Schumann Conrad, Kugler Amit, Shah Bhavik Ashwin, Berggren Gustav, Land Henrik, Blikstad Cecilia, Stensjö Karin
Department of Chemistry-Ångström Laboratory, Uppsala University, 751 20, Uppsala, Sweden.
Microb Cell Fact. 2025 Apr 23;24(1):93. doi: 10.1186/s12934-025-02708-x.
Isobutene is a promising precursor for jet fuel due to its high energy density and favorable combustion properties. Light-driven bioproduction of isobutene has recently been investigated as an alternative strategy to crude oil refinement or fermentation-based manufacturing processes by harnessing the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the α-ketoisocaproate dioxygenase (RnKICD) from Rattus norvegicus. However, the obtained production level was not sufficient, partially due to the promiscuous activity of RnKICD. The enzyme catalyzes both the reaction with ρ-hydroxyphenylpyruvate (HPP) for homogentisate formation, as well as the reaction with α-ketoisocaproate (KIC), the precursor for isobutene synthesis. Here, to overcome this bottleneck step in the isobutene biosynthesis, protein engineering was employed to improve RnKICD activity and in vivo isobutene production. Purified RnKICD variants were characterized by measuring in vitro KIC and HPP consumption rates, as well as isobutene formation rate. The active site mutations F336V, N363A altered the KIC and HPP consumption rates, while the KIC-to-isobutene conversion ratio was only marginally affected. Besides, the RnKICD variants F336V, N363A and F336V/N363A exhibited a substantially enhanced substrate selectivity for KIC over HPP. Among the examined engineered Synechocystis strains, Syn-F336V showed a 4-fold improvement in isobutene production, compared to the base strain (Syn-RnKICD). Our findings reveal that residues F336 and N363 play a crucial role in substrate interactions, as targeted mutations at these sites shifted the substrate selectivity towards KIC while F336V elevated the in vivo isobutene production levels significantly. We conclude that engineering the active site of RnKICD is a potent tool for improving isobutene bioproduction in Synechocystis.
异丁烯因其高能量密度和良好的燃烧特性，是一种很有前景的喷气燃料前驱体。最近，通过利用单细胞蓝藻集胞藻PCC 6803和来自褐家鼠的α-酮异己酸双加氧酶（RnKICD），对光驱动生物生产异丁烯进行了研究，作为原油精炼或基于发酵的制造工艺的替代策略。然而，所获得的产量水平并不足够，部分原因是RnKICD的混杂活性。该酶既催化与ρ-羟基苯丙酮酸（HPP）反应生成尿黑酸，也催化与异丁烯合成前体α-酮异己酸（KIC）的反应。在此，为克服异丁烯生物合成中的这一瓶颈步骤，采用蛋白质工程来提高RnKICD活性和体内异丁烯产量。通过测量体外KIC和HPP消耗速率以及异丁烯形成速率，对纯化的RnKICD变体进行了表征。活性位点突变F336V、N363A改变了KIC和HPP消耗速率，而异丁烯转化率仅受到轻微影响。此外，RnKICD变体F336V、N363A和F336V/N363A对KIC的底物选择性比对HPP有显著增强。在所检测的工程化集胞藻菌株中，与基础菌株（Syn-RnKICD）相比，Syn-F336V的异丁烯产量提高了4倍。我们的研究结果表明，F336和N363残基在底物相互作用中起关键作用，因为这些位点的靶向突变将底物选择性转向KIC，而F336V显著提高了体内异丁烯产量水平。我们得出结论，对RnKICD活性位点进行工程改造是提高集胞藻中异丁烯生物生产的有效工具。
