Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany.
Dipartimento Di Biotecnologie E Bioscienze, Università Degli Studi Di Milano-Bicocca, Piazza della Scienza, 4, 67056, Milan, Italy.
Microb Cell Fact. 2022 May 28;21(1):102. doi: 10.1186/s12934-022-01817-1.
The microbial production of succinic acid (SA) from renewable carbon sources via the reverse TCA (rTCA) pathway is a process potentially accompanied by net-fixation of carbon dioxide (CO). Among reduced carbon sources, glycerol is particularly attractive since it allows a nearly twofold higher CO-fixation yield compared to sugars. Recently, we described an engineered Saccharomyces cerevisiae strain which allowed SA production in synthetic glycerol medium with a maximum yield of 0.23 Cmol Cmol. The results of that previous study suggested that the glyoxylate cycle considerably contributed to SA accumulation in the respective strain. The current study aimed at improving the flux into the rTCA pathway accompanied by a higher CO-fixation and SA yield.
By changing the design of the expression cassettes for the rTCA pathway, overexpressing PYC2, and adding CaCO to the batch fermentations, an SA yield on glycerol of 0.63 Cmol Cmol was achieved (i.e. 47.1% of the theoretical maximum). The modifications in this 2nd-generation SA producer improved the maximum biomass-specific glycerol consumption rate by a factor of nearly four compared to the isogenic baseline strain solely equipped with the dihydroxyacetone (DHA) pathway for glycerol catabolism. The data also suggest that the glyoxylate cycle did not contribute to the SA production in the new strain. Cultivation conditions which directly or indirectly increased the concentration of bicarbonate, led to an accumulation of malate in addition to the predominant product SA (ca. 0.1 Cmol Cmol at the time point when SA yield was highest). Off-gas analysis in controlled bioreactors with CO-enriched gas-phase indicated that CO was fixed during the SA production phase.
The data strongly suggest that a major part of dicarboxylic acids in our 2nd-generation SA-producer was formed via the rTCA pathway enabling a net fixation of CO. The greatly increased capacity of the rTCA pathway obviously allowed successful competition with other pathways for the common precursor pyruvate. The overexpression of PYC2 and the increased availability of bicarbonate, the co-substrate for the PYC reaction, further strengthened this capacity. The achievements are encouraging to invest in future efforts establishing a process for SA production from (crude) glycerol and CO.
通过反三羧酸（rTCA）途径，利用可再生碳源微生物生产琥珀酸（SA）是一个潜在的过程，伴随着二氧化碳（CO）的净固定。在还原碳源中，甘油特别有吸引力，因为与糖相比，它可以使 CO 固定的产率提高近两倍。最近，我们描述了一种经过工程改造的酿酒酵母菌株，该菌株可以在合成甘油培养基中生产 SA，最大产率为 0.23Cmol Cmol。先前研究的结果表明，乙醛酸循环对该菌株中 SA 的积累有很大贡献。本研究旨在通过提高 rTCA 途径的通量，同时提高 CO 固定和 SA 产率。
通过改变 rTCA 途径表达盒的设计，过表达 PYC2，并在分批发酵中添加 CaCO，实现了甘油上 SA 的产率为 0.63Cmol Cmol（即理论最大值的 47.1%）。与仅配备二羟丙酮（DHA）途径用于甘油分解的同基因基线菌株相比，第二代 SA 生产菌的这些改进将最大生物质特异性甘油消耗率提高了近四倍。数据还表明，乙醛酸循环并没有为新菌株的 SA 生产做出贡献。直接或间接增加碳酸氢盐浓度的培养条件，除了主要产物 SA 外，还会积累苹果酸（在 SA 产率最高时约为 0.1Cmol Cmol）。在富含 CO 的气相控制生物反应器中的废气分析表明，CO 在 SA 生产阶段被固定。
数据强烈表明，我们第二代 SA 生产菌中的大部分二羧酸是通过 rTCA 途径形成的，从而实现了 CO 的净固定。rTCA 途径的容量大大增加，显然使它能够成功与其他途径竞争共同的前体丙酮酸。PYC2 的过表达和碳酸氢盐（PYC 反应的共底物）可用性的增加进一步增强了这种能力。这些成果令人鼓舞，值得投入未来的努力，建立从（粗）甘油和 CO 生产 SA 的工艺。
