The gene encoding translation elongation factor 1-alpha from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P (TEF1)) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P (GAP)) by using a bacterial lipase gene as a reporter gene. P (TEF1) demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P (GAP). The sequence of the gene was deposited in GenBank under accession no. EF014948.

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na ؉ )-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P PHO89 ) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P PHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1␣ and the glyceraldehyde-3-phosphate dehydrogenase promoter, P PHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P PHO89 -based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P PHO89 for controlled production of recombinant proteins in P. pastoris.Pichia pastoris is a methylotrophic yeast species that is increasingly used as a host system for heterologous protein expression for both academic and industrial purposes. To date, more than 500 proteins have been cloned and expressed using this system (19; http://faculty.kgi.edu/cregg/index htm), and it has provided the protein production platform for several structural genomics programs (25,35).In this system, most recombinant proteins have been produced using the alcohol oxidase I promoter (P AOX1 ), which is completely repressed when cells are grown on glucose and is induced in the presence of methanol (31). However, there are circumstances in which the promoter may not be suitable; the principal problem is that the highly volatile and inflammable compound methanol is required for transcription (29). Use of methanol for the induction of gene expression is often not permitted in the production of food products since methanol is a petroleum by-product (13, 33). Also, P AOX1 -based fermentation requires relatively long times and sophisticated feeding strategies for maintenance of the induction phase. These properties hamper the industrial applicability of this approach. Therefore, a promoter that does not require methanol is attractive for expression of foreign genes in P. pastoris. Potential promoters that are alternatives to P AOX1 include the glutathione-dependent formaldehyde dehydrogenase promoter (P FLD1 ) (26), for which either methanol or methylamine can act as an inducer, and the glyceraldehyde-3-phosphate dehydrogenase promoter (P GAP ) (33), which is a strong constitutive promoter in various microorganisms. Recently, we also developed the translation elongation factor 1␣ promoter (P TEF1 ), with more highly growth-associated expression characteristics, as an alternative to P GAP (2). The current study describes the use of a phosphate-responsive promoter as an alternative to constitutive and inducible promoters to drive expres...

We explored the physiological and metabolic effects of different carbon sources (glucose, fructose, and glucose/fructose mixture) in phosphoglucose isomerase (pgi) knockout Escherichia coli mutant producing shikimic acid (SA). It was observed that the pgi(-) mutant grown on glucose exhibited significantly lower cell growth compared with the pgi(+) strain and its mixed glucose/fructose fermentation grew well. Interestingly, when fructose was used as a carbon source, the pgi(-) mutant showed the enhanced SA production compared with the pgi(+) strain. In silico analysis of a genome-scale E. coli model was then conducted to characterize the cellular metabolism and quantify NAPDH regeneration, which allowed us to understand such experimentally observed attenuated cell growth and enhanced SA production in glucose- and fructose-consuming pgi(-) mutant, respectively with respect to cofactor regeneration.

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