Properties of the -derived constitutive promoter, assessed using an HSA reporter gene

Abstract

The glyceraldehyde-3-phosphate dehydrogenase promoter, P(GAP), was employed to direct the constitutive expression of recombinant human serum albumin (HSA) in Hansenula polymorpha. A set of integration vectors containing the HSA cDNA under the control of P(GAP) was constructed and the elemental parameters affecting the expression of HSA from P(GAP) were analyzed. The presence of a 5'-untranslated region derived from the HSA cDNA and the integration of the expression vector into the GAP locus were shown to improve the expression of HSA under P(GAP). Glycerol supported a higher level of HSA expression from P(GAP) along with a higher cell density than either glucose or methanol. The growth at high glycerol concentrations up to 12% did not cause any significant repression of the cell growth. A high cell density culture, up to 83 g l(-1) dry cell weight with a HSA production of 550 mg l(-1), was obtained in less than 32 h of cultivation in a fed-batch fermentation employing intermittent feeding with 12% glycerol. The GAP promoter-based HSA expression system showed a higher specific production rate and required a much simpler fermentation process than the MOX promoter-based system, demonstrating that P(GAP) can be a practical alternative of the MOX promoter in the large-scale production of HSA from H. polymorpha.

Effects of different glycerol feeding strategies on S-adenosyl-l-methionine biosynthesis by PGAP-driven Pichia pastoris overexpressing methionine adenosyltransferase

“…Among the promoters employed, alcohol oxidase 1 promoter (P AOX ) was utilized initially and applied widely. However, there existed several marked disadvantages, such as usage of explosive and toxic inducer of methanol, and a long cultivation cycle (Heo et al, 2003).…”

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae

“…However, when the recombinant strain was grown in different condition, at which glucose was added as the carbon source instead of glycerol, the enzyme activities varied, showing 2.9-fold increase of DhaB activity but 7.2-fold decrease of DhaT activity compared to those obtained cells grown at glycerol. This is an interesting result regarding the fact that activity of GAPDH promoter directing expression of the genes on pYH-dhaBT was known to be constitutive in H. polymorpha [12]. The recombinant yeast strain Hp DL1-L/pYH-dhaBT showed poor growth compared with the control strain during cultivation in the medium supplemented with glycerol (2%) or glucose (2%) as carbon source (Fig.…”

“…pGEM-T Easy (Promega) was used for cloning and sequencing the amplified DNA fragments. pYHSA161 [12] was used to express the genes in H. polymorpha. LB medium [yeast extract (Difco), 0.5% (w/v); Bacto-tryptone, 1% (w/v); and NaCl, 1% (w/v)] was used to grow E. coli stains.…”

Development of an α-amylase reporter system for efficient screening of clones with highly expressed heterologous protein in Hansenula polymorpha

“…But as for this correlation of the a-amylase activity and the yield of recombinant target protein, the same integration sites might be an important factor. The over-expression of a-amylase in yeast cell might overload the transcription machinery because the Amy gene under the control of a strong constitutive GAP promoter (Heo et al 2003). To solve this problem, the a-amylase reporter system cassette was Fig.…”