Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a ‘real case’, i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in PAOX1-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new PAOX1 synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.

The current interest in microalgae as a sustainable source of next generation biofuels and other valuable substances is driving exploration of their use as unique biotechnological production systems. To design and optimise appropriate production strategies, the behaviour of particular microalgal species should be well characterised under different culture conditions. Thus, flow cytometric (FCM) methods, which are already well established in environmental and toxicological studies of microalgae, are also useful for analysing the physiological state of microalgae, and have the potential to contribute to the rapid development of feasible bioprocesses. These methods are commonly based on the examination of intrinsic features of individual cells within a population (such as autofluorescence or size). Cells possessing the desired physiological or morphological features, which are detectable with or without fluorescent staining, are counted or isolated (sorted) using an FCM device. The options for implementation of FCM in the development of biotechnological processes detailed in this review are (i) analysing the chemical composition of biomass, (ii) monitoring cellular enzyme activity and cell viability, and (iii) sorting cells to isolate those overproducing the target compound or for the preparation of axenic cultures.

Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.

Microalgae of numerous heterotrophic genera (obligate or facultative) exhibit considerable metabolic versatility and flexibility but are currently underexploited in the biotechnological manufacturing of known plant-derived compounds, novel high-value biomolecules or enriched biomass. Highly efficient production of microalgal biomass without the need for light is now feasible in inexpensive, well-defined mineral medium, typically supplemented with glucose. Cell densities of more than 100 g l−1 cell dry weight have been achieved with Chlorella, Crypthecodinium and Galdieria species while controlling the addition of organic sources of carbon and energy in fedbatch mode. The ability of microalgae to adapt their metabolism to varying culture conditions provides opportunities to modify, control and thereby maximise the formation of targeted compounds with non-recombinant microalgae. This review outlines the critical aspects of cultivation technology and current best practices in the heterotrophic high-cell-density cultivation of microalgae. The primary topics include (1) the characteristics of microalgae that make them suitable for heterotrophic cultivation, (2) the appropriate chemical composition of mineral growth media, (3) the different strategies for fedbatch cultivations and (4) the principles behind the customisation of biomass composition. The review confirms that, although fundamental knowledge is now available, the development of efficient, economically feasible large-scale bioprocesses remains an obstacle to the commercialisation of this promising technology.

No abstract

Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.

No abstract