Fina Albert, Heux Stephanie, Albiol Joan, Ferrer Pau
Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Catalonia, Spain.
TBI, Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France.
Front Bioeng Biotechnol. 2022 Jul 22;10:942304. doi: 10.3389/fbioe.2022.942304. eCollection 2022.
Production of 3-hydroxypropionic acid (3-HP) in () the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase ( ) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L of 3-HP at 0.712 g L h with a final product yield on glycerol of 0.194 Cmol in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for . Overall, the combination of glycerol as carbon source and a metabolically engineered strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.
最近已证明利用甘油作为碳源，通过丙二酸单酰辅酶A途径生产3-羟基丙酸（3-HP），但所报道的指标与商业应用无关。从丙二酸单酰辅酶A到3-HP的异源途径通量被认为是主要瓶颈。在本研究中，已将不同的代谢工程方法结合起来以提高原始3-HP生产菌株的生产力。为此，引入了编码该途径潜在限速步骤（即丙二酸单酰辅酶A还原酶的C末端结构域）的基因的额外拷贝。此外，过表达内源性乙酰辅酶A羧化酶的一个变体，目的是增加丙二酸单酰辅酶A的供应。此外，分别过表达编码丙酮酸脱羧酶、醛脱氢酶和乙酰辅酶A合酶的基因，以增强丙酮酸向胞质乙酰辅酶A的转化，并删除负责副产物D-阿拉伯糖醇产生的主要基因。使用三种不同的筛选条件对不同菌株的性能进行分类：24孔深孔板分批培养、使用FeedBeads®（即甘油随时间缓慢释放）在离心管中进行小规模培养以及微型生物反应器分批培养。在FeedBeads®筛选中表现最佳的两个菌株PpHP8和PpHP18，在生物反应器补料分批培养中使用预先设定的指数增长补料速率进行测试。菌株PpHP18在补料分批培养中以0.712 g L h的速度产生高达37.05 g L的3-HP，最终甘油产物得率为0.194 Cmol。值得注意的是，PpHP18在深孔板和微型生物反应器中的小规模分批培养中并非前两名高产菌株，这突出了多重筛选条件对于充分评估代谢工程策略的重要性。与之前获得的指标相比，这些结果代表产物得率和最终浓度提高了50%，体积生产力提高了30%以上。总体而言，甘油作为碳源与经过代谢工程改造的菌株相结合，产生了迄今为止酵母中报道的最高3-HP浓度和生产力。
