Dataset Information

The Safety of Soy Leghemoglobin Protein Preparation Derived from Pichia pastoris Expressing a Soy Leghemoglobin Gene from Glycine max: In Vitro and In Vivo Studies.

ABSTRACT: Soy leghemoglobin (LegH) protein derived from soy (Glycine max) produced in Pichia pastoris (reclassified as Komagataella phaffii) as LegH Prep is a novel food ingredient that provides meat-like flavor and aroma to plant-derived food products. The safety of LegH Prep has been previously assessed in a battery of in vivo and in vitro testing and found no adverse effects under the conditions tested. In this new work, we present the results of new in vivo and in vitro tests evaluating the safety of LegH Prep. LegH Prep was nonmutagenic in a bacterial reverse mutation assay and nonclastogenic in an in vitro micronucleus assay in human lymphocytes. Systemic toxicity was evaluated in the 90 day dietary study in male and female Sprague-Dawley® rats that included a 28 day recovery period. The study resulted in no animal deaths associated with the administration of LegH Prep at the highest dose (90,000 ppm). There were no significant adverse clinical or physical changes attributed to LegH Prep administration, and no observed adverse effects on either male or female rats over the course of the 28 day recovery phase study. The new 90 day dietary toxicity study established a no observed adverse effect level (NOAEL) of 4798.3 and 5761.5 mg/kg/day, the maximum level tested for male and female rats, respectively. Thus, the results of the studies demonstrate that under the conditions tested, LegH Prep is not toxic for consumption in meat analog products.

SUBMITTER: Reyes TF

PROVIDER: S-EPMC10581837 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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The Safety of Soy Leghemoglobin Protein Preparation Derived from Pichia pastoris Expressing a Soy Leghemoglobin Gene from Glycine max: In Vitro and In Vivo Studies.

Reyes Teresa F TF Agrawal Puja P Chan Teresa T Green Richard R Matulka Ray A RA

Journal of toxicology 20231010

Soy leghemoglobin (LegH) protein derived from soy (Glycine max) produced in Pichia pastoris (reclassified as Komagataella phaffii) as LegH Prep is a novel food ingredient that provides meat-like flavor and aroma to plant-derived food products. The safety of LegH Prep has been previously assessed in a battery of in vivo and in vitro testing and found no adverse effects under the conditions tested. In this new work, we present the results of new in vivo an ...[more]

PMID: 37854042

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Project description:The transition metal tungsten (W) finds increasing application in military, aviation and household appliance industry, opening new paths for the heavy metal into the environment. Since tungsten shares certain chemical properties with the essential plant micro nutrient molybdenum, it is proposed to inhibit enzymatic activity of molybdoenzymes by replacing the Mo-ion bound to the co-factor. However, recent studies suggest the inhibition of enzymatic activity might not be the only effect W has on plants and that, much like other heavy metals, tungsten exerts toxicity on its own. Still, our understanding of the mechanisms behind the apparent phytotoxicity remains limited.This study investigates the effects of W on growth, nutrient levels (ionome, ICP-MS), starch levels and nitrogen nutrition (IRMS, enzyme activity assays) as well as root and nodule proteome (LC-MS/MS) of Glycine max cv. Primus. Plants were inoculated with Bradyrhizobium japonicum and grown in a semi hydroponic set up using three different tungsten concentrations (zero, 0.1 mM and 0.5 mM). To identify possible benefits of a functional bacterial symbiosis on W induced stress response, two different environmental growth conditions, one with suppressed N-fixation, supplied with Nitrate (10 mM KNO3) and one solely relying on symbiotic N-fixation (zero KNO3), were applied. Glycine max was able to take up considerable amounts of W (703 ± 136 mg kg-1). However, high W resulted in a strong reduction of shoot biomass of both N regimes and of root biomass (N fed only). Irrespective of N regime, NR activity and total N decreased with increasing W concentrations. Similarly, nitrogenase precursor levels as well as N2-fixation, nodule fixation activity (mg N g-1 nodule dry weight) and nodulation were reduced, indicating that nitrogenase synthesis and activity were negatively affected by W stress. However, along a N fix nodule specific induction of the secondary and phytohormone metabolism, it appears that N2 fixation of remaining nodules was not as severely affected by increasing levels of tungsten than nitrate reduction. Besides N metabolism, plants exhibited an imbalance in nutrient and a failure of carbon metabolic pathways accompanied by an accumulation of starch at high tungsten concentrations, independent of N-regime. Proteomic data demonstrated that the response to high W concentrations is independent of nodule functionality and dominated by several peroxidases and other general stress related proteins. Based on an evaluation of several W responsive proteotypic peptides, we identified a set of protein markers of W stress and possible targets for improved stress tolerance.

Project description:BackgroundCurrently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy.ResultsA scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3'hydroxyibuprofen.ConclusionsFor the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Dataset Information

The Safety of Soy Leghemoglobin Protein Preparation Derived from Pichia pastoris Expressing a Soy Leghemoglobin Gene from Glycine max: In Vitro and In Vivo Studies.

Publications

The Safety of Soy Leghemoglobin Protein Preparation Derived from <i>Pichia pastoris</i> Expressing a Soy Leghemoglobin Gene from <i>Glycine max</i>: <i>In Vitro</i> and <i>In Vivo</i> Studies.

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