Increase in copy number of an integrated vector during continuous culture of Hansenula polymorpha expressing functional human haemoglobin

Abstract

Recombinant human haemoglobin A (rHbA) was produced by a leucine-requiring strain of Hansenula polymorpha which had been transformed with an integration vector containing the Saccharomyces cerevisiae LEU2 gene and cDNAs for the expression of alpha and beta globin each driven by the H. polymorpha MOX promoter. After 40 generations in a chemostat it was found that the integrated vector had become amplified in the host strain. In some cases this led to an increase in LEU2 gene dosage, but a loss of globin expression cassettes. In other cases the globin gene dosage also increased. These changes coincided with an increase in rHbA production in the culture, which was reversed when the dilution rate was increased. Isolates from a chemostat culture producing elevated levels of rHbA were grown in fed-batch fermentations, resulting in higher productivities than when inoculated with the parent strain. The rHbA produced was purified and characterized. Oxygen binding studies and electrospray mass spectrometry showed that the rHbA had been processed and assembled correctly, and behaved as a fully functional co-operative tetramer.

Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL‐1

“…The present systems have a certain biotechnological advantage in that they can avoid the possible penalty of instability, which results from high-level expression as a result of tandem multiple integration (Gilbert et al, 1994). In particular, the development of the mox⌬ transformant would alleviate the operational problems associated with the large amounts of methanol feeding required to grow the MOX transformants of H. polymorpha, such as the need for explosion-proof equipment and special technical infrastructure.…”

Recombinant Glucoamylase Production byAspergillus nigerB1 in Chemostat and pH Auxostat Cultures

“…The loss of recombinant DNA due to segregational (Moreno et al, 1994) or structural instability (Gilbert et al, 1994) may release a 'metabolic burden' on the cell and consequently confer a growth rate advantage on the mutant strain, relative to the parental strain. Since the extra glaA genes are integrated into the chromosome of A. niger B1 (Verdoes et al, 1993), their loss would only occur as a result of mutation, deletion, or chromosomal rearrangement, rather than by segregation.…”

“…During growth in continuous flow culture, recombinant plasmid DNA is subject to structural (Adams et al, 1992) and segregational (Nugent et al, 1983) instability. The development of vectors which can integrate directly into the genome has improved segregational stability in strains of yeast (Hensing et al, 1995) and fungi (Withers et al, 1995) although the inserted DNA is still prone to structural instability (Gilbert et al, 1994).…”

Autocloning and Amplification of LIP2 in Yarrowia lipolytica

“…Amplification is usually based on drug resistance markers (18) or a defective marker such as poorly expressed heterologous auxotrophic markers (5,11) and promoter-defective alleles (ura3d, leu2d, and trp1d) (3,20,22,29). For promoterdefective alleles, amplification depends upon localization of the marker to the integrative cassette (29).…”