Macroscopic modeling of the growth and substrate consumption of wild type and genetically modified Pichia pastoris.

Pichia pastoris is a popular yeast platform to generate several industrially relevant products which have applications in a wide range of sectors. The complexities in the processes due to the addition of a foreign gene are not widely explored. Since these complexities can be dependent on the strain characteristics, promoter, and type of protein produced, it is vital to investigate the growth and substrate consumption patterns of the host to facilitate customized process optimization. In this study, the growth rates of P. pastoris GS115 wild type (WT) and genetically modified (GM) strains grown on glycerol and methanol in batch cultivation mode were estimated and the model providing the best representation of the true growth kinetics based on substrate consumption was identified. It was observed that the growth of P. pastoris exhibits Haldane kinetics on glycerol rather than the most commonly used Monod kinetics due to the inability of the latter to describe growth inhibition at high concentrations of glycerol. Whereas, the cardinal parameter model, a newly proposed model for this application, was found to be the best fitting to describe the growth of P. pastoris on methanol due to its ability to describe methanol toxicity. Interestingly, the findings from this study concluded that in both substrates, the genetically engineered strain exhibited a higher growth rate compared to the WT strain. Such an observation has not been established yet in other published works, indicating an opportunity to further optimize the carbon source feeding strategies when the host is grown in fed-batch mode.

Processing Method for the Quantification of Methanol and Ethanol from Bioreactor Samples Using Gas Chromatography-Flame Ionization Detection.

Methanol, a simple polar solvent, has been widely identified as an attractive carbon source to produce chemicals and fuels in bioprocesses. Specifically, to achieve recombinant protein production from methylotrophic yeasts, such as Pichia pastoris, this organic solvent can be used as a sole carbon source for growth and maintenance as well as an inducer for protein expression. However, if methanol feeding is not controlled well in such a fermentation process, accumulation of the solvent in the growth media will have a detrimental effect on the cells. Hence, monitoring the levels of methanol in these fermentation processes is a crucial step to ensure a healthy culture and maximum protein production. There are various techniques elaborated in the literature for monitoring methanol in cell cultures, but often, they appear to be expensive methods that are less affordable for many laboratories. This is because, in addition to the sophisticated equipment that is required for the analysis, the complexity of the samples retrieved from the bioprocesses necessitates laborious processing steps often involving expensive tools. In this study, a fast, simple, and sensitive method is developed to process biological samples by using the salting-out-assisted liquid-liquid extraction technique to quantify the concentration of methanol and ethanol using gas chromatography. On comparing the combinations of widely available salts and solvents, it was noticed that salting out using potassium carbonate followed by the liquid-liquid extraction of the analyte using ethyl acetate showed the best recovery. Followed by this, a validation test for the developed method was performed, which resulted in good peak resolution, linearity, and limit of detection for the quantitation of methanol and ethanol. By further assessing the tested combination, it was confirmed that its application could be extended to other matrices. Such an approach facilitates the possibility to monitor and control the methanol levels in fermentation and aids in bioprocess optimization.

Effects of Temperature and pH on Recombinant Thaumatin II Production by Pichia pastoris.

The sweet protein thaumatin is emerging as a promising sugar replacer in the market today, especially in the food and beverage sector. Rising demand for its production necessitates the large-scale extraction of this protein from its natural plant source, which can be limited in terms of raw material availability and production costs. Using a recombinant production technique via a yeast platform, specifically, Pichia pastoris, is more promising to achieve the product economically while maintaining batch-to-batch consistency. However, the bioproduction of recombinant proteins requires the identification of optimal process variables, constituting the maximal yield of the product of interest. These variables have a direct effect on the growth of the host organism and the secretion levels of the recombinant protein. In this study, two important environmental factors, pH, and temperature were assessed by cultivating P. pastoris in shake flasks to understand their influence on growth and the production levels of thaumatin II protein. The results from the pH study indicate that P. pastoris attained a higher viable cell density and secretion of protein at pH 6.0 compared to 5.0 when grown at 30 °C. Furthermore, within the three levels of temperatures investigated when grown at pH 6.0, the protein levels were the highest at 30 °C compared to 20 and 25 °C, whereas 25 °C exhibited the highest viable cell density. Interestingly, the trend observed from the qualitative effects of temperature and pH occurred in all the media that was investigated. These results broaden our understanding of how pH and temperature adjustment during P. pastoris cultivation aid in enhancing the production yields of thaumatin II prior to optimising the fed batch bioreactor operation.

Bioproduction of the Recombinant Sweet Protein Thaumatin: Current State of the Art and Perspectives.

There is currently a worldwide trend to reduce sugar consumption. This trend is mostly met by the use of artificial non-nutritive sweeteners. However, these sweeteners have also been proven to have adverse health effects such as dizziness, headaches, gastrointestinal issues, and mood changes for aspartame. One of the solutions lies in the commercialization of sweet proteins, which are not associated with adverse health effects. Of these proteins, thaumatin is one of the most studied and most promising alternatives for sugars and artificial sweeteners. Since the natural production of these proteins is often too expensive, biochemical production methods are currently under investigation. With these methods, recombinant DNA technology is used for the production of sweet proteins in a host organism. The most promising host known today is the methylotrophic yeast, Pichia pastoris. This yeast has a tightly regulated methanol-induced promotor, allowing a good control over the recombinant protein production. Great efforts have been undertaken for improving the yields and purities of thaumatin productions, but a further optimization is still desired. This review focuses on (i) the motivation for using and producing sweet proteins, (ii) the properties and history of thaumatin, (iii) the production of recombinant sweet proteins, and (iv) future possibilities for process optimization based on a systems biology approach.