【作者】 张倩；

【导师】 周景文；

【作者基本信息】 江南大学 ， 发酵工程， 2022， 博士

【摘要】 乙酰辅酶A(Acetyl-CoA)是碳代谢、脂质代谢和氨基酸分解代谢的关键代谢中间体,参与细胞内众多的生化反应和代谢途径,为细胞生长和代谢提供碳源和能量。在酿酒酵母中,乙酰辅酶A是丙二酰辅酶A的直接合成前体,乙酰辅酶A的合成受到磷脂合成的严格调控,细胞内乙酰辅酶A和丙二酰辅酶A维持在较低水平,调控乙酰辅酶A代谢是调控丙二酰辅酶A代谢的基础。许多高附加值化合物是以丙二酰辅酶A为合成前体,然而,细胞质内低水平的丙二酰辅酶A限制了相关化合物的合成。黄酮类化合物如柚皮素是一种简单的丙二酰辅酶A衍生物,其合成过程需要以3分子的丙二酰辅酶A为前体;蒽酮类化合物如胭脂红酸是一种复杂的丙二酰辅酶A衍生物,其合成过程需要以1分子的乙酰辅酶A和7分子的丙二酰辅酶A为前体,此外,目前胭脂红酸在酿酒酵母中的合成途径尚未解析。在本研究中,首先解析了胭脂红酸完整的合成途径及其相关的酶,在酿酒酵母中构建了从头合成胭脂红酸的底盘细胞。在此基础上,对胭脂红酸合成过程进行了深层解析,分析了胭脂红酸合成途径的关键节点。此外,为了进一步优化脂肪酸β氧化过程的疏水性底物的吸收,构建了一种不依赖于转运蛋白的油脂内吞体系,随后,通过脂肪酸β氧化过程的调控,优化了柚皮素和胭脂红酸的合成。本论文的主要研究内容如下:(1)鉴定了酿酒酵母中胭脂红酸完整的合成途径和相关的酶。首先,通过在酿酒酵母中表达文献报道的构巢曲霉中胭脂红酸的合成途径相关的酶:芦荟(Aloe arborescens)来源的聚酮合酶OKS,链霉菌(Streptomyces sp.R1128)来源的环化酶Zhu I和芳香酶Zhu J,胭脂虫来源的C-葡萄糖基转移酶UGT2,然而,在酿酒酵母中并未检测到胭脂红酸,推测这并不是胭脂红酸的完整合成途径,构巢曲霉中可能有涉及参与胭脂红酸合成的酶;通过对构巢曲霉的基因组分析,挖掘了构巢曲霉中参与胭脂红酸合成的相关酶:4’-磷酸戊二烯基转移酶Npg A和单加氧酶Apt C(在本论文中定义为黄胭脂酮酸蒽酮氧化酶);然后,通过将胭脂红酸合成途径的异源基因整合于酿酒酵母基因组的多拷贝转座子位点Ty2,构建了一株胭脂红酸的生产菌株,胭脂红酸在摇瓶上的产量达到2664.6μg/L。(2)解析了酿酒酵母中胭脂红酸合成途径的关键节点。基于合成胭脂红酸的底盘细胞,对胭脂红酸的羟基化过程进行了分析,通过敲除酿酒酵母内源的羟基化酶编码基因SCS7,SUR2,TPA1和CAT5,其中,CAT5的敲除导致了胭脂红酸合成能力的丧失,CAT5的回补表达恢复了胭脂红酸的合成,鉴定了酿酒酵母内源羟化酶Cat5(在本论文中定义为黄胭脂酮酸羟化酶)参与了胭脂红酸的合成;通过分别过量表达胭脂红酸合成途径的基因,确定了成环过程是胭脂红酸合成的关键限速步骤;通过对成环过程的聚酮合酶及其辅酶的融合表达,以及环化酶与芳香酶的融合表达,分析其对胭脂红酸合成的影响;通过敲除内源的三个关键水解酶、强化UDP-葡萄糖和表达不同来源的糖基转移酶,分析糖基化过程对胭脂红酸合成的影响;进一步通过添加不同浓度的抗坏血酸,分析了中间产物的氧化水平对胭脂红酸合成的影响。(3)基于小窝蛋白的表达,构建了不依赖于转运蛋白的油脂内吞体系。为了解除脂肪酸依赖于转运蛋白的机制和油脂不能跨膜转运的限制,强化细胞对底物的吸收和利用,为了解决这一挑战,首先,通过将来源于人的小窝蛋白与绿色荧光蛋白的融合表达,解析了小窝蛋白可在酿酒酵母中形成微囊泡结构;然后通过添加5,6-羧酸荧光素,验证了小窝对外源物质的内吞能力;为了进一步验证小窝对疏水性底物的内吞能力,通过在培养基中添加大豆油和薄层色谱分析,鉴定了小窝对油脂的内吞能力;此外,将小窝蛋白应用于柚皮素和胭脂红酸合成的底盘细胞,考察了基于小窝蛋白的油脂内吞体系对柚皮素和胭脂红酸的促进作用。(4)基于脂肪酸β氧化过程的调控,优化了丙二酰辅酶A衍生物的合成。首先,以简单的丙二酰辅酶A衍生物柚皮素的合成为研究对象,通过提高脂肪酸的底物水平和强化脂肪酸β氧化过程,考察了脂肪酸β氧化的调控对乙酰辅酶A和丙二酰辅酶A含量以及柚皮素产量的影响;为了进一步分析脂肪酸β氧化过程的调控对复杂丙二酰辅酶A衍生物胭脂红酸合成的影响,将脂肪酸β氧化的调控策略应用于胭脂红酸合成的底盘细胞;通过协同强化脂肪酸β氧化的关键基因和胭脂红酸合成途径基因的表达,进一步促进胭脂红酸的合成;将关键的组合表达框整合于基因组多拷贝转座子位点Ty1cons1,经过初筛和复筛,胭脂红酸在摇瓶上的产量是4092.0μg/L;最后,最优柚皮素工程菌株L07和胭脂红酸工程菌株CA133分别在5 L发酵罐上进行发酵优化和放大培养,柚皮素的产量达到1129.4 mg/L,胭脂红酸的产量达到7983.8μg/L。更多还原

【Abstract】 Acetyl-CoA is a key metabolic intermediate of carbon metabolism,lipid metabolism and amino acid catabolism,which participates in many metabolic processes and biochemical reactions in cells and provides carbon source and energy for cell growth and metabolism.In Saccharomyces cerevisiae,acetyl-CoA is as the precursor of malonyl-CoA.The biosynthesis of acetyl-CoA is strictly regulated by phospholipid biosynthesis.Generally,the intracellular acetyl-CoA and malonyl-CoA are maintained at a low level.Regulating the metabolism of acetyl-CoA is the basis for regulating the metabolism of malonyl-CoA.Many high value-added compounds use malonyl-CoA as the biosynthetic precursor.However,the low level of malonyl-CoA in the cytoplasm limits the biosynthesis of compounds.Flavonoids such as naringenin