TY - JOUR
T1 - A SWEET surprise
T2 - Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast
AU - Podolsky, Igor A.
AU - Seppälä, Susanna
AU - Xu, Haiqing
AU - Jin, Yong Su
AU - O'Malley, Michelle A.
N1 - Funding Information:
The authors wish to acknowledge funding support from the National Science Foundation (NSF) (MCB-1553721). This work was part of the Department of Energy (DOE) Joint BioEnergy Institute (http://www.jbei.org) supported by the Office of Biological and Environmental Research of the DOE Office of Science through contract DE-AC02–05CH11231 between Lawrence Berkeley National Laboratory and the DOE and Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) supported by the DOE, Office of Science, Office of Biological and Environmental Research under Award Number(s) DE-SC0018420. We also acknowledge the use of the UC Santa Barbara NRI-MCDB Microscopy Facility. We thank Dr. Eckhard Boles for the EBY.VW4000 strain, and Dr. Morten Nørholm for the PfuX7 polymerase.
Funding Information:
The authors wish to acknowledge funding support from the National Science Foundation (NSF) ( MCB-1553721 ). This work was part of the Department of Energy (DOE) Joint BioEnergy Institute ( http://www.jbei.org ) supported by the Office of Biological and Environmental Research of the DOE Office of Science through contract DE-AC02–05CH11231 between Lawrence Berkeley National Laboratory and the DOE and Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) supported by the DOE, Office of Science, Office of Biological and Environmental Research under Award Number(s) DE-SC0018420. We also acknowledge the use of the UC Santa Barbara NRI-MCDB Microscopy Facility. We thank Dr. Eckhard Boles for the EBY.VW4000 strain, and Dr. Morten Nørholm for the PfuX7 polymerase.
Publisher Copyright:
© 2021 The Authors
PY - 2021/7
Y1 - 2021/7
N2 - In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.
AB - In the yeast Saccharomyces cerevisiae, microbial fuels and chemicals production on lignocellulosic hydrolysates is constrained by poor sugar transport. For biotechnological applications, it is desirable to source transporters with novel or enhanced function from nonconventional organisms in complement to engineering known transporters. Here, we identified and functionally screened genes from three strains of early-branching anaerobic fungi (Neocallimastigomycota) that encode sugar transporters from the recently discovered Sugars Will Eventually be Exported Transporter (SWEET) superfamily in Saccharomyces cerevisiae. A novel fungal SWEET, NcSWEET1, was identified that localized to the plasma membrane and complemented growth in a hexose transporter-deficient yeast strain. Single cross-over chimeras were constructed from a leading NcSWEET1 expression-enabling domain paired with all other candidate SWEETs to broadly scan the sequence and functional space for enhanced variants. This led to the identification of a chimera, NcSW1/PfSW2:TM5-7, that enhanced the growth rate significantly on glucose, fructose, and mannose. Additional chimeras with varied cross-over junctions identified residues in TM1 that affect substrate selectivity. Furthermore, we demonstrate that NcSWEET1 and the enhanced NcSW1/PfSW2:TM5-7 variant facilitated novel co-consumption of glucose and xylose in S. cerevisiae. NcSWEET1 utilized 40.1% of both sugars, exceeding the 17.3% utilization demonstrated by the control HXT7(F79S) strain. Our results suggest that SWEETs from anaerobic fungi are beneficial tools for enhancing glucose and xylose co-utilization and offers a promising step towards biotechnological application of SWEETs in S. cerevisiae.
KW - Lignocellulose
KW - Membrane proteins
KW - Metabolic engineering
KW - Sugar
KW - Transporter
KW - Yeast
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UR - http://www.scopus.com/inward/citedby.url?scp=85104718754&partnerID=8YFLogxK
U2 - 10.1016/j.ymben.2021.04.009
DO - 10.1016/j.ymben.2021.04.009
M3 - Article
C2 - 33887459
AN - SCOPUS:85104718754
SN - 1096-7176
VL - 66
SP - 137
EP - 147
JO - Metabolic Engineering
JF - Metabolic Engineering
ER -