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.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout fry syndrome, and hence this bacterium is placed among the most important salmonid pathogens in the freshwater aquaculture industry. Since bacteria in biofilms differ substantially from free-living counterparts, this study sought to find the main differences in gene expression between sessile and planktonic states of F. psychrophilum LM-02-Fp and NCMB1947^T, with focus on stress-related changes in gene expression occurring during biofilm formation. To this end, biofilm and planktonic samples were analyzed by RNA sequencing to detect differentially expressed candidate genes (DECGs) between the two growth states, and decreasing the effects of interstrain variation by considering only genes with log₂-fold changes ≤ −2 and ≥ 2 at Padj-values ≤ 0.001 as DECGs. Overall, 349 genes accounting for ~15% of total number of genes expressed in transcriptomes of F. psychrophilum LM-02-Fp and NCMB1947^T (n = 2327) were DECGs between biofilm and planktonic states. Approximately 83 and 81% of all up- and down-regulated candidate genes in mature biofilms, respectively, were assigned to at least one gene ontology term; these were primarily associated with the molecular function term “catalytic activity.” We detected a potential stress response in mature biofilms, characterized by a generalized down-regulation of DECGs with roles in the protein synthesis machinery (n = 63, primarily ribosomal proteins) and energy conservation (seven ATP synthase subunit genes), as well as an up-regulation of DECGs involved in DNA repair (ruvC, recO, phrB1, smf, and dnaQ) and oxidative stress response (cytochrome C peroxidase, probable peroxiredoxin, and a probable thioredoxin). These results support the idea of a strategic trade-off between growth-related processes and cell homeostasis to preserve biofilm structure and metabolic functioning. In addition, LDH-based cytotoxicity assays and an intraperitoneal challenge model for rainbow trout fry agreed with the transcriptomic evidence that the ability of F. psychrophilum to form biofilms could contribute to the virulence. Finally, the reported changes in gene expression, as induced by the plankton-to-biofilm transition, represent the first transcriptomic guideline to obtain insights into the F. psychrophilum biofilm lifestyle that could help understand the prevalence of this bacterium in aquaculture settings.

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.