Heterologous protein production in Kluyveromyces lactis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biotechnology and Bioprocess Engineering at Massey University, Palmerston North, New Zealand

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In this study, the recombinant yeast Kluyveromyces lactis CBS 683 : pCR1 was investigated as a model system for the production of a heterologous protein in a whey-based medium. The plasmid pCR1 has been constructed to express a wheat α-amylase enzyme in K lactis strains. The construct is based on the vector pCXJ-kan1, which has been derived from pKD1, a native plasmid of K lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Construct pCR1 produces an α-amylase from DNA isolated from a wheat cDNA clone which is controlled by a Saccharomyces cerevisiae PG K promoter. An electroporation method using a Bio-Rad Gene Pulser has been optimized for introducing heterologous DNA into K lactis yeasts. Selection of transform ants can be made using either the biosynthetic marker URAA or the G418 resistance gene, depending on whether the yeast is an auxotrophic mutant or a wild-type strain, respectively. Transformation was optimal at 4500 V cm-1, 25 J.LF, and oo n with 0.2 J.Lg plasmid DNA. Transformation efficiencies were comparable to those obtained using a PS1 0 Electropulsator, and were in the range 104-105 transformants per 107 cells per J.Lg DNA. Twenty-nine Kluyveromyces strains were examined for efficiency of transformation and fermentation performance on rich glucose and rich lactose media under high and low aeration in batch culture. Of these, K. lactis CBS 141 and CBS 683 were chosen for recombinant studies. The transformed yeasts K lactis CBS 141 : pCR1 and CBS 683 : pCR1 were qualitatively shown to produce an active α-amylase enzyme. The α-amylase was produced at a low level but could be measured using a modified starch-iodine assay. A typical yield of 6 U mr1 was obtained for batch growth of K Jactis CBS 683 : pCR1 in a rich lactose medium, where one unit is the amount of enzyme that will hydrolyze 0.1 mg starch in 30 minutes at 40°C when 4.0 mg starch is present. Both batch and continuous cultivation were used to investigate growth of the recombinant yeasts and, in particular, plasmid stability and protein production were examined. Three methods for measuring the stability of plasmid pCR1 in recombinant K /actis were statistically analyzed and compared, and two, the plate ratio and clearing zones methods, were chosen for use in the fermentation studies. Initial batch fermentation studies indicated plasmid pCR1 to be extremely unstable in K lactis CBS 141 : pCR1 and so only K. lactis CBS 683 : pCR1 was investigated further. Plasmid instability was also high in this latter yeast, with 50 - 60 % of cells becoming plasmid-free after 1 0 generations of non-selective growth in high aeration batch culture using a whey-based medium. In batch culture the stability of the plasmid pCXJ-kan1 was much higher, with minimal plasmid loss detected, and this indicated that the low stability of the plasmid pCR1 was probably due to the PGK-α-amylase DNA insert. The stability of plasmid pCR1 was shown to improve by using low aeration conditions, selective medium, or a growth temperature of 20°C in both batch and continuous culture. The use of a selective medium and a lower temperature also allowed the level of α-amylase to be maintained for an increased fermentation time, and the latter also gave an increased specific yield of α-amylase in continuous culture. Thus, this study has demonstrated the successful production of a wheat α- amylase from a K. lactis strain grown in a whey-based medium.
Kluyveromyces lactis, α-Amylase, Enzymes, Yeast