Vikromvarasiri Nunthaphan, Noda Shuhei, Shirai Tomokazu, Kondo Akihiko
RIKEN Center for Sustainable Resource Science, 1‑7‑22 Suehiro‑cho, Tsurumi‑ku, Yokohama, Kanagawa, 230‑0045, Japan.
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
J Biol Eng. 2023 Jan 10;17(1):3. doi: 10.1186/s13036-022-00320-w.
Flux Balance Analysis (FBA) is a well-known bioinformatics tool for metabolic engineering design. Previously, we have successfully used single-level FBA to design metabolic fluxes in Bacillus subtilis to enhance (R,R)-2,3-butanediol (2,3-BD) production from glycerol. OptKnock is another powerful technique for devising gene deletion strategies to maximize microbial growth coupling with improved biochemical production. It has never been used in B. subtilis. In this study, we aimed to compare the use of single-level FBA and OptKnock for designing enhanced 2,3-BD production from glycerol in B. subtilis.
Single-level FBA and OptKnock were used to design metabolic engineering approaches for B. subtilis to enhance 2,3-BD production from glycerol. Single-level FBA indicated that deletion of ackA, pta, lctE, and mmgA would improve the production of 2,3-BD from glycerol, while OptKnock simulation suggested the deletion of ackA, pta, mmgA, and zwf. Consequently, strains LM01 (single-level FBA-based) and MZ02 (OptKnock-based) were constructed, and their capacity to produce 2,3-BD from glycerol was investigated. The deletion of multiple genes did not negatively affect strain growth and glycerol utilization. The highest 2,3-BD production was detected in strain LM01. Strain MZ02 produced 2,3-BD at a similar level as the wild type, indicating that the OptKnock prediction was erroneous. Two-step FBA was performed to examine the reason for the erroneous OptKnock prediction. Interestingly, we newly found that zwf gene deletion in strain MZ02 improved lactate production, which has never been reported to date. The predictions of single-level FBA for strain MZ02 were in line with experimental findings.
We showed that single-level FBA is an effective approach for metabolic design and manipulation to enhance 2,3-BD production from glycerol in B. subtilis. Further, while this approach predicted the phenotypes of generated strains with high precision, OptKnock prediction was not accurate. We suggest that OptKnock modelling predictions be evaluated by using single-level FBA to ensure the accuracy of metabolic pathway design. Furthermore, the zwf gene knockout resulted in the change of metabolic fluxes to enhance the lactate productivity.
通量平衡分析（FBA）是一种用于代谢工程设计的著名生物信息学工具。此前，我们已成功使用单级FBA设计枯草芽孢杆菌中的代谢通量，以提高甘油生产（R，R）-2,3-丁二醇（2,3-BD）的产量。OptKnock是另一种用于设计基因删除策略以最大化微生物生长并提高生化产物产量的强大技术。它从未在枯草芽孢杆菌中使用过。在本研究中，我们旨在比较单级FBA和OptKnock在设计提高枯草芽孢杆菌中甘油生产2,3-BD方面的应用。
使用单级FBA和OptKnock设计枯草芽孢杆菌的代谢工程方法以提高甘油生产2,3-BD的产量。单级FBA表明，删除ackA、pta、lctE和mmgA将提高甘油生产2,3-BD的产量，而OptKnock模拟表明应删除ackA、pta、mmgA和zwf。因此，构建了菌株LM01（基于单级FBA）和MZ02（基于OptKnock），并研究了它们从甘油生产2,3-BD的能力。多个基因的删除对菌株生长和甘油利用没有负面影响。在菌株LM01中检测到最高水平的2,3-BD产量。菌株MZ02产生的2,3-BD水平与野生型相似，表明OptKnock预测有误。进行了两步FBA以检查OptKnock预测错误的原因。有趣的是，我们新发现菌株MZ02中zwf基因的删除提高了乳酸产量，这在迄今为止尚未见报道。菌株MZ02的单级FBA预测与实验结果一致。
我们表明，单级FBA是一种有效的代谢设计和操作方法，可提高枯草芽孢杆菌中甘油生产2,3-BD的产量。此外，虽然这种方法能够高精度地预测所构建菌株的表型，但OptKnock预测并不准确。我们建议通过使用单级FBA评估OptKnock建模预测，以确保代谢途径设计的准确性。此外，zwf基因敲除导致代谢通量变化，提高了乳酸生产率。
