黑曲霉高产柠檬酸机制及代谢调控研究

发布时间：2018-08-12 13:58

【摘要】：柠檬酸作为生产量最大的有机酸,广泛应用于食品、医药、洗涤剂和化妆品等领域。目前柠檬酸主要通过黑曲霉进行深层有氧发酵来生产,产量和转化率均已达到较高水平,但根据Alvarez-Vasquez的模型,仍然有提高空间,要进一步加强柠檬酸的生产需要从基因组和转录组水平探索黑曲霉高产柠檬酸机制,以此来指导代谢调控。此外,柠檬酸生产菌株经过多轮诱变形成短粗菌丝且细胞壁增厚,遗传转化困难且缺少有力的代谢调控工具,需要研究适用于柠檬酸生产菌株的转化方法和代谢调控元件。本论文对黑曲霉柠檬酸工业生产菌株H915-1建立了遗传转化方法,并以H915-1及其诱变株为研究对象,通过比较基因组学和转录组学,探讨了黑曲霉高产柠檬酸的机制,进而发现了低pH诱导的启动子Pgas可以作为动态调控的基因元件,最后通过调整葡萄糖转运蛋白的表达提高了柠檬酸的产量。主要研究结果如下:(1)对黑曲霉H915-1的原生质体形成条件进行优化并建立了遗传转化系统。最优酶解液配比为5 mg×m L~(-1)溶壁酶、0.2 U×m L~(-1)几丁质酶和460 U×m L~(-1)葡萄糖醛酸酶;优化后的原生质体制备条件:渗透压稳定剂为0.7 M KCl,菌体量15 mg,酶解温度37°C,菌球直径50μm。采用PEG介导法,利用共转化的方式,可以使2个表达框整合到黑曲霉基因组中,共整合概率为58%。在未敲除非同源末端连接(non-homologous end joining,NHEJ)基因Ku-70的情况下,利用2.3 kb同源臂对oah进行敲除,同源整合的概率为65%,基因敲除菌株在整个发酵过程中不再合成草酸。(2)以黑曲霉H915-1为出发菌株,利用等离子诱变和高通量筛选获得2株低产菌株A1和L2,它们的柠檬酸产量分别由出发菌株的157 g×L~(-1)降为117 g×L~(-1)和76 g×L~(-1)。对生产菌株和诱变株A1和L2进行基因组测序、拼接和注释,它们的基因组大小分别为35.98 Mb、34.64 Mb和36.45 Mb,共发现59个基因家族存在差异,单核苷酸多态性(Single nucleotide polymorphism,SNP)和插入缺失(insertion-deletion,INDEL)位点1210处,结构性变异(Structural variation,SV)52处,共涉及35个基因的表达。中心代谢通路的顺乌头酸酶和γ-氨基丁酸(γ-aminobutyric acid,GABA)通路的琥珀酸半醛脱氢酶基因发生变异。(3)对黑曲霉H915-1在柠檬酸合成阶段的4个时间点和菌体生长阶段的转录组数据进行分析,发现479个基因的表达发生变化。确定了黑曲霉中心代谢通路的主效基因。糖酵解通路的大部分酶的表达没有变化,磷酸丙糖异构酶表达上调,丙酮酸激酶表达下调,TCA循环大部分酶的表达下调;发现GABA通路关键酶的表达上调;ATP-柠檬酸裂解酶表达上调,与TCA循环一起构成了一条消耗ATP的无效循环;鉴定到35个转运蛋白表达持续上调,包含3个有机阴离子转运蛋白,以及1个单羧酸转运蛋白。(4)通过转录组分析,筛选到低pH诱导表达的基因gas并进行启动子预测,利用报告基因荧光蛋白(s GFP)进行启动子表达强度的验证,Pgas在pH 2.0时被诱导而强烈表达s GFP,表达强度和Pgpd A在pH 2.0时启动表达的能力一致。利用Pgas启动顺乌头酸脱羧酶(s CAD)基因的表达赋予黑曲霉H915-1合成衣康酸的能力,发酵24 h和108 h的s CAD的表达量比8 h的表达量分别增加了2.37和3.23倍,转化子的衣康酸产量达到4.92 g×L~(-1),为Pgpd A-CAD转化子产量的5倍。利用q PCR对Pgas的诱导能力进行验证,发现Pgas仅受pH的诱导,受酸种类的影响很小,酸根离子浓度对Pgas没有影响,且pH与Pgas的启动能力存在线性关系。通过DNA pull-down技术鉴定到2个与Pgas特异结合的转录调节因子XP_001388781.2和XP_001396281。(5)基于转录组分析,对假定的葡萄糖转运蛋白进行进化树分析和序列比对分析,获得与Kl HGT1亲缘关系较近的evm.model.unitig_0.1770序列,经跨膜预测该蛋白含有11个跨膜区域,N端在细胞膜内,C端在胞内,命名为An HGT1。在限制性葡萄糖培养基上进行生长实验,HGT转化子的菌落直径比对照增加50%~150%。在发酵后期补加30 g×L~(-1)葡萄糖后HGT1转化子完全消耗葡萄糖的时间比H915-1减少12 h。HGT1转化子的柠檬酸产量比对照增加了14.7%,发酵时间缩短了6 h,最大比产酸速率提升了29.5%,提高了发酵生产强度。
[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%.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ921.1

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