is a kind of simple malonyl-CoA-derived compound,which requires three molecules of malonyl-CoA as the precursors.Anthraquinones such as carminic acid belongs to complex malonyl-CoA-derived compounds,which requires one molecule of acetyl-CoA and seven molecules of malonyl-CoA as precursors.In addition,the biosynthetic pathway of carminic acid in S.cerevisiae has not been resolved yet.In this paper,the complete biosynthetic pathway of carminic acid and its related enzymes were analyzed and de novo biosynthesis of carminic acid were reconstructed.Moreover,the key points of the biosynthetic pathway of carminic acid were analysed.Then,a lipid endocytosis mechanism of independent-transporters has been developed to further optimize the hydrophobic substrate absorption of fatty acid β-oxidation.Finally,the production of naringenin and carminic acid was optimized by regulating the fatty acid β-oxidation process.The main results of this paper are as follows:(1)The complete biosynthetic pathway and related enzymes of carminic acid in S.cerevisiae were identified.First,the enzymes related to the biosynthetic pathway of carminic acid in A.nidulans reported were expressed in S.cerevisiae: polyketone synthase OKS from Aloe arborescens,cyclase Zhu I and aromatase Zhu J from Streptomyces sp.R1128,and C-glucosyltransferase UGT2 from Dactylopius coccus.However,carminic acid was not detected.It was speculated that this was not a complete pathway for the biosynthesis of carminic acid in S.cerevisiae,and there may be enzymes involved in the biosynthesis of carminic acid in A.nidulans.By analyzing the genome of A.nidulans,the related enzymes involved in the biosynthesis of carminic acid in A.nidulans were discovered: 4’-phosphopantetheinyl transferases Npg A and monooxygenase Apt C(defined as flavokermesic acid anthrone oxidase in this paper).By integrating the heterologous genes of carminic acid biosynthetic pathway into the multiple copy site Ty2 of S.cerevisiae genome,a carminic acid-producing strain was constructed.The carminic acid production was 2664.6 μg/L in shake flask.(2)The key points of carminic acid biosynthetic pathway in S.cerevisiae were analysed.the hydroxylation process of carminic acid was analysed by knocking out the genes(SCS7,SUR2,TPA1 and CAT5)of hydroxylase in S.cerevisiae,when the knock out of CAT5 led to the loss of carminic acid biosynthesis,and the complementary expression of CAT5 restored the biosynthesis of carminic acid,showing that the endogenous hydroxylase Cat5 of S.cerevisiae(defined as flavokermesic acid hydroxylase in this paper)was involved in the biosynthesis of carminic acid.By overexpressing the genes of the biosynthetic pathway of carminic acid separately,it was concluded that the cyclization process was the key rate-limiting step of carminic acid biosynthesis.Through