Àvila-Cabré Sílvia, Albiol Joan, Ferrer Pau
Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Barcelona, Catalonia, Spain.
J Biol Eng. 2025 Feb 20;19(1):19. doi: 10.1186/s13036-025-00488-x.
Bioconversion of methanol derived from CO reduction into value-added chemicals provides a unique approach for mitigating global warming and reducing fossil fuels dependence. Production of 3-hydroxypropionic acid (3-HP), a key building block for the development of biobased products such as acrylates and 1,3-propanediol, has been successfully achieved using methanol as the sole carbon and energy source in the methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris). However, challenges remain in meeting commercially relevant concentrations, yields and productivities of 3-HP, prompting further strain optimization. In the present study, we have combined metabolic engineering strategies aiming at increasing metabolic precursors supply and redirecting carbon flux towards 3-HP production.
A combinatorial metabolic engineering strategy targeting precursors supply and 3-HP export was applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a mutated NADP-dependent formate dehydrogenase from Pseudomonas sp. 101 (PseFDH(V9)). To do so, several genes encoding enzymes catalyzing reactions immediately upstream of the β-alanine pathway were overexpressed to enhance precursors availability. However, only the overexpression of the pyruvate carboxylase PYC2 gene significantly increased the 3-HP yield on biomass (Y) in small-scale cultivations. Co-overexpression of PYC2 and the lactate permeases ESBP6 and JEN1 genes led to a 55% improvement in 3-HP titer and product yield in methanol deep-well plate cultures compared to the reference strain, mostly due to Esbp6 activity, proving its effectiveness as a 3-HP transporter. Deletion of the native formate dehydrogenase gene FDH1 did not increase methanol flux entering the assimilatory pathway. Instead, knockout strains showed severe growth defects due to toxic intermediates accumulation. Co-expression of the PseFDH(V9) encoding gene in these strains failed to compensate for the loss of the native FDH. The strain combining PYC2, ESBP6, and JEN1 overexpression was further tested in fed-batch cultures at pH 5, achieving a 3-HP concentration of 27.0 g l, with a product yield of 0.19 g g, and a volumetric productivity of 0.56 g l h for the methanol feeding phase of the cultivations. These results represent a 42% increase in final concentration and over 20% improvement in volumetric productivity compared to the original 3-HP-producing strain. Furthermore, bioreactor-scale cultivations at pH 3.5 revealed increased robustness of the strains overexpressing monocarboxylate transporters.
Our results point out the potential of lactate transporters to efficiently drive 3-HP export in K. phaffii, leading to higher titers, yields, and productivities, even at lower pH conditions.
将一氧化碳还原产生的甲醇生物转化为高附加值化学品，为缓解全球变暖及减少对化石燃料的依赖提供了一种独特方法。3-羟基丙酸（3-HP）是用于生产生物基产品（如丙烯酸酯和1,3-丙二醇）的关键组成部分，利用甲醇作为甲基营养型酵母毕赤酵母（同义词：巴斯德毕赤酵母）的唯一碳源和能源，已成功实现了3-HP的生产。然而，要达到与商业相关的3-HP浓度、产量和生产率仍面临挑战，这促使进一步优化菌株。在本研究中，我们结合了代谢工程策略，旨在增加代谢前体供应并将碳通量重定向至3-HP生产。
针对前体供应和3-HP输出的组合代谢工程策略应用于原始的3-HP生产毕赤酵母菌株，该菌株含有合成β-丙氨酸途径和来自假单胞菌属101的突变型NADP依赖性甲酸脱氢酶（PseFDH(V9)）。为此，过表达了几个编码催化β-丙氨酸途径上游紧邻反应的酶的基因，以提高前体的可用性。然而，在小规模培养中，只有丙酮酸羧化酶PYC2基因的过表达显著提高了基于生物量的3-HP产量（Y）。与参考菌株相比，甲醇深孔板培养中PYC2与乳酸通透酶ESBP6和JEN1基因的共过表达使3-HP滴度和产物产量提高了55%，这主要归因于Esbp6的活性，证明了其作为3-HP转运蛋白的有效性。天然甲酸脱氢酶基因FDH1的缺失并未增加进入同化途径的甲醇通量。相反，敲除菌株由于有毒中间体的积累而表现出严重的生长缺陷。在这些菌株中共表达PseFDH(V9)编码基因未能弥补天然FDH的损失。在pH 5的补料分批培养中进一步测试了过表达PYC2、ESBP6和JEN1的菌株，在培养的甲醇补料阶段实现了3-HP浓度为27.0 g/L，产物产量为0.19 g/g，体积生产率为0.56 g/L·h。与原始的3-HP生产菌株相比，这些结果表明最终浓度提高了42%，体积生产率提高了20%以上。此外，在pH 3.5的生物反应器规模培养中发现，过表达单羧酸转运蛋白的菌株具有更高的稳健性。
我们的结果指出了乳酸转运蛋白在毕赤酵母中有效驱动3-HP输出的潜力，即使在较低pH条件下也能实现更高的滴度、产量和生产率。
