Elsevier

Gene Reports

Volume 17, December 2019, 100497
Gene Reports

Current status of genetic & metabolic engineering and novel QTL mapping-based strategic approach in bioethanol production

https://doi.org/10.1016/j.genrep.2019.100497Get rights and content

Abstract

Since many decades, strains of Saccharomyces genus are being employed in several bioprocess set-ups for the production of bioethanol. Recently, osmo/halotolerant strains of Zygosaccharomyces has been identified and recognized as a potential model system for bioethanol production owing to the ability of the strains to withstand stringently harsh environment conditions, the same that yeasts face in any bioprocessing set-up. Zygosaccharomyces strains have been bestowed with a variety of industrially beneficial physiological traits that enable Zygosaccharomyces strains to withstand low pH, high osmolarity, high ethanol, high temperature and other conditions. Unlike Mendelian traits, these physiological capabilities are governed by multiple genes (polygenes) and are, therefore, regarded as polygenic or quantitative traits. The characterization of genes/loci associated with these traits is called as quantitative trait loci (QTL) mapping. To date, for improved bioethanol production, researchers have mainly used genetic/metabolic engineering, and media/process optimization strategies using mainly Saccharomyces as a model system. However, such strategies have only showed limited success. In this work, we wish to present methodological approach for generating F1, F2 and recombinant progeny population through genetic crossing of two divergent yeast strains in order to achieve descendants improved phenotypic robustness, improved adaptation to hostile bioprocess conditions (such as low pH, low nitrogen, high osmolarity, high ethanol, high temperature and high inhibitor concentration) and increase bioethanol production. So far as our knowledge is concerned, no one has ever applied this approach in India or globally for improving bioethanol production at large-scale. We believe that the presented methodological approach holds high promise for technology transfer, commercialization and also for meeting the future increasing demands of the biofuel/bioenergy in India.

Introduction

For decades, Saccharomyces cerevisiae and other yeast strains have been used in fermentative elaboration of a number of food, beverages and biofuels (Solieri et al., 2006, Solieri et al., 2013a, Solieri et al., 2014a, Solieri et al., 2014b; Swinnen et al., 2012a, Swinnen et al., 2012b; Hubmann et al., 2013; Dakal et al., 2014, Dakal et al., 2016). In Europe and some other countries, industrial potential of Zygosaccharomyces strains have been explored at large-batch high cell density cultivation (HCDC) and in winemaking as well (Escott et al., 2018; Li et al., 2018). Zygosaccharomyces strains have been identified and recognized as a promising biological/model system for various industrial applications owing to their high osmo/halotolerant and sugar fermentative abilities (Dakal et al., 2014). Industrial bioprocess conditions often represent multi-stress conditions for yeast survival and growth and the ability of yeast to grow under such multi-stress conditions is governed by multiple genes called as polygenes. These polygenes genes regulate physiological capabilities (known as quantitative traits) of yeasts, i.e., yeasts' ability to withstand harsh bioprocess conditions such as low pH, low nitrogen, high osmolarity, high ethanol content, extreme temperature, and presence of inhibitors such as furfural, HMF and acetic acid (Steinmetz et al., 2002; Goddard et al., 2005; Zeyl, 2006; Miles and Wayne, 2008; Liti and Louis, 2012; Tesfaw and Assefa, 2014). The genetic determinants of yeast robustness and tolerance to multi-stress conditions in relation to bioethanol production are largely unknown. Of several ways, one is to identify, characterize and map genetic loci (or DNA regions) in the chromosomes that contain the genes affecting these complex polygenic/quantitative traits (traits that explain yeast tolerance to multi-stress conditions and improved bioethanol production in Zygosaccharomyces). The characterization of such loci or DNA regions is globally referred to as quantitative trait loci (QTL) mapping, which is a statistical approach for linking genotypic data with a trait value, i.e., phenotypic data (Falconer and Mackay, 1996; Lynch and Walsh, 1998; Miles and Wayne, 2008). One can take the advantage of the availability of some novel state-of-the-art techniques, such as selective breeding (Zorgo et al., 2012; Timberlake et al., 2011; Shapira et al., 2014), genome-wide methods for scoring SNPs as genetic markers (Zimmer et al., 2014), next-generation sequencing platforms (Morozova and Marra, 2008; Solieri et al., 2013b; Buermans and den Dunnen, 2014), QTL mapping (Marullo et al., 2007a, Marullo et al., 2007b; Zimmer et al., 2014) and highly versatile CRISPR/cas9 gene editing system (Sander and Joung, 2014), for generating and identifying attractive hybrid Zygosaccharomyces yeasts variants with phenotypic robustness, multi-stress tolerance and improved bioethanol production. The generated strains are non-GMOs as the approach involves genetic crossing and not genetic engineering. To the best of our knowledge, only few studies have addressed this issue at single-nucleotide resolution using selective breeding techniques coupled with QTL mapping for improved bioethanol production in India and across the globe. As a future prospect, some candidate strains can be further improved by means of evolutionary, genetic and metabolic flux engineering to generate novel recombinant yeast variants with inherent properties for increased bioethanol production to meet growing biofuel/bioenergy demands in future.

