發(fā)布時間：2018-08-12 13:58

【摘要】：檸檬酸作為生產(chǎn)量最大的有機酸,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黑曲霉進行深層有氧發(fā)酵來生產(chǎn),產(chǎn)量和轉(zhuǎn)化率均已達到較高水平,但根據(jù)Alvarez-Vasquez的模型,仍然有提高空間,要進一步加強檸檬酸的生產(chǎn)需要從基因組和轉(zhuǎn)錄組水平探索黑曲霉高產(chǎn)檸檬酸機制,以此來指導(dǎo)代謝調(diào)控。此外,檸檬酸生產(chǎn)菌株經(jīng)過多輪誘變形成短粗菌絲且細胞壁增厚,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象,通過比較基因組學(xué)和轉(zhuǎn)錄組學(xué),探討了黑曲霉高產(chǎn)檸檬酸的機制,進而發(fā)現(xiàn)了低pH誘導(dǎo)的啟動子Pgas可以作為動態(tài)調(diào)控的基因元件,最后通過調(diào)整葡萄糖轉(zhuǎn)運蛋白的表達提高了檸檬酸的產(chǎn)量。主要研究結(jié)果如下:(1)對黑曲霉H915-1的原生質(zhì)體形成條件進行優(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個表達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oah進行敲除,同源整合的概率為65%,基因敲除菌株在整個發(fā)酵過程中不再合成草酸。(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)。對生產(chǎn)菌株和誘變株A1和L2進行基因組測序、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1210處,結(jié)構(gòu)性變異(Structural variation,SV)52處,共涉及35個基因的表達。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)對黑曲霉H915-1在檸檬酸合成階段的4個時間點和菌體生長階段的轉(zhuǎn)錄組數(shù)據(jù)進行分析,發(fā)現(xiàn)479個基因的表達發(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沒有變化,磷酸丙糖異構(gòu)酶表達上調(diào),丙酮酸激酶表達下調(diào),TCA循環(huán)大部分酶的表達下調(diào);發(fā)現(xiàn)GABA通路關(guān)鍵酶的表達上調(diào);ATP-檸檬酸裂解酶表達上調(diào),與TCA循環(huán)一起構(gòu)成了一條消耗ATP的無效循環(huán);鑒定到35個轉(zhuǎn)運蛋白表達持續(xù)上調(diào),包含3個有機陰離子轉(zhuǎn)運蛋白,以及1個單羧酸轉(zhuǎn)運蛋白。(4)通過轉(zhuǎn)錄組分析,篩選到低pH誘導(dǎo)表達的基因gas并進行啟動子預(yù)測,利用報告基因熒光蛋白(s GFP)進行啟動子表達強度的驗證,Pgas在pH 2.0時被誘導(dǎo)而強烈表達s GFP,表達強度和Pgpd A在pH 2.0時啟動表達的能力一致。利用Pgas啟動順烏頭酸脫羧酶(s CAD)基因的表達賦予黑曲霉H915-1合成衣康酸的能力,發(fā)酵24 h和108 h的s CAD的表達量比8 h的表達量分別增加了2.37和3.23倍,轉(zhuǎn)化子的衣康酸產(chǎn)量達到4.92 g×L~(-1),為Pgpd A-CAD轉(zhuǎn)化子產(chǎn)量的5倍。利用q PCR對Pgas的誘導(dǎo)能力進行驗證,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Pgas沒有影響,且pH與Pgas的啟動能力存在線性關(guān)系。通過DNA pull-down技術(shù)鑒定到2個與Pgas特異結(jié)合的轉(zhuǎn)錄調(diào)節(jié)因子XP_001388781.2和XP_001396281。(5)基于轉(zhuǎn)錄組分析,對假定的葡萄糖轉(zhuǎn)運蛋白進行進化樹分析和序列比對分析,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)測該蛋白含有11個跨膜區(qū)域,N端在細胞膜內(nèi),C端在胞內(nèi),命名為An HGT1。在限制性葡萄糖培養(yǎng)基上進行生長實驗,HGT轉(zhuǎn)化子的菌落直徑比對照增加50%~150%。在發(fā)酵后期補加30 g×L~(-1)葡萄糖后HGT1轉(zhuǎn)化子完全消耗葡萄糖的時間比H915-1減少12 h。HGT1轉(zhuǎn)化子的檸檬酸產(chǎn)量比對照增加了14.7%,發(fā)酵時間縮短了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)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TQ921.1

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