Strategies for Continuous Processing in Microbial Systems

Kopp, Julian; Slouka, Christoph; Delvigne, Frank; Herwig, Christoph

doi:10.1002/9783527827343.ch1

Cited by 1 publication

(2 citation statements)

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“…While P GAP and P UPP (commercially available variant of the K. phaffii P GCW14 ) are referred to as strong constitutive regulatory elements, P HHT1 is considered a medium to intermediate constitutive strength promoter. Importantly, constitutive recombinant protein production does not allow for a separation of the process in dedicated growth-and production-phases, resulting in decreasing biomass yields and, thus, lower volumetric target protein expression [28][29][30]. To avoid the inability of switching the induction mechanism on and off, derepressive activated promoters are a sustainable alternative to methanol-based induction.…”

Section: Introductionmentioning

confidence: 99%

“…Still, methanol is a toxic reductant with a high heat of combustion, causing high oxygen consumption [35] and being lethal to K. phaffii cells at concentrations between 2 and 5% v/v [36]. Hence, implementation, of methanol-based induction mechanisms on an industrial scale demands complex infrastructure, such as an explosion-safe environment and strong cooling facilities to reduce the heat caused by methanol consumption [30]. Even though the cooling issue can be circumvented by utilizing a Mut S strain, methanol-based induction triggers the misincorporation of O-methyl-L-homoserine instead of methionine in Mut S strains, demanding different induction mechanisms [37][38][39].…”

Section: Introductionmentioning

confidence: 99%

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Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii

Besleaga,

Zimmermann,

Ebner

et al. 2024

Microb Cell Fact

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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.

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Section: Introductionmentioning

confidence: 99%