背景:整合血红素的过氧化酶负责多种生物体中的电子传输。然而,由于它们具有挑战性的重组生产,它们在生物催化中的应用受到阻碍。先前的研究表明,Komagataellaphaffi是含血红素酶的合适生产宿主。此外,辅助蛋白质的共表达已被证明有助于酵母中的蛋白质折叠。为了促进非特异性过氧化物酶(AnoUPO)的重组蛋白表达,我们的目标是将双向表达策略应用于Komagataellaphafii。
结果:在初始筛查中,发现蛋白质二硫键异构酶的共表达有助于K.phaffi中表达的非特异性过氧化物酶的正确折叠。筛选了多种不同的双向启动子组合。将具有最有希望的启动子组合的克隆扩大到生物反应器培养,并与单向构建体(仅表达过氧化物酶)进行比较。在动态物质中筛选了菌株的目标酶生产率,研究诱导的去抑制和混合进料(甲醇-甘油)。生物反应器筛选的设定值,导致最高的过氧化物酶生产率,对于去阻抑和基于甲醇的诱导,我们选择进行专门的过氧合酶生产运行,并用RT-qPCR进行分析。结果表明,在细胞特异性酶生产率方面,无甲醇培养优于混合饲喂。RT-qPCR分析证实,混合饲喂导致宿主细胞的高应激,阻碍高生产率。此外,双向化的构建体导致比单向表达系统高得多的比酶活性。
结论:在这项研究中,我们展示了一种无甲醇的生物反应器生产策略,用于非特异性过氧化物酶,但没有在文献中显示。因此,K.phafii的双向辅助蛋白表达,在抑制条件下种植,表明是含血红素的氧化还原酶的有效生产策略。这种生产策略可能会为生物催化开辟更多机会。
BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii.
RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (
methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and
methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that
methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system.
CONCLUSIONS: In this study, we demonstrate a
methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.