Directed evolution of a cellodextrin transporter for improved biofuel production under anaerobic conditions in Saccharomyces cerevisiae

Introduction of a cellobiose utilization pathway consisting of a cellodextrin transporter and a β‐glucosidase into Saccharomyces cerevisiae enables co‐fermentation of cellobiose and xylose. Cellodextrin transporter 1 (CDT1) from Neurospora crassa has been established as an effective transporter for the engineered cellobiose utilization pathways. However, cellodextrin transporter 2 (CDT2) from the same species is a facilitator and has the potential to be more efficient than CDT1 under anaerobic conditions due to its energetic benefits. Currently, CDT2 has a very low activity and is considered rate‐limiting in cellobiose fermentation. Here, we report the directed evolution of CDT2 with an increased cellobiose uptake activity, which results in improved cellobiose fermentation under anaerobic conditions. After three rounds of directed evolution, the cellobiose uptake activity of CDT2 was increased by 2.2‐fold, which resulted from both increased specific activity and transporter expression level. Using high cell density fermentation under anaerobic conditions, the evolved mutant conferred 4.0‐ and 4.4‐fold increase in the cellobiose consumption rate and ethanol productivity, respectively. In addition, although the cellobiose uptake activity was still lower than that of CDT1, the engineered CDT2 showed significantly improved cellobiose consumption and ethanol production under anaerobic conditions, representing the energetic benefits of a sugar facilitator for anaerobic cellobiose fermentation. This study demonstrated that anaerobic biofuel production could be significantly improved via directed evolution of a sugar transporter protein in yeast. Biotechnol. Bioeng. 2014;111: 1521–1531. © 2014 Wiley Periodicals, Inc.

[1]  H. Alper,et al.  A molecular transporter engineering approach to improving xylose catabolism in Saccharomyces cerevisiae. , 2012, Metabolic engineering.

[2]  Huimin Zhao,et al.  Recent advances in biocatalysis by directed enzyme evolution. , 2006, Combinatorial chemistry & high throughput screening.

[3]  K. J. Wise,et al.  Directed evolution of bacteriorhodopsin for device applications. , 2004, Methods in enzymology.

[4]  Zhanglin Lin,et al.  An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli , 2009, Microbial cell factories.

[5]  Jee Loon Foo,et al.  Directed evolution of an E. coli inner membrane transporter for improved efflux of biofuel molecules , 2013, Biotechnology for Biofuels.

[6]  Huimin Zhao,et al.  Iowa State University From the SelectedWorks of Zengyi Shao 2012 Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae , 2017 .

[7]  M. Nardini,et al.  Directed evolution of an enantioselective lipase. , 2000, Chemistry & biology.

[8]  István Simon,et al.  The HMMTOP transmembrane topology prediction server , 2001, Bioinform..

[9]  Huimin Zhao,et al.  Customized optimization of metabolic pathways by combinatorial transcriptional engineering , 2012, Nucleic acids research.

[10]  E. Bokma,et al.  Directed evolution of a bacterial efflux pump: Adaptation of the E. coli TolC exit duct to the Pseudomonas MexAB translocase , 2006, FEBS letters.

[11]  L. Lynd,et al.  Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum , 2004, Applied and Environmental Microbiology.

[12]  M. Inui,et al.  Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook , 2009, Applied Microbiology and Biotechnology.

[13]  Venkatesh Balan,et al.  Continuous SSCF of AFEX™ pretreated corn stover for enhanced ethanol productivity using commercial enzymes and Saccharomyces cerevisiae 424A (LNH‐ST) , 2013, Biotechnology and bioengineering.

[14]  L. Giver,et al.  Engineered enzymes for chemical production. , 2008, Biotechnology and bioengineering.

[15]  Jamie H. D. Cate,et al.  Single Amino Acid Substitutions in HXT2.4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae , 2012, Applied and Environmental Microbiology.

[16]  Gunnar Lidén,et al.  Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae. , 2003, Biotechnology and bioengineering.

[17]  Y. Jang,et al.  Bio‐based production of C2–C6 platform chemicals , 2012, Biotechnology and bioengineering.

[18]  Stavros J. Hamodrakas,et al.  TMRPres2D: high quality visual representation of transmembrane protein models , 2004, Bioinform..

[19]  Huimin Zhao,et al.  Directed evolution of a highly efficient cellobiose utilizing pathway in an industrial Saccharomyces cerevisiae strain , 2013, Biotechnology and bioengineering.

[20]  Jamie H. D. Cate,et al.  Enhanced xylitol production through simultaneous co-utilization of cellobiose and xylose by engineered Saccharomyces cerevisiae. , 2013, Metabolic engineering.

