Abstract
BackgroundACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes. Apart from ACEII, transcription factors such as XYR1, CRE1, HAP2/3/5 complex and ACEI function in a coordinated pattern for regulating the gene expression of cellulases and hemicellulases. Studies have demonstrated that ACEII gene deletion results in decreased total cellulase and xylanase activities with reduced transcript levels of lignocellulolytic enzymes.ResultsIn this study, we have successfully transformed the ACEII transcription factor encoding gene in Trichoderma reesei to significantly improve its degrading abilities. Transformation experiments on parental strain T. reesei QM9414 has resulted in five genetically engineered strains T/Ace2-2, T/Ace2-5, T/Ace2-8, T/Ace5-4 and T/Ace10-1. Among which, T/Ace2-2 has exhibited significant increase in enzyme activity by twofolds, when compared to parental strain. The T/Ace2-2 was cultured on growth substrates containing 2% bark supplemented with (a) sugar free + MA medium (b) glucose + MA medium and (c) xylose + MA medium. The bark degradation efficiency of genetically modified T/Ace2-2 strain was assessed by analyzing the xylitol production yield using HPAEC. By 6th day, about 10.52 g/l of xylitol was produced through enzymatic conversion of bark (2% bark + MA + xylose) by the T/Ace2-2 strain and by 7th day the conversion rate was found to be 0.21 g/g. Obtained results confirmed that bark growth medium supplemented with d-xylose has profoundly increased the conversion rate of bark by T/Ace2-2 strain when compared to sugar free and glucose supplemented growth media. Results obtained from scanning electron microscopy has endorsed our current results. Bark samples inoculated with T/Ace2-2 strain has showed large number of degraded cells with clearly visible cavities and fractures, by exposing the microfibrillar interwoven complex.ConclusionWe propose a cost effective and ecofriendly method for the degradation of lignocellulosic biomass such as bark to produce xylitol by using genetically modified T. reesei. Efficient conversion rate and production yield obtained in our current study provides a great scope for the xylitol industries, as our method bypasses the pretreatment of bark achieving clean and low-cost xylitol production.
Highlights
ACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes
Construction of expression vector ace2‐hph‐PRIM In order to increase the production of xylanase and overall cellulolytic and hemicellulolytic activities of T. reesei, we have constructed and overexpressed ace2 gene incorporated in an expression vector pace2-hph-PRIM
Xylanase regulator 1 (XYR1) is the main activator for the cellulase and hemicellulase encoding genes as the XYR1 gene deletion resulted in impaired induction of cellulases and hemicellulases [16, 17]
Summary
ACEII transcription factor plays a significant role in regulating the expression of cellulase and hemicellulase encoding genes. Apart from ACEII, transcription factors such as XYR1, CRE1, HAP2/3/5 complex and ACEI function in a coordinated pattern for regulating the gene expression of cellulases and hemicellulases. Studies have demonstrated that ACEII gene deletion results in decreased total cellulase and xylanase activities with reduced transcript levels of lignocellulolytic enzymes. Xylitol a naturally occurring five-carbon sugar alcohol, popularly known as a low-calorie sugar substituent. It was reported that xylitol maintains the acid–base balance and prevents the formation of dental caries and oral cavities [2]. Zabner et al [3], have reported that xylitol prevents bacteria from binding to the human cells protecting us from respiratory tract infections [3]. Due to its wide range of applications in medicine and food sectors, there is a tremendous demand for xylitol production, it was expected that by 2020, global consumption of xylitol will reach to 242 thousand metric tons with a market value of 1 billion US$ [4]
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