Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440.
Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440.
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DOI：
10.1016/j.meteno.2017.05.002
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发表时间：
2017-12
影响因子：
5.2
通讯作者：
Beckham GT
中科院分区：
文献类型：
--
作者：
Johnson CW;Abraham PE;Linger JG;Khanna P;Hettich RL;Beckham GT
碳分解代谢物抑制是指微生物在多种调节机制下对某些生长底物的代谢优于其他底物。这种偏好对生物体在自然环境中的适应性很重要，但可能会阻碍它们作为驯化微生物细胞工厂的表现。在一株腐臭假单胞菌KT2440菌株中，通过工程改造将木质素衍生的芳香单体（如对香豆酸酯和阿威酸酯）转化为木酸酯，这是生物基尼龙和其他化学物质的前体，代谢中间体包括4-羟基苯甲酸酯和香草酸酯积累并随后降低生产力。我们假设，这些代谢瓶颈可能至少部分是葡萄糖或醋酸引起的碳分解代谢抑制的影响，这是必须提供给菌株补充能量和细胞生长的更优选底物。利用基于质谱的蛋白质组学，我们已经确定了4-羟基苯甲酸羟化酶PobA和香草酸去甲基化酶VanAB是分解代谢物抑制蛋白（Crc）的靶标，这是碳分解代谢物抑制的全球调节剂。通过从该菌株中删除编码Crc的基因，减少了4-羟基苯甲酸盐和香草酸盐的积累，从而提高了黏液酸盐的产量。在葡萄糖培养基中，由于缺失了编码Crc的基因，36 h后对香豆酸酯的产率提高了近70%(94.6±0.6%比56.0±3.0% (mol/mol))， 72 h后阿魏酸酯的产率提高了一倍多（28.3±3.3%比12.0±2.3% (mol/mol)）。消除结直肠癌的效果与乙酸培养基相似，Crc缺失菌株在24 h后产率略高（47.7±0.6%比40.7±3.6% (mol/mol)），在72 h后产率提高60%以上（16.9±1.4%比10.3±0.1% (mol/mol)）。这些结果是减少碳分解代谢物抑制对微生物细胞工厂转化复杂原料的好处的一个例子，我们认为这一概念可以被广泛认为是代谢工程中将可再生原料转化为增值化学品的一种策略。Crc是假单胞菌中碳分解代谢抑制的全球调节剂。编码Crc的基因从产生恶臭杆菌的株中被删除。根据我们的蛋白质组学数据，PobA和VanAB的表达受Crc的调控。在葡萄糖或乙酸存在的情况下，删除Crc可改善转化为黏液酸盐。这可能是开发假单核细胞工厂的一个有用策略。
Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a Pseudomonas putida KT2440 strain engineered to convert lignin-derived aromatic monomers such as p-coumarate and ferulate to muconate, a precursor to bio-based nylon and other chemicals, metabolic intermediates including 4-hydroxybenzoate and vanillate accumulate and subsequently reduce productivity. We hypothesized that these metabolic bottlenecks may be, at least in part, the effect of carbon catabolite repression caused by glucose or acetate, more preferred substrates that must be provided to the strain for supplementary energy and cell growth. Using mass spectrometry-based proteomics, we have identified the 4-hydroxybenzoate hydroxylase, PobA, and the vanillate demethylase, VanAB, as targets of the Catabolite Repression Control (Crc) protein, a global regulator of carbon catabolite repression. By deleting the gene encoding Crc from this strain, the accumulation of 4-hydroxybenzoate and vanillate are reduced and, as a result, muconate production is enhanced. In cultures grown on glucose, the yield of muconate produced from p-coumarate after 36 h was increased nearly 70% with deletion of the gene encoding Crc (94.6 ± 0.6% vs. 56.0 ± 3.0% (mol/mol)) while the yield from ferulate after 72 h was more than doubled (28.3 ± 3.3% vs. 12.0 ± 2.3% (mol/mol)). The effect of eliminating Crc was similar in cultures grown on acetate, with the yield from p-coumarate just slightly higher in the Crc deletion strain after 24 h (47.7 ± 0.6% vs. 40.7 ± 3.6% (mol/mol)) and the yield from ferulate increased more than 60% after 72 h (16.9 ± 1.4% vs. 10.3 ± 0.1% (mol/mol)). These results are an example of the benefit that reducing carbon catabolite repression can have on conversion of complex feedstocks by microbial cell factories, a concept we posit could be broadly considered as a strategy in metabolic engineering for conversion of renewable feedstocks to value-added chemicals. Crc is a global regulator of carbon catabolite repression in pseudomonads. The gene encoding Crc was deleted from muconate a producing P. putida strain. Based on our proteomics data, expression of PobA and VanAB are regulated by Crc. Deleting Crc improved conversion to muconate in the presence of glucose or acetate. This may be a useful strategy toward developing pseudomonad cell factories.