Cold shock induction of recombinant Arctic environmental genes

Abstract

BackgroundHeterologous expression of psychrophilic enzymes in E. coli is particularly challenging due to their intrinsic instability. The low stability is regarded as a consequence of adaptation that allow them to function at low temperatures. Recombinant production presents a significant barrier to their exploitation for commercial applications in industry.MethodsAs part of an enzyme discovery project we have investigated the utility of a cold-shock inducible promoter for low-temperature expression of five diverse genes derived from the metagenomes of marine Arctic sediments. After evaluation of their production, we further optimized for soluble production by building a vector suite from which the environmental genes could be expressed as fusions with solubility tags.ResultsWe found that the low-temperature optimized system produced high expression levels for all putatively cold-active proteins, as well as reducing host toxicity for several candidates. As a proof of concept, activity assays with one of the candidates, a putative chitinase, showed that functional protein was obtained using the low-temperature optimized vector suite.ConclusionsWe conclude that a cold-shock inducible system is advantageous for the heterologous expression of psychrophilic proteins, and may also be useful for expression of toxic mesophilic and thermophilic proteins where properties of the proteins are deleterious to the host cell growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0185-1) contains supplementary material, which is available to authorized users.

Engineering low-temperature expression systems for heterologous production of cold-adapted enzymes

“…This has recently allowed us to perform preliminary characterization of the selected enzymes. For example, activity assays on the His-SUMO fused chitinase described in our paper 1 suggests that the enzyme is truly psychrophilic with an optimal temperature around 20°C ( Fig. 1 ).…”

“…By expressing the putatively psychrophilic genes in this E. coli system, we found that expression levels were generally high, and comparable to the commonly used T7 system which functions optimally at 37°C. 1 In contrast to the T7 system some soluble proteins were detected, however in most cases the yield was still too low for further experimentation. We therefore optimized cspA -driven expression by fusing the maltose-binding protein (MBP), thioredoxin (TRX), the small ubiquitin-like modifier (SUMO) and trigger factor (TF) N-terminally to these enzymes, in an attempt to improve solubility.…”

“…Our latest work involves the investigation of low-temperature expression of putatively cold-adapted (psychrophilic) gene candidates. 1 Psychrophilic enzymes present significant economic potential for industrial applications. For example, introduction of psychrophilic proteases, lipases, cellulases and amylases into laundry detergents can decrease the demand for both chemical additives and enzyme concentrations during production, but can also reduce the need for heating and the water consumption for end-users.…”

“…In our case, co-expression of cold-adapted chaperonines was not sufficient for soluble expression of genes derived from Arctic-sources metagenomes in combination with the commonly used T7 expression system (T7 RNA polymerase/ T7 promoter). 1 We therefore targeted optimization of low-temperature expression at the transcription level by investigating the utility of a cold-shock inducible promoter for low-temperature expression. 1 In this E. coli based system, expression of recombinant proteins is regulated by a temperature-inducible promoter, the E. coli cold shock protein A ( cspA) promoter, which allows induction by a downshift in growth temperature.…”

“… 1 We therefore targeted optimization of low-temperature expression at the transcription level by investigating the utility of a cold-shock inducible promoter for low-temperature expression. 1 In this E. coli based system, expression of recombinant proteins is regulated by a temperature-inducible promoter, the E. coli cold shock protein A ( cspA) promoter, which allows induction by a downshift in growth temperature. 12,13 Analogous systems have been investigated for Bacillus subtilis where the cold-inducible promoter of the desaturase encoding gene ( des ) in combination with other 5′ UTR regulatory elements was shown to generate higher expression levels.…”