Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA.
Department of Cell and Molecular Biology, College of the Environment and Life Sciences, University of Rhode Island, Kingston, Rhode Island, USA.
Appl Environ Microbiol. 2024 Jan 24;90(1):e0195123. doi: 10.1128/aem.01951-23. Epub 2023 Dec 22.
The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in , an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C and C sugars. In addition grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of .IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.
平台化学品 2,3-丁二醇（2,3-BDO）用于衍生产品，如 1,3-丁二烯和甲基乙基酮，用于化工和燃料生产行业。由于各种原因，微生物在工业规模上高效生产 2,3-BDO 尚未实现，包括产物对宿主生物的抑制、产物形成中的混合立体特异性以及对昂贵底物（即葡萄糖）的依赖。在这项研究中，我们探索了在 ，一种极其耐热（最佳生长温度=78°C）和厌氧细菌中工程化 2,3-BDO 途径，该细菌可以将结晶纤维素和半纤维素分解成可发酵的 C 和 C 糖。此外，它还可以在未经预处理的植物生物质上生长，如柳枝稷。2,3-BDO 的生物合成涉及三个步骤：两个丙酮酸分子缩合形成乙酰乳酸；乙酰乳酸脱羧形成乙酰酮，最后，乙酰酮还原为 2,3-BDO。天然产生乙酰酮；因此，为了完成 2,3-BDO 生物合成途径，工程菌产生了一种次级醇脱氢酶（sADH）来催化最后一步。两种以前表征的、对乙酰酮具有高亲和力的耐热 sADH 酶，一种来自细菌，一种来自古菌，分别进行了测试。当重组菌株中存在任何一种 sADH 时，都能够从结晶纤维素和木聚糖中生产高达 2.5-mM 的 2,3-BDO，并且能够直接从未经预处理的柳枝稷中生产 0.2-mM 的 2,3-BDO。这为更高的产量和生产力奠定了基础，为此，使用 的基因组规模代谢模型评估了限制因素和进一步优化的潜在遗传靶点。重要性以木质纤维素植物生物质为底物，通过微生物合成 2,3-丁二醇是实现该化学品在工业规模上具有成本效益的生物生产的主要关键之一。然而，为了释放微生物生长所需的糖，对生物质进行解构目前需要昂贵的热化学和酶预处理。在这项研究中，成功地对热纤维素分解细菌 进行了工程化改造，使其能够从纤维素、木聚糖和未经预处理的柳枝稷中生产 2,3-丁二醇。应用 的基因组规模代谢模型来调整碳和氧化还原通量，以最大限度地提高 2,3-丁二醇的生产力，从而揭示需要遗传修饰的瓶颈。
