RESUMO
Production of psychrophilic enzymes in the commonly used mesophilic expression systems is hampered by low intrinsic stability of the recombinant enzymes at the optimal host growth temperatures. Unless strategies for low-temperature expression are advanced, research on psychrophilic enzymes may end up being biased toward those that can be stably produced in commonly used mesophilic host systems. Two main strategies are currently being explored for the development of low-temperature expression in bacterial hosts: (i) low-temperature adaption of existing mesophilic expression systems, and (ii) development of new psychrophilic hosts. These developments include genetic engineering of the expression cassettes to optimize the promoter/operator systems that regulate heterologous expression. In this addendum we present our efforts in the development of such low-temperature expression systems, and speculate about future advancements in the field and potential applications.
Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial , Pseudoalteromonas/genética , Pseudomonas/genética , Trichoderma/genética , Regiões Árticas , Proteínas de Bactérias/genética , Temperatura Baixa , Engenharia Metabólica/métodos , Estabilidade Proteica , Pseudoalteromonas/metabolismo , Pseudomonas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Trichoderma/metabolismoRESUMO
BACKGROUND: Heterologous 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. METHODS: As 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. RESULTS: We 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. CONCLUSIONS: We 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.
Assuntos
Adaptação Biológica/genética , Temperatura Baixa , Resposta ao Choque Frio/fisiologia , Indução Enzimática/fisiologia , Enzimas/metabolismo , Sedimentos Geológicos/microbiologia , Metagenoma/genética , Regiões Árticas , Quitinases/metabolismo , Clonagem Molecular , Enzimas/genética , Escherichia coli , Vetores Genéticos/genética , Oceanos e Mares , Proteínas Recombinantes/metabolismoRESUMO
Here we report the 8 Mb high quality draft genome of Streptomyces sp. strain AW19M42, together with specific properties of the organism and the generation, annotation and analysis of its genome sequence. The genome encodes 7,727 putative open reading frames, of which 6,400 could be assigned with COG categories. Also, 62 tRNA genes and 8 rRNA operons were identified. The genome harbors several gene clusters involved in the production of secondary metabolites. Functional screening of the isolate was positive for several enzymatic activities, and some candidate genes coding for those activities are listed in this report. We find that this isolate shows biotechnological potential and is an interesting target for bioprospecting.
