TY - JOUR

T1 - In-depth analysis of the Aspergillus niger glucoamylase (glaA) promoter performance using high-throughput screening and controlled bioreactor cultivation techniques

AU - Ganzlin, Markus

AU - Rinas, Ursula

N1 - Funding Information: Financial support by the EC grant BIO4CT96-0535 is gratefully acknowledged.

PY - 2008/6/30

Y1 - 2008/6/30

N2 - An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.

AB - An in-depth characterization of the Aspergillus niger glucoamylase (glaA) promoter performance was carried out on defined medium employing multi-well high-throughput screening as well as controlled batch and fed-batch bioreactor culture techniques with GFP as a fluorescent reporter protein. A variety of metabolizable carbon substrates and non-metabolizable analogs were screened with regard to their effect on the glaA expression system. The results clearly demonstrate that only starch and its hydrolytic products, including glucose, act as inducers. However, induction of the glaA expression system through the monosaccharide glucose is significantly lower compared to starch and the higher molecular weight starch degradation products. All other 26 carbon substrates tested do not induce, or even, as in the case of the easily metabolizable monosaccharide xylose, repress glaA-promoter controlled gene expression in the presence of the inducing disaccharide maltose with an increase of repression strength by increasing xylose concentrations. The complex effect of glucose on glaA-promoter controlled expression was also analyzed using non-metabolizable glucose analogs, namely 5-thio-glucose and 2-deoxyglucose, which were identified as novel and potent inducers of the glaA expression system. The results show that the induction strength depends on the inducer concentration with a maximum at defined concentrations and lower induction or even repression at concentrations above. Moreover, controlled fed-batch cultivations using a high maltose feed rate with concomitant extracellular accumulation of glucose resulted in lower levels of the reporter protein compared to cultures with a low-maltose feed rate without extracellular glucose accumulation, thus supporting the conclusion that increasing the glucose concentration beyond a critical point reduces the induction strength or may even cause repression. This way, the speed of polymer hydrolysis, glucose uptake and intracellular breakdown can be fine-tuned for optimal fungal growth and the metabolic burden for glucoamylase synthesis can be limited adequately in response to nutrient availability.

KW - Aspergillus niger

KW - GFP

KW - glaA-Promoter

KW - High-throughput screening

UR - http://www.scopus.com/inward/record.url?scp=45449105751&partnerID=8YFLogxK

U2 - 10.1016/j.jbiotec.2008.04.005

DO - 10.1016/j.jbiotec.2008.04.005

M3 - Article

C2 - 18501461

AN - SCOPUS:45449105751

VL - 135

SP - 266

EP - 271

JO - Journal of biotechnology

JF - Journal of biotechnology

SN - 0168-1656

IS - 3

ER -