[21]  Z. P. Çakar,et al.  Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties. , 2012, FEMS yeast research.

[22]  Nathan B. Gillespie,et al.  Optimization of bacteriorhodopsin for bioelectronic devices. , 2002, Trends in biotechnology.

[23]  J. Lian,et al.  Preparative Scale Production of Functional Mouse Aquaporin 4 Using Different Cell-Free Expression Modes , 2010, PloS one.

[24]  Huimin Zhao,et al.  Directed evolution as a powerful synthetic biology tool. , 2013, Methods.

[25]  Huimin Zhao,et al.  Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis , 2008, Biotechnology and bioengineering.

[26]  Yong-Su Jin,et al.  Engineered Saccharomyces cerevisiae capable of simultaneous cellobiose and xylose fermentation , 2010, Proceedings of the National Academy of Sciences.

[27]  Jens Nielsen,et al.  Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries , 2012, Cellular and Molecular Life Sciences.

[28]  Jamie H. D. Cate,et al.  Energetic benefits and rapid cellobiose fermentation by Saccharomyces cerevisiae expressing cellobiose phosphorylase and mutant cellodextrin transporters. , 2013, Metabolic engineering.

[29]  J. Lian,et al.  High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system , 2009, Applied Microbiology and Biotechnology.

[30]  Huimin Zhao,et al.  Engineering microbial factories for synthesis of value-added products , 2011, Journal of Industrial Microbiology & Biotechnology.

[31]  Jamie H. D. Cate,et al.  Single Amino Acid Substitutions in HXT 2 . 4 from Scheffersomyces stipitis Lead to Improved Cellobiose Fermentation by Engineered Saccharomyces cerevisiae , 2015 .

[32]  Huimin Zhao,et al.  Directed evolution: an evolving and enabling synthetic biology tool. , 2012, Current opinion in chemical biology.

[33]  Jeffrey M. Skerker,et al.  Rational and Evolutionary Engineering Approaches Uncover a Small Set of Genetic Changes Efficient for Rapid Xylose Fermentation in Saccharomyces cerevisiae , 2013, PloS one.

[34]  C. Roca,et al.  Engineering of carbon catabolite repression in recombinant xylose fermenting Saccharomyces cerevisiae , 2004, Applied Microbiology and Biotechnology.

[35]  D. Clark,et al.  An evaluation of cellulose saccharification and fermentation with an engineered Saccharomyces cerevisiae capable of cellobiose and xylose utilization , 2012, Biotechnology journal.

[36]  R. Schiestl,et al.  High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method , 2007, Nature Protocols.

[37]  Zengyi Shao,et al.  DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways , 2008, Nucleic acids research.

[38]  Yong-Su Jin,et al.  Simultaneous co-fermentation of mixed sugars: a promising strategy for producing cellulosic ethanol. , 2012, Trends in biotechnology.

[39]  Huimin Zhao,et al.  Directed evolution of a cellobiose utilization pathway in Saccharomyces cerevisiae by simultaneously engineering multiple proteins , 2013, Microbial Cell Factories.

[40]  Huimin Zhao,et al.  Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis. , 2010, Molecular bioSystems.

[41]  J. Swartz,et al.  Cell‐free synthesis of functional aquaporin Z in synthetic liposomes , 2009, Biotechnology and bioengineering.

[42]  Huimin Zhao,et al.  Overcoming glucose repression in mixed sugar fermentation by co-expressing a cellobiose transporter and a β-glucosidase in Saccharomyces cerevisiae. , 2010, Molecular bioSystems.

[43]  J. Lian,et al.  Improving aquaporin Z expression in Escherichia coli by fusion partners and subsequent condition optimization , 2009, Applied Microbiology and Biotechnology.

[44]  Huimin Zhao,et al.  Protein engineering in designing tailored enzymes and microorganisms for biofuels production. , 2009, Current opinion in biotechnology.

[45]  W. A. Scheffers,et al.  Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. , 1990, Journal of general microbiology.

[46]  Albert J. Vilella,et al.  Cellodextrin Transport in Yeast for Improved Biofuel Production , 2010, Science.

[47]  Huimin Zhao,et al.  Protein design for pathway engineering. , 2014, Journal of structural biology.

[48]  Bärbel Hahn-Hägerdal,et al.  Metabolic engineering for pentose utilization in Saccharomyces cerevisiae. , 2007, Advances in biochemical engineering/biotechnology.

[49]  Yong-Su Jin,et al.  Cofermentation of Cellobiose and Galactose by an Engineered Saccharomyces cerevisiae Strain , 2011, Applied and Environmental Microbiology.