the fusion expression of polyketide synthase and its coenzyme,and the fusion expression of cyclase and aromatase,the effects on the biosynthesis of carminic acid were analysed.In addition,the effects of glycosylation on carminic acid biosynthesis were analysed by knocking out key endogenous hydrolases,enhancing UDP-glucose and expressing glycosyltransferases from different sources.In addition,the influence of oxidation level of intermediate products on the biosynthesis of carminic acid was analysed by adding different concentrations ascorbic acid.(3)Based on the expression of caveolin generated from human,a lipid endocytosis system of independent-transporters was constructed.To remove the dependence of transporters and the transport restriction of hydrophobic substrates,a new mechanism was required to strengthen the absorption and utilization of hydrophobic substrates by cells.First,caveolin was confirmed to form a microbubble structure in S.cerevisiae by the fusion expression of CAV1 and green fluorescent protein.Then,the endocytosis ability of the caveolin was verified by adding 5,6-carboxyfluorescein.In order to further verify the endocytosis ability of caveolin on hydrophobic substrate,adding soybean oil pre-stained with Nile Red and thin layer chromatography was used to identify the endocytosis ability of caveolin on oils.In addition,caveolin was applied to the chassis cells for biosynthesis of naringenin and carminic acid,and the role of oil endocytosis mechanism of caveolin in promoting naringenin and carminic acid was analysed.(4)The biosynthesis of malonyl-CoA derivatives was optimized by regulating the fatty acid β-oxidation process.First,the simple malonyl-CoA derivative(naringenin)-producing strain was as the chassis cell,and the effects of improvement of substrate level and the enhancement of fatty acid β-oxidation on the level of acetyl-CoA and malonyl-CoA and the production of naringenin was analysed.To further analyse the effect of fatty acid β-oxidation process on the biosynthesis of more complex malonyl-CoA derivative(carminic acid),the regulation strategy of fatty acid β-oxidation was applied to carminic acid-producing strain.To further strengthen the biosynthesis of carminic acid,the key genes of fatty acid β-oxidation and carminic acid biosynthetic pathway were combined overexpression,and the key combination expression frames were integrated into the genome multiple copy site Ty1cons1.Through the preliminary screening and re-screening,the carminic acid production was enhanced to 4092.0μg/L in the shake flask.Finally,the engineered naringenin-producing strain L07 and engineered carminic acid-producing strain CA133 were fermented in 5 L bioreactor,resulting in 1129.4 mg/L of naringenin production and 7983.8 μg/L of carminic acid production,respectively.更多还原