Department of Bioengineering and Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal.
Department of Chemical Engineering, Loughborough University, Leicestershire, LE11 3TU Loughborough, United Kingdom.
ACS Synth Biol. 2021 Apr 16;10(4):724-736. doi: 10.1021/acssynbio.0c00518. Epub 2021 Mar 25.
Levulinic acid is a versatile platform molecule with potential to be used as an intermediate in the synthesis of many value-added products used across different industries, from cosmetics to fuels. Thus far, microbial biosynthetic pathways having levulinic acid as a product or an intermediate are not known, which restrains the development and optimization of a microbe-based process envisaging the sustainable bioproduction of this chemical. One of the doors opened by synthetic biology in the design of microbial systems is the implementation of new-to-nature pathways, that is, the assembly of combinations of enzymes not observed , where the enzymes can use not only their native substrates but also non-native ones, creating synthetic steps that enable the production of novel compounds. Resorting to a combined approach involving complementary computational tools and extensive manual curation, in this work, we provide a thorough prospect of candidate biosynthetic pathways that can be assembled for the production of levulinic acid in or . Out of the hundreds of combinations screened, five pathways were selected as best candidates on the basis of the availability of substrates and of candidate enzymes to catalyze the synthetic steps (that is, those steps that involve conversions not previously described). Genome-scale metabolic modeling was used to assess the performance of these pathways in the two selected hosts and to anticipate possible bottlenecks. Not only does the herein described approach offer a platform for the future implementation of the microbial production of levulinic acid but also it provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of value-added chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology.
乙酰丙酸是一种多功能的平台分子，有可能被用作许多不同行业（从化妆品到燃料）中使用的许多增值产品的合成中间体。到目前为止，还不知道微生物生物合成途径中以乙酰丙酸为产物或中间体，这限制了基于微生物的过程的开发和优化，该过程设想了这种化学物质的可持续生物生产。合成生物学在微生物系统设计中开辟的一扇门是实施新天然途径，即组合使用未观察到的酶，这些酶不仅可以使用它们的天然底物，还可以使用非天然底物，从而创造出可以合成新化合物的合成步骤。在这项工作中，我们采用了一种综合方法，涉及互补的计算工具和广泛的人工策展，全面展望了可以组装用于在 或 中生产乙酰丙酸的候选生物合成途径。在筛选的数百种组合中，根据底物的可用性和候选酶来催化合成步骤（即涉及以前未描述的转化的步骤），选择了五种途径作为最佳候选途径。使用基因组规模的代谢建模来评估这些途径在两个选定宿主中的性能，并预测可能的瓶颈。本文描述的方法不仅为未来微生物生产乙酰丙酸提供了一个平台，而且还提供了一个有条理的研究策略，可作为实施其他用于生产增值化学品的新天然生物合成途径的框架，从而促进新兴的合成工业微生物技术领域。
