开发高效的生物过程需要选择最佳的生物合成途径,由于数据库和文献中可用的大量数据,这可能是具有挑战性和耗时的。用于生物合成商业上有吸引力的分子的莽草酸途径的延伸通常涉及混杂的酶或缺乏成熟的途径。为了应对这些挑战,我们开发了一个集成枚举/逆向综合算法的计算工作流,途径分析的工具箱,酶选择工具,和一个基因发现管道,由人工策展和文献综述支持。我们的重点是实施酪氨酸衍生化合物的生物合成途径,特别是L-3,4-二羟基苯丙氨酸(L-DOPA)和多巴胺,在健康和营养方面具有重要应用。我们选择了一种产生L-DOPA的途径和两种不同的多巴胺途径-一种已经在文献中描述的途径和一种新的途径。我们的目标是确定最适合在大肠杆菌中表达的已知途径的候选基因,或者发现创新途径。虽然并非所有实施的途径都导致目标化合物的积累,在我们的摇瓶实验中,对于已知和新的途径,我们实现了最大L-DOPA滴度为0.71g/L,多巴胺滴度为0.29和0.21g/L。分别。在L-DOPA的情况下,我们利用,第一次,一种来自青枯雷尔氏菌的酪氨酸酶的突变版本。通过已知的生物合成途径生产多巴胺是通过将L-DOPA途径与恶臭假单胞菌的DOPA脱羧酶的表达偶联来完成的,产生了以前文献中从未报道过的独特的生物合成途径。在新的途径的背景下,多巴胺是使用酪胺作为中间化合物生产的。为了实现这一点,酪氨酸最初是通过表达短小杆菌的TDC转化为酪胺,which,反过来,通过Mucunapruriens的ppoMP编码的酶的作用转化为多巴胺。这标志着多巴胺的替代生物合成途径首次在微生物中得到验证。这些发现强调了我们的计算工作流程在促进途径枚举和选择方面的有效性,提供了发现新的生物合成路线的潜力,从而为生物技术感兴趣的其他目标化合物铺平了道路。
Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of the shikimate pathway for the biosynthesis of commercially attractive molecules often involves promiscuous enzymes or lacks well-established routes. To address these challenges, we developed a computational workflow integrating enumeration/retrosynthesis algorithms, a toolbox for pathway analysis, enzyme selection tools, and a gene discovery pipeline, supported by manual curation and literature review. Our focus has been on implementing biosynthetic pathways for tyrosine-derived compounds, specifically L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine, with significant applications in health and nutrition. We selected one pathway to produce L-DOPA and two different pathways for dopamine-one already described in the literature and a novel pathway. Our goal was either to identify the most suitable gene candidates for expression in Escherichia coli for the known pathways or to discover innovative pathways. Although not all implemented pathways resulted in the accumulation of target compounds, in our shake-flask experiments we achieved a maximum L-DOPA titer of 0.71 g/L and dopamine titers of 0.29 and 0.21 g/L for known and novel pathways, respectively. In the case of L-DOPA, we utilized, for the first time, a mutant version of tyrosinase from Ralstonia solanacearum. Production of dopamine via the known biosynthesis route was accomplished by coupling the L-DOPA pathway with the expression of DOPA decarboxylase from Pseudomonas putida, resulting in a unique biosynthetic pathway never reported in literature before. In the context of the novel pathway, dopamine was produced using tyramine as the intermediate compound. To achieve this, tyrosine was initially converted into tyramine by expressing TDC from Levilactobacillus brevis, which, in turn, was converted into dopamine through the action of the enzyme encoded by ppoMP from Mucuna pruriens. This marks the first time that an alternative biosynthetic pathway for dopamine has been validated in microbes. These findings underscore the effectiveness of our computational workflow in facilitating pathway enumeration and selection, offering the potential to uncover novel biosynthetic routes, thus paving the way for other target compounds of biotechnological interest.