Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered.

The yeast Komagataella phaffii (formerly called Pichia pastoris) is used widely as a host for secretion of heterologous proteins, but only a few isolates of this species exist and all the commonly used expression systems are derived from a single genetic background, CBS7435 (NRRL Y-11430). We hypothesized that other genetic backgrounds could harbor variants that affect yields of secreted proteins. We crossed CBS7435 with 2 other K. phaffii isolates and mapped quantitative trait loci (QTLs) for secretion of a heterologous protein, β-glucosidase, by sequencing individual segregant genomes. A major QTL mapped to a frameshift mutation in the mannosyltransferase gene HOC1, which gives CBS7435 a weaker cell wall and higher protein secretion than the other isolates. Inactivation of HOC1 in the other isolates doubled β-glucosidase secretion. A second QTL mapped to an amino acid substitution in IRA1 that tripled β-glucosidase secretion in 1-week batch cultures but reduced cell viability, and its effects are specific to this heterologous protein. Our results demonstrate that QTL analysis is a powerful method for dissecting the basis of biotechnological traits in nonconventional yeasts, and a route to improving their industrial performance.

Chemical amination of the enzyme was demonstrated to favor immobilization onto polydopamine (PDA)-coated magnetic nanoparticles (MNPs) for the first time, to the best of the author’s knowledge. MNPs prepared via hydrothermal synthesis were coated with PDA for the immobilization of naringinase. X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy showed that the MNPs were composed mainly of Fe₃O₄ with an average size of 38.9 nm, and coated with a 15.1 nm PDA layer. Although the specific activities of α-L-rhamnosidase (RAM) and β-D-glucosidase (GLU) of free naringinase decreased with amination, the immobilization yields of the aminated enzyme increased by more than 40% for RAM and more than 10-fold for GLU. The immobilization improved the enzyme’s thermal stability (at 50 °C), reaching a half-life of 40.7 and 23.1 h for RAM and GLU activities, respectively. The biocatalyst was successfully used for the debittering of grapefruit juice, detecting a reduction in naringin of 56% after 24 h. These results demonstrate that the enzyme amination is an effective strategy to enhance the immobilization on a PDA coating and could be applied to other enzymes in order to obtain an easily recoverable biocatalyst using a simple immobilization methodology.

This research characterized a novel Komagataella phaffii strain generated through intraspecific crossing between a wild isolate and a laboratory strain. This segregant, called S467, expressed 2.2-fold more secreted recombinant β-glucosidase than its parental strains in microtiter scale, which suggested that S467 could be an attractive host for bioprocess optimization. S467 was grown alongside the laboratory strain CBS7435 expressing β-glucosidase (CBS_BGL9), as a control, in a 1.5 L bioreactor to determine kinetics parameters, and similar cell growth rate (0.12 h⁻¹) but higher recombinant protein activity, measured as enzymatic activity, was observed in S467. The effect of specific cell growth rate was studied using continuous cultures (chemostat) at different dilution rates, identifying conditions that provided up to a twofold increase in enzymatic activity in S467. RT-qPCR was conducted on key genes associated with the genetic background of S467, in order to clarify differences at the transcriptomic level that render S467 as a potential superior host for recombinant protein production. Overall, this study provides quantitative evidence of the positive effect of the natural isolate IRA1 allele for the generation of recombinant β-glucosidase and highlights the usability of natural genetic diversity in K. phaffii.

Cultivation under hypothermia has emerged as an interesting strategy for enhancing recombinant protein production in Komagataella phaffii, yet predictive a