Controlling gene expression is often the foremost goal of most biological endeavors like the production of industrial enzymes and expression of heterologous metabolic pathway genes. The components of the entire "expression cassette" exert control on net protein output. This control is primarily achieved through altering the promoter driving expression and by changing the copy number of the gene. However, there are only a few recent studies on terminators. Terminators are essential components in expression cassettes that influence the 3' end processing of mRNA, mRNA stability, and translational efficiency, which can modulate protein production. In Pichia pastoris (Komagataella phaffi), little attention has been paid to the selection of terminator regions in efforts to increase heterologous gene expression. To explore the potential application of the terminator regions on increased secretory production of Candida antarctica lipase B (CALB), we assessed the ability of three different classes of terminator regions: (1) terminator regions of methanol oxidation pathway genes of P. pastoris; (2) terminator regions of well-expressed and housekeeping genes of P. pastoris; and (3) terminators of other yeast genes like Saccharomyces cerevisiae. The terminator of dihydroxyacetone synthase (DHAS TT), a high expressing gene in the methanol utilization pathway, shows inducible CALB expression levels similar to the AOX1 terminator (AOX1 TT) under the control of AOX1 promoter and threefold higher in constitutive expression of CALB under the control of GAP promoter. The Calb transcript abundance was also found to correlate with protein expression. Furthermore, mRNA half-life determination showed a direct correlation between the stability of transcripts and increased transcription rate. Together, our results emphasize that enhancing transcript stability using the correct choice of transcription terminators (TT) will help in developing robust production strains suitable for scale-up.Key points• Influence of transcription terminators on Calb gene expression• Modulation of gene expression by enhancing transcript stability.

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Journal Article

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Ramakrishnan K, Prattipati M, Samuel P, Sankaranarayanan M

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