dá)框整合到黑曲霉基因組中,共整合概率為58%。在未敲除非同源末端連接(non-homologous end joining,NHEJ)基因Ku-70的情況下,利用2.3 kb同源臂對(duì)oah進(jìn)行敲除,同源整合的概率為65%,基因敲除菌株在整個(gè)發(fā)酵過(guò)程中不再合成草酸。(2)以黑曲霉H915-1為出發(fā)菌株,利用等離子誘變和高通量篩選獲得2株低產(chǎn)菌株A1和L2,它們的檸檬酸產(chǎn)量分別由出發(fā)菌株的157 g×L~(-1)降為117 g×L~(-1)和76 g×L~(-1)。對(duì)生產(chǎn)菌株和誘變株A1和L2進(jìn)行基因組測(cè)序、拼接和注釋,它們的基因組大小分別為35.98 Mb、34.64 Mb和36.45 Mb,共發(fā)現(xiàn)59個(gè)基因家族存在差異,單核苷酸多態(tài)性(Single nucleotide polymorphism,SNP)和插入缺失(insertion-deletion,INDEL)位點(diǎn)1210處,結(jié)構(gòu)性變異(Structural variation,SV)52處,共涉及35個(gè)基因的表達(dá)。中心代謝通路的順烏頭酸酶和γ-氨基丁酸(γ-aminobutyric acid,GABA)通路的琥珀酸半醛脫氫酶基因發(fā)生變異。(3)對(duì)黑曲霉H915-1在檸檬酸合成階段的4個(gè)時(shí)間點(diǎn)和菌體生長(zhǎng)階段的轉(zhuǎn)錄組數(shù)據(jù)進(jìn)行分析,發(fā)現(xiàn)479個(gè)基因的表達(dá)發(fā)生變化。確定了黑曲霉中心代謝通路的主效基因。糖酵解通路的大部分酶的表達(dá)沒(méi)有變化,磷酸丙糖異構(gòu)酶表達(dá)上調(diào),丙酮酸激酶表達(dá)下調(diào),TCA循環(huán)大部分酶的表達(dá)下調(diào);發(fā)現(xiàn)GABA通路關(guān)鍵酶的表達(dá)上調(diào);ATP-檸檬酸裂解酶表達(dá)上調(diào),與TCA循環(huán)一起構(gòu)成了一條消耗ATP的無(wú)效循環(huán);鑒定到35個(gè)轉(zhuǎn)運(yùn)蛋白表達(dá)持續(xù)上調(diào),包含3個(gè)有機(jī)陰離子轉(zhuǎn)運(yùn)蛋白,以及1個(gè)單羧酸轉(zhuǎn)運(yùn)蛋白。(4)通過(guò)轉(zhuǎn)錄組分析,篩選到低pH誘導(dǎo)表達(dá)的基因gas并進(jìn)行啟動(dòng)子預(yù)測(cè),利用報(bào)告基因熒光蛋白(s GFP)進(jìn)行啟動(dòng)子表達(dá)強(qiáng)度的驗(yàn)證,Pgas在pH 2.0時(shí)被誘導(dǎo)而強(qiáng)烈表達(dá)s GFP,表達(dá)強(qiáng)度和Pgpd A在pH 2.0時(shí)啟動(dòng)表達(dá)的能力一致。利用Pgas啟動(dòng)順烏頭酸脫羧酶(s CAD)基因的表達(dá)賦予黑曲霉H915-1合成衣康酸的能力,發(fā)酵24 h和108 h的s CAD的表達(dá)量比8 h的表達(dá)量分別增加了2.37和3.23倍,轉(zhuǎn)化子的衣康酸產(chǎn)量達(dá)到4.92 g×L~(-1),為Pgpd A-CAD轉(zhuǎn)化子產(chǎn)量的5倍。利用q PCR對(duì)Pgas的誘導(dǎo)能力進(jìn)行驗(yàn)證,發(fā)現(xiàn)Pgas僅受pH的誘導(dǎo),受酸種類的影響很小,酸根離子濃度對(duì)Pgas沒(méi)有影響,且pH與Pgas的啟動(dòng)能力存在線性關(guān)系。通過(guò)DNA pull-down技術(shù)鑒定到2個(gè)與Pgas特異結(jié)合的轉(zhuǎn)錄調(diào)節(jié)因子X(jué)P_001388781.2和XP_001396281。(5)基于轉(zhuǎn)錄組分析,對(duì)假定的葡萄糖轉(zhuǎn)運(yùn)蛋白進(jìn)行進(jìn)化樹(shù)分析和序列比對(duì)分析,獲得與Kl HGT1親緣關(guān)系較近的evm.model.unitig_0.1770序列,經(jīng)跨膜預(yù)測(cè)該蛋白含有11個(gè)跨膜區(qū)域,N端在細(xì)胞膜內(nèi),C端在胞內(nèi),命名為An HGT1。在限制性葡萄糖培養(yǎng)基上進(jìn)行生長(zhǎng)實(shí)驗(yàn),HGT轉(zhuǎn)化子的菌落直徑比對(duì)照增加50%~150%。在發(fā)酵后期補(bǔ)加30 g×L~(-1)葡萄糖后HGT1轉(zhuǎn)化子完全消耗葡萄糖的時(shí)間比H915-1減少12 h。HGT1轉(zhuǎn)化子的檸檬酸產(chǎn)量比對(duì)照增加了14.7%,發(fā)酵時(shí)間縮短了6 h,最大比產(chǎn)酸速率提升了29.5%,提高了發(fā)酵生產(chǎn)強(qiáng)度。
[Abstract]:Citric acid, as the most productive organic acid, is widely used in food, medicine, detergent, cosmetics and other fields. At present, citric acid is mainly produced by Aspergillus Niger deep aerobic fermentation. The yield and conversion rate have reached a higher level. However, according to the Alvarez-Vasquez model, there is still room for improvement, and citric acid should be further strengthened. The production of Aspergillus Niger requires exploring the mechanism of high citric acid production at the genome and transcriptome levels to guide metabolic regulation. In addition, citric acid producing strains undergo multiple rounds of mutagenesis to form short thick mycelia with thickened cell walls, difficult genetic transformation and lack of powerful metabolic control tools. Therefore, it is necessary to study the transformation of citric acid producing strains. METHODS AND METABOLISM REGULATING ELEMENTS.A genetic transformation method was established for Aspergillus Niger citric acid producing strain H915-1. Taking H915-1 and its mutants as research objects, the mechanism of citric acid production by Aspergillus niger was explored by comparative genomics and transcriptome, and the promoter Pgas induced by low pH was found to be a dynamic regulator. The main results are as follows: (1) The protoplast formation conditions of Aspergillus Niger H915-1 were optimized and a genetic