MINI-REVIEW Developmental strategies and regulation of cell-free enzyme system for ethanol production: a molecular prospective Waleed Ahmad Khattak & Muhammad Wajid Ullah & Mazhar Ul-Islam & Shaukat Khan & Minah Kim & Yeji Kim & Joong Kon Park Received: 17 July 2014 /Revised: 9 October 2014 /Accepted: 12 October 2014 /Published online: 31 October 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Most biomanufacturing systems developed for the production of biocommodities are based on whole-cell sys- tems. However, with the advent of innovative technologies, the focus has shifted from whole-cell towards cell-free en- zyme system. Since more than a century, researchers are using the cell-free extract containing the required enzymes and their respective cofactors in order to study the fundamental aspects of biological systems, particularly fermentation. Although yeast cell-free enzyme system is known since long ago, it is rarely been studied and characterized in detail. In this review, we hope to describe the major pitfalls encountered by whole- cell system and introduce possible solutions to them using cell-free enzyme systems. We have discussed the glycolytic and fermentative pathways and their regulation at both tran- scription and translational levels. Moreover, several strategies employed for development of cell-free enzyme system have been described with their potential merits and shortcomings associated with these developmental approaches. We also described in detail the various developmental approaches of synthetic cell-free enzyme system such as compartmentaliza- tion, metabolic channeling, protein fusion, and co- immobilization strategies. Additionally, we portrayed the nov- el cell-free enzyme technologies based on encapsulation and immobilization techniques and their development and com- mercialization. Through this review, we have presented the basics of cell-free enzyme system, the strategies involved in development and operation, and the advantages over conven- tional processes. Finally, we have addressed some potential directions for the future development and industrialization of cell-free enzyme system. Keywords Cell-free enzyme system . Whole-cell system . Glycolyticand fermentationenzymes . Ethanol . Conventional fermentation Introduction Fossil fuels have remained the major energy source since last few centuries; however, the rapid depletion of their reservoirs has been brought under spotlight only recently (Khattak et al. 2014). Besides energy concerns, serious environmental com- plications have originated from fossil fuel depletion (Saptoro et al. 2014; Prasetyo and Park 2013; Wernick and Liao 2013). These concerns have been intensely debated on several plat- forms and demands have been put forward for strenuous efforts for an urgent resolution. In this scenario, renewable energy technology has been proposed as the only ultimate solution to overcome issues associated with fossil fuels (Johansson et al. 1993; Wang et al. 2013, Edwards and Doran-Peterson 2012). Considering the significance of renew- able energy sources, a number of biofuels, including ethanol, have been produced from renewable sources through micro- bial fermentation (Shirsat et al. 2013; He et al. 2014; Ayeni et al. 2014 ). Among the fermentative microbiota, Saccharomyces cerevisiae is viewed as a common and attrac- tive organism adopted for ethanol production and is highly valued in biochemical, genetic, pharmacological, and post- genomic studies (Mager and Winderickx 2005). Over the past few decades, ethanol has emerged as the most promising and attractive alternative energy resource, holding potential advantages both from ecological and environmental aspects (Prasad et al. 2007), despite past criticism of the use of food reservoirs for ethanol production as being responsible for possible future food crises. However, the eventual shift of ethanol production sources from food reservoirs to secondary food reservoirs and waste resources resolved the issue (Mai W. A. Khattak : M. W. Ullah : M. Ul-Islam : S. Khan : M. Kim : Y. Kim : J. K. Park (*) Department of Chemical Engineering, Kyungpook National University, Daegu 7020-701, Korea e-mail: parkjk@knu.ac.kr Appl Microbiol Biotechnol (2014) 98:9561–9578 DOI 10.1007/s00253-014-6154-0