Section snippets

Current status of research and development in the field of bioethanol production: background and outlines

A key question to address in coming future is to unravel the molecular and genetic determinants of phenotypic robustness, multi-stress tolerance and improved bioethanol production in both Saccharomyces and non-Saccharomyces yeasts. Yeasts ability to withstand multi-stress conditions such as harsh pH, osmolarity, temperature, ethanol, and inhibitors (such as furfural, HMF and acetic acid) etc. has a great impact on their growth and viability in any bioprocess condition, that in turn,

Bioethanol production: global status

Bioethanol derived from sugar-based biomass in a fermentation process is a potential way of generating energy-rich transportation fuels (Karakashev et al., 2007). First generation feedstock materials such as corn and sugarcane have already been widely exploited (Bothast and Schlicher, 2005). One of the most widely adopted S. cerevisiae strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil (Argueso et al., 2009). Other S. cerevisiae

Bioethanol production: current status in India

In context to research in the field of bioethanol production in India, we are still in the developmental stage. For improved bioethanol production, researchers in India have mainly adopted or used: 1) isolation and screening of yeast strains from different extreme environments for their use in bioethanol production (Gupta et al., 2009; Sridhar et al., 2002; Priya et al., 2016); 2) chemical and UV mutagenesis of yeast strains for improved bioethanol production (Sridhar et al., 2002; Yuvraj et

Importance of QTL mapping-based research in bioethanol production

Although, there is adequate knowledge available on basic physiology, biochemistry and genetics related to industrially beneficial traits in Zygosaccharomyces and other yeast strains; however, their biotechnological and biopharmaceutical applications is often limited (both nation-wide and world-wide) owing to unavailability of genotypic-phenotypic data at single nucleotide resolution level. Here, we have presented few strategies used to production of bioethanol from Zygosaccharomyces rouxii

Conclusion

The presented work comprises of many key questions that will be dealt or addressed in the using the methodological approach presented and shown in Fig. 1. The presented methodological approach is expected to provide a novel platform for development of yeast strains for various biotechnological and biopharmaceutical applications, including bioethanol/biofuel production. We anticipate that the strains developed from the presented approach will have direct application in biofuel, biotechnological

Acknowledgements

Authors are thankful to the Mohanlal Sukhadia University, Udaipur (Raj.) for the facilities. This study was funded by DST-SERB ECR Grant through the Project no. ECR/2017/000454/LS.

Declaration of competing interest

There is no conflict of interest.

Funding information

This work is funded and supported by Society for Engineering and Research Board, India ECR Grant (ECR/2017/000454/LS) sanctioned to Dr. Tikam Chand Dakal.

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