transformation system was established. The optimal ratio of enzymatic hydrolysate was 5 mg (-1) lysozyme, 0.2 U (-1) chitinase and 460 U 65507 (-1) glucuronidase; optimized conditions for protoplast preparation: osmotic stabilizer 0.7 M KCl, cell mass 15 mg, enzymatic hydrolysis temperature 37 In the case of NHEJ gene Ku-70, the 2.3 KB homologous arm was used to knock out oah, and the probability of homologous integration was 65%. Oxalic acid was not synthesized in the whole fermentation process. (2) Two low-yield strains A1 and L2 were obtained by plasma mutation and high throughput screening with Aspergillus Niger H915-1 as the starting strain. Citric acid production decreased from 157 g (-1) to 117 g (-1) and 76 g (-1), respectively. Genome sequencing, splicing and annotation were performed on the production strain and mutant strains A1 and L2. Their genome sizes were 35.98 Mb, 34.64 Mb and 36.45 Mb, respectively. A total of 59 gene families were found to be different and single nucleotide polymorphisms (SNPs) were detected. Polymorphism, SNP, and insertion-deletion (INDEL) loci were 1210, and structural variation (SV) 52, involving 35 genes. Cis-aconitase and gamma-aminobutyric acid (GABA) pathways in the central metabolic pathway were mutated in succinic hemialdehyde dehydrogenase genes. The transcriptome data of 15-1 were analyzed at four time points during citric acid synthesis and at the growth stage of the bacteria, and 479 genes were found to have changed. The main genes in the central metabolic pathway of Aspergillus niger were identified. The expression of most enzymes in the glycolysis pathway remained unchanged, the expression of triose phosphate isomerase was up-regulated, and pyruvate kinase was down-regulated. The expression of most of the enzymes in the TCA cycle was down-regulated; the expression of the key enzymes in the GABA pathway was up-regulated; the expression of ATP-citrate lyase was up-regulated, which together with the TCA cycle constituted an ineffective ATP-depleting cycle; 35 transporters were identified to be up-regulated continuously, including three organic anion transporters and one monocarboxylate transporter. Transcriptome analysis showed that low-pH-induced gene gas was screened and its promoter was predicted. Promoter expression intensity was verified by reporter gene fluorescent protein (s GFP). Pgas was induced to express s GFP strongly at pH 2.0, and the expression intensity was consistent with that of PgpdA at pH 2.0. The expression of CAD gene endowed Aspergillus Niger H915-1 with the ability to synthesize itaconic acid. The expression of s CAD at 24 h and 108 h increased 2.37 and 3.23 times than that at 8 h, respectively. The yields of itaconic acid reached 4.92 g (-1) and 5 times that of Pgpd A-CAD. The induction ability of Pgas was verified by q-PCR. Two transcription regulators, XP_001388781.2 and XP_001396281, specifically binding to Pgas were identified by DNA pull-down technique. Evm. model. unitig_0. 1770 sequence, which was closely related to Kl HGT1, was obtained by chemical tree analysis and sequence alignment analysis. It was predicted that the protein contained 11 transmembrane regions, N-terminal in the cell membrane and C-terminal in the cell membrane, named ANHGT1. The total glucose consumption time of HGT1 transformer was 12 h less than that of H915-1. The citric acid yield of HGT1 transformer was increased by 14.7%, fermentation time was shortened by 6 h, and the maximum specific acid production rate was increased by 29.5%.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ921.1

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