Production of a recombinant protein using Pichia pastoris grown in evaporator condensate from a Kraft pulp mill

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Abstract

The feasibility of cultivating a recombinant strain of the methylotrophic yeast, Pichia pastoris, on combined evaporator condensate from a Kraft mill was investigated. The use of recombinant yeast in this application is novel and the reasoning behind this research is two-fold. Firstly, to facilitate complete methanol removal from a waste stream, rendering it suitable for re-use within the mill or for discharge. Secondly, to generate a product of potential economic value in the pulp and paper industry. A literature review was conducted to generate a list of Pichia transformants expressing potentially useful products. Through a selection process that rated each candidate based on a criteria set, a recombinant Pichia strain expressing lipase from Geotrichum candidum was selected.

A series of shake flask experiments were performed to gauge the effect of various media compositions on yeast growth. This was followed by fed-batch cultivations in a 1.8 L reactor, which was automated via a control program written in LabVIEW (National Instruments, Austin, Texas). Cell densities of 8–12 g/L (dry weight) were reached within the reactor grown on a condensate/methanol feed. Lipase activity was determined titrimetrically and was found to occur only in the presence of yeast peptone.

Introduction

Methanol is a ubiquitous substrate in nature, and is present in many industrial waste streams including those from Kraft pulp mills. The low concentration of methanol in combined Kraft mill effluent precludes its economic use as a dedicated substrate for microbial growth. However, internal process streams, such as combined and foul condensates, contain much higher concentrations of methanol which must be removed prior to re-use to meet methanol emission standards US EPA Cluster rules [1]. Energy intensive steam stripping is currently the method of choice to achieve this end.

Biological treatment of condensate is an alternative means to remove the methanol fraction. Its advantage over steam stripping is a much-reduced energy cost as well as complete removal of the compound, although it is a slower and more complex process to operate. Biological treatment processes are typically run in either aerobic or anaerobic mode with mixed bacterial cultures adapted to methanol consumption [1], [2]. The use of a methylotrophic yeast in this application allows the potential to generate a valuable product.

Over the past two decades, the methylotrophic yeast Pichia pastoris has been developed as a commercial expression system for recombinant proteins. The promoter for the alcohol oxidase gene (AOX1) is exceptionally active, allowing for the production of high concentrations of foreign proteins. Unlike bacterial expression systems, the yeast is capable of exercising post-translational modifications, which can be crucial to the activity of the expressed protein [3]. This active expression system provides the potential for production of valuable products from methanol-bearing streams, such as Kraft mill condensates. The purpose of this study was to determine whether P. pastoris could be grown in condensates, and whether engineered products could be produced in such an industrial media using engineered strains of the yeast.

During the late 1960's, P. pastoris was identified as one of a select group of yeasts that had the ability to metabolize methanol as a sole carbon source [3]. In P. pastoris, methanol metabolism requires the presence of several unique enzymes. First among these is alcohol oxidase (AOX), which catalyzes the primary step in the methanol utilization pathway. Though this enzyme is virtually undetectable when a rich carbon source (i.e. glucose or glycerol) is present in the substrate, it has been observed that AOX is hyper-produced when methanol is introduced in growth-limiting concentrations [4], [5]. The discovery of the presence of a strong AOX promoter was the driving force for developing P. pastoris into an effective expression system.

Expression of the alcohol oxidase enzyme is controlled by two genes, AOX1 and AOX2, with AOX1 contributing to the large majority of enzyme activity [3], [5]. In turn, regulation of these genes is performed on the level of transcription through a joint repression/derepression and induction mechanism. As suggested above, the presence of rich carbon sources favors a metabolic pathway, which does not result in the synthesis of key enzymes required for methanol consumption, thus transcription of AOX1 (and AOX2) is repressed. Upon depletion or removal of these carbon sources, transcription of the alcohol oxidase genes may still not commence until induced by the presence of methanol.

A large volume of work has been done over the years in investigating the ability of P. pastoris to express a multitude of products. For this study, it was preferable to select transformed yeast yielding a product with potential value for the pulp and paper industry.

A literature review (Table 1) was conducted and a number of prospective strains were short listed and evaluated based on a set of criteria. The criteria set included: quality and level of expressed protein, ease of the enzyme assay, type of equipment required for the assay, ability to express extracellularly and potential applicability to the pulp and paper industry. Ranking of the candidate transformants was done via a points system, where each recombinant was assigned a score out of a maximum value for each criteria. The Pichia strain with the highest total points was judged the best overall candidate for the study. Based on this procedure, the Pichia strain expressing a lipase from Geotrichum candidum was chosen [8].

The selected Pichia transformant contained two G. candidum lipase isoenzymes (GCL) genes, both of which were highly expressed (to 60 mg/L protein) virtually free from contaminating proteins [8]. Lipase activity assays further demonstrated the expressed product to be fully active. Although Holmquist et al. [8] isolated and analyzed the two lipase enzymes (GCL I and GCL II) independently, the strain obtained for this study exclusively expressed the GCL I enzyme only.

Section snippets

Origin of yeast strains

The P. pastoris wild-type strain was obtained from the Northern Regional Research Laboratory (NRRL-1603), Peoria, Illinios. The G. candidum recombinant Pichia strain (GS115 (his4)) was obtained from M. Holmquist and D.C. Tessier of the Biotechnology Research Institute, National Research Council of Canada, Montreal, Que.

Culture media

Yeast cultures were grown in rich media or defined media made up in either distilled water or combined evaporator condensate obtained from a local Kraft pulp mill (Western Pulp,

Effect of media composition on shake flask growth of wild-type P. pastoris

Shake flask experiments were performed to observe the influence of different variables on growth of the yeasts. One of the first objectives of the preliminary trials was to attempt P. pastoris cultivation in a condensate-based medium. A majority of the work involving this yeast had been done in ‘clean’ media supplemented with rich nutrients. This is understandably the environment of choice for maximizing protein expression. However, given the scope of this project, it was desired to observe the

Conclusions

The growth of a recombinant strain of P. pastoris bearing lipase from G. candidum was successfully carried out in various media compositions. Yeast growth in combined evaporator condensate was strongly inhibited, an inhibition that could be overcome by air-stripping. Yeast peptone was required for expression of lipase. In scaling up to fed-batch reactor cultures, a sophisticated control system was created that automated methanol feeding based on measuring and maintaining the dissolved oxygen

Acknowledgements

The authors would like to acknowledge Mats Holmquist and Daniel C. Tessier for their gracious donation of the Geotrichum recombinant Pichia strain, which allowed this study to commence. Thanks as well to Norman Woo, Jason Prichett and Dr. Susan Baldwin for their aid and insight during the early stages of this project. The authors gratefully acknowledge NSERC, which funded the work through their Discovery Grants program.

A special thanks goes to Mr. Karam S. Manhas and Shalimar Engineering Ltd.,

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