Downregulation by organic nitrogen of AOX1 promoter used for controlled expression of foreign genes in the yeast Pichia pastoris

Pichia pastoris is a well-established cell factory for recombinant protein synthesis. Various optimization strategies of processes based on AOX1 promoter have been investigated, including methanol co-feeding with glycerol or sorbitol during the induction stage. Compared with carbon sources, comparatively little research has been devoted to the effects of nitrogen sources. Several reports have described the benefits of adding casamino acids (CA) to the recombinant protein production medium, however, without considering its effects at the gene expression level. Using enhanced green fluorescent protein as a reporter protein, monitored using flow cytometry, CA was shown to downregulate AOX1 promoter induction. Despite higher growth rates, cultures containing CA exhibited slower transition to the induced state, whereas metabolite analysis revealed that methanol consumption was reduced in the presence of CA compared with its absence. The repressive effect of CA was further confirmed by analysing the synthesis of extracellular recombinant Candida antarctica lipase under control of the AOX1 promoter. These findings highlight nitrogen source selection as an important consideration for AOX1-based protein production.     

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop‐out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5‐fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter‐selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co‐transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1), FJ226453 (HpMET2) and FJ493241 (HpADE2), respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Cytotoxic effects of geranyl flavonoid derivatives from the fruit of Artocarpus communis in SK-Hep-1 human hepatocellular carcinoma cells

Breadfruit (Artocarpus communis) is cultivated in tropical and subtropical regions as a traditional starch crop. This study investigated the cytotoxic activity of geranyl flavonoid derivatives isolated from the fruit of A. communis. Two new geranyl flavonoid derivatives, arcommunol A and arcommunol B, together with four known compounds, were isolated from the fruit of A. communis. The effects of these compounds on the viability of HepG2, Hep3B, PLC5 and SK-Hep-1 cells were investigated. Arcommunol A showed the highest inhibitory activity with 2.05 μM IC₅₀ in SK-Hep-1 cells. Treatment with arcommunol A changed the ratio of expression levels of pro- and anti-apoptotic Bcl-2 family members and subsequently induced the activation of apoptosis-inducing factor, apoptotic protease activating factor-1, caspase-9 and caspase-3, which may result in a cleavage of poly(ADP-ribose) polymerase. These findings provide critical information regarding the chemopreventive potential of arcommunol A from the fruit of A. communis.