Jones J Andrew, Vernacchio Victoria R, Collins Shannon M, Shirke Abhijit N, Xiu Yu, Englaender Jacob A, Cress Brady F, McCutcheon Catherine C, Linhardt Robert J, Gross Richard A, Koffas Mattheos A G
Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA.
Department of Chemistry, Hamilton College, Clinton, New York, USA.
mBio. 2017 Jun 6;8(3):e00621-17. doi: 10.1128/mBio.00621-17.
Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies. To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts.
基于发酵的化学生产策略为快速、安全且可持续地生产从燃料到药品等多种重要化学产品提供了一条可行途径。由于这些策略在经济上无法与传统提取和化学生产方法竞争，它们尚未得到广泛的工业应用。在此，我们首次从糖开始，对花青素这种植物天然产物的复杂微生物生物合成进行了工程改造。这是通过开发一种合成的、由4个菌株组成的混合培养物实现的，该混合培养物共同表达了来自不同植物和其他微生物的15种外源或经过修饰的途径酶。这种基于合成菌群的方法能够实现长途径的功能性表达和连接，同时有效管理伴随的代谢负担。十多年来，生产特定的花青素分子，如翠菊苷，一直是一个难以实现的代谢工程目标。我们的混合培养策略实现了每升毫克级的生产滴度。这项研究还为通过非传统方法开发具有成本竞争力的生化生产宿主的菌株和工艺设计取得重大进展奠定了基础。要在微生物宿主中高效表达具有高通量的活性广泛重组途径，需要仔细平衡和分配代谢资源，如三磷酸腺苷（ATP）、还原当量和丙二酰辅酶A（丙二酰 - CoA），以及各种其他途径依赖性辅因子和前体。为了解决这个问题，我们报告了首个合成4菌株混合培养物的设计、表征和实施。将15种酶和转录因子的过表达分配到4个独立的菌株模块中，使得代谢负担得以分担，并能够针对模块特异性代谢物需求对菌株进行独立优化。这项研究代表了迄今为止构建的用于代谢工程应用的最复杂的合成菌群，并为在非天然宿主中重构广泛代谢途径的代谢工程提供了新的范例。
