Herein, autotrophic metabolism of Cupriavidus necator H16 growing on CO₂, H₂ and O₂ gas mixture was analyzed by metabolic pathway analysis tools, specifically elementary mode analysis (EMA) and flux balance analysis (FBA). As case studies, recombinant strains of C. necator H16 for the production of short-chain (isobutanol) and long-chain (hexadecanol) alcohols were constructed and examined by a combined tools of EMA and FBA to comprehensively identify the cell’s metabolic flux profiles and its phenotypic spaces for the autotrophic production of recombinant products. The effect of genetic perturbations via gene deletion and overexpression on phenotypic space of the organism was simulated to improve strain performance for efficient bioconversion of CO₂ to products at high yield and high productivity. EMA identified multiple gene deletion together with controlling gas input composition to limit phenotypic space and push metabolic fluxes towards high product yield, while FBA identified target gene overexpression to debottleneck rate-limiting fluxes, hence pulling more fluxes to enhance production rate of the products. A combination of gene deletion and overexpression resulted in designed mutant strains with a predicted yield of 0.21–0.42 g/g for isobutanol and 0.20–0.34 g/g for hexadecanol from CO₂. The in silico-designed mutants were also predicted to show high productivity of up to 38.4 mmol/cell-h for isobutanol and 9.1 mmol/cell-h for hexadecanol under autotrophic cultivation. The metabolic modeling and analysis presented in this study could potentially serve as a valuable guidance for future metabolic engineering of C. necator H16 for an efficient CO₂-to-biofuels conversion.

中文翻译：

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用于高效自养高级生物燃料的计算机设计的代谢途径分析

在此，通过代谢途径分析工具，特别是基本模式分析（EMA）和通量平衡分析（FBA），分析了在CO ₂，H ₂和O ₂气体混合物上生长的Cupriavidus necator H16的自养代谢。作为案例研究，C。necator的重组菌株使用EMA和FBA的组合工具构建并检查了用于生产短链（异丁醇）和长链（十六醇）的H16，以全面鉴定细胞的代谢通量谱及其表型空间，以自养性生产重组产物。模拟了通过基因缺失和过度表达引起的遗传扰动对生物表型空间的影响，从而改善了菌株性能，从而实现了有效的CO ₂生物转化。以高产量和高生产率生产产品。EMA确定了多个基因缺失以及控制气体输入成分以限制表型空间并将代谢通量推向高产品产量，而FBA确定了目标基因的过表达达到了瓶颈速率限制通量，因此拉动了更多通量以提高产品的生产率。基因缺失和过表达的结合导致了设计突变株的产生，CO ₂预测的异丁醇产量为0.21-0.42 g / g，十六烷醇的产量为0.20-0.34 g / g。。在自养栽培中，还预测了计算机设计的突变体在异丁醇中显示出高达38.4 mmol / cell-h的效率，在十六烷醇中显示出高达9.1 mmol / cell-h的高生产率。这项研究中提出的代谢建模和分析可能会为C. necator H16的未来代谢工程学提供有价值的指导，以实现从CO ₂到生物燃料的有效转化。