Improving the catalytic performance of <i>Pichia pastoris</i> whole-cell biocatalysts by fermentation process

Wang, Denggang; Chen, Meiqi; Xin, Zeng; Li, Wenjie; Liang, Shuli; Lin, Ying

doi:10.1039/d1ra06253k

Cited by 5 publications

(4 citation statements)

References 55 publications

(61 reference statements)

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“…By overexpressing the transcription factor Yap1p to downregulate ROS level, twofold enhanced secretion of trypsinogen was achieved in P. pastoris (Delic et al 2014). It has been reported that overexpression of the HAC1 gene leads to a reduced ROS level in yeast, which is consistent with our results (Wang et al 2021). We also discovered a significant decrease in rhPH-20 secretion after supplementing H 2 O 2, ROS precursor, which might indicate ScHac1p overexpression exerts a protective effect on rhPH-20 secretion by reducing ROS level.…”

Section: Discussionsupporting

confidence: 92%

“…We also discovered a significant decrease in rhPH-20 secretion after supplementing H 2 O 2, ROS precursor, which might indicate ScHac1p overexpression exerts a protective effect on rhPH-20 secretion by reducing ROS level. However, while several studies have also shown that Hac1p overexpression can reduce ROS levels and relieve oxidative stress in P. pastoris (Graf et al 2008;Wang et al 2021), how the ROS level was downregulated by Hac1p overexpression is still unclear. Therefore, future investigations are needed to explore the exact mechanism of ROS reduction in Hac1p-overexpressing yeasts.…”

Section: Discussionmentioning

confidence: 99%

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Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris

Zhang,

Gong,

Jiang

et al. 2024

Appl Microbiol Biotechnol

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Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter P GCW14 . Then, two endoplasmic reticulum-related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. Key points• Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time.• Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion.• A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris

Zhang,

Gong,

Jiang

et al. 2024

Appl Microbiol Biotechnol

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“…Methanol concentration was determined by high performance liquid chromatography [ 16 ]. A 1 mL sample from the fermentation was centrifuged at 6000 rpm for 5 min and the supernatant was filtered through a 0.2 μm filter using a syringe and analyzed on an LC-16 high performance liquid chromatograph (Shimadzu) equipped with a differential refractive index detector (RID-20 A) using a Carbomix H-NP10: 8% column (Sepax Technologies, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol

Wang,

Li,

Zhao

et al. 2024

Microb Cell Fact

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Background Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO2. However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase—an essential enzyme in the dissimilation pathway—displayed growth defects when exposed to a methanol-containing medium. Results Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD+/NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD+/NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4+) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol. Conclusions The reduction of formaldehyde accumulation, the increase of NAD+/NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii.

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“…The tubes containing centrifuged cells were dried at 105 • C to a constant weight to determine the DCW. The relationship between the OD 600 and DCW was represented via empirical Equation (1), OD 600 L −1 = 0.24 g DCW L −1 [54]. The nitrogen content of the homogenized residues was analyzed using the Kjeldahl method [55].…”

Section: Biomass and Crude Protein Analysismentioning

confidence: 99%

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies

Dai,

Dong,

Zhang

et al. 2023

IJMS

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Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process.

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Improving the catalytic performance of Pichia pastoris whole-cell biocatalysts by fermentation process

Abstract: Fermentation process was applied to relieve the substrate transport-limitation of P. pastoris whole-cell biocatalysts, which was much simpler, more energy-saving and greener than c traditional permeabilizing reagent and ultrasonication treatment.

Cited by 5 publications

References 55 publications

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris

Metabolic engineering of Komagataella phaffii for the efficient utilization of methanol

Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies

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