The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

/pdf/heterologous-protein-production-using-the-pichia-pastoris-22lqcx2i7v.pdf

Heterologous protein production using the Pichia pastoris expression system.

The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.

Recombinant protein expression in Pichia pastoris.

Production of recombinant proteins by microbes and higher organisms.

Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

https://link.springer.com/content/pdf/10.1007%2Fs00253-014-5732-5.pdf

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.

Microbial enzymes are of great importance in the development of industrial bioprocesses. Current applications are focused on many different markets including pulp and paper, leather, detergents and textiles, pharmaceuticals, chemical, food and beverages, biofuels, animal feed and personal care, among others. Today there is a need for new, improved or/and more versatile enzymes in order to develop more novel, sustainable and economically competitive production processes. Microbial diversity and modern molecular techniques, such as metagenomics and genomics, are being used to discover new microbial enzymes whose catalytic properties can be improved/modified by different strategies based on rational, semi-rational and random directed evolution. Most industrial enzymes are recombinant forms produced in bacteria and fungi.

https://www.mdpi.com/2218-273X/4/1/117/pdf

Microbial Enzymes: Tools for Biotechnological Processes

Recombinant non-hydroxylated gelatins based on mouse type I and rat type III collagen sequences were secreted from the methylotrophic yeast Pichia pastoris, using the Saccharomyces cerevisiae alpha-mating factor prepro signal. Proteolytic degradation could be minimized to a large extent by performing fermentations at pH 3.0 and by adding casamino acids to the medium, even though gelatin is extremely susceptible to proteolysis due to its open, unfolded structure. Proteolytic cleavage at specific mono-arginylic sites, by a putative Kex2-like protease, could be successfully abolished by site-directed mutagenesis of these sites. Production levels as high as 14.8 g/l clarified both were obtained, using multicopy tranformants. To our knowledge, this represents the highest level of heterologous protein secretion reported to date for P. pastoris.

High-yield secretion of recombinant gelatins by Pichia pastoris.

A tabular silver halide emulsion wherein the tabular grains account for more than 75% of the total grain projected area said emulsion comprising silver halide grains nucleated in the presence of nucleation peptizer and thereafter grown in the presence of growth peptizer, wherein at least one of the peptizers is substantially pure collagen like material prepared by genetic engineering, said peptizer having an amino acid sequence comprising more than 4 different amino acids. A process of preparing the AgX emulsion. A process of producing recombinant collagen like polypeptide comprising expression of a collagen like polypeptide encoding nucleic acid sequence by a microorganism to a degree exceeding 0.95 grammes/liter, said recombinant collagen being free of helix structure and preferably the expression occurring in a microorganism other than E. coli or Saccharomyces cerevisiae.

Silver halide emulsions with recombinant collagen suitable for photographic application and also the preparation thereof

A composition comprising a purified collagen-like polypeptide suitable as a peptizer, said polypeptide comprising [Gly-X-Y]n repeats, wherein Gly stands for glycine, X and Y represent any amino acid and n is an integer and selected such that the length of the [Gly-X-Y]n repeat is at least 2.5 kDa and wherein the amino acid sequence of said [Gly-X-Y]n repeats comprises more than 4 different amino acids and wherein said purified polypeptide is free of helix structure.

Method for recombinant microorganism expression and isolation of collagen-like polypeptides

The invention provides oil-in-water emulsions comprising recombinant collagen-like polymer in an amount sufficient to act as stabiliser of the emulsion. The polymer is especially a polypeptide which is free of helix structure, has an isoelectric point at least 0.5 pH units removed from the pH of the oil-in-water emulsion. Furthermore, amphiphilic recombinant collagen-like polymers are provided for use in oil-in-water emulsions. The amphiphilic polymers are polar at one end as a result of a relative abundance of polar amino acids, and apolar at the other end as a result of a relative abundance of apolar amino acids,

Oil-in-water emulsions stabilised with recombinant collagen-like material

PROBLEM TO BE SOLVED: To provide a low-cost high quality gelatin to be used for the production of a silver halide emulsion by using a recombination DNA technique. SOLUTION: This silver halide emulsion and the preparation of the AgX emulsion have the following features. The emulsion is a planer silver halide emulsion in which planer particles possess >75% of the projected area of the whole particles. The emulsion contains silver halide particles which are produced as nuclei in the presence of a nuclear forming peptizer and grown in the presence of a growing peptizer. The peptizer is a substantially pure collagen material prepared by genetic engineering, and the peptizer has an amino acid sequence having more than four different amino acids. By the producing method of the recombinant collagen polypeptide, the expression of the nucleic acid sequence which codes collagen polypeptide is obtd. by microorganisms to a degree over 0.95 g/l, the recombinant collagen has no helical structure, and the expression is obtd. in microorganisms except for Escherichia coli or saccharomyces cerevisiae.

Richele Deodata Wind

Papers

High-yield secretion of recombinant gelatins by Pichia pastoris.

Silver halide emulsions with recombinant collagen suitable for photographic application and also the preparation thereof

Method for recombinant microorganism expression and isolation of collagen-like polypeptides

Oil-in-water emulsions stabilised with recombinant collagen-like material

Silver halide emulsion having recombinant collagen suitable for photographic use and its preparation