BioChem - DOS

Directed evolution reveals unexpected epistatic interactions that alter metabolic regulation and enable anaerobic xylose use by Saccharomyces cerevisiae

PLoS genetics

Sato, Trey K.; Tremaine, Mary; Parreiras, Lucas S.; Hebert, Alexander S.; Myers, Kevin S.; Higbee, Alan J.; Sardi, Maria; McIlwain, Sean J.; Ong, Irene M.; Breuer, Rebecca J.; Avanasi Narasimhan, Ragothaman; McGee, Mick A.; Dickinson, Quinn; La Reau, Alex; Xie, Dan; Tian, Mingyuan; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert

<▼1><P>The inability of native <I>Saccharomyces cerevisiae</I> to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram <I>S</I>. <I>cerevisiae</I> to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved <I>S</I>. <I>cerevisiae</I> strains depends upon epistatic interactions among genes encoding a xylose reductase (<I>GRE3</I>), a component of MAP Kinase (MAPK) signaling (<I>HOG1</I>), a regulator of Protein Kinase A (PKA) signaling (<I>IRA2</I>), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (<I>ISU1</I>). Interestingly, the mutation in <I>IRA2</I> only impacted anaerobic xylose consumption and required the loss of <I>ISU1</I> function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in <I>HOG1</I> and <I>ISU1</I> unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.</P></▼1><▼2><P><B>Author Summary</B></P><P>The yeast <I>Saccharomyces cerevisiae</I> is being genetically engineered to produce renewable biofuels from sustainable plant material. Efficient biofuel production from plant material requires conversion of the complex suite of sugars found in plant material, including the five-carbon sugar xylose. Because it does not efficiently metabolize xylose, <I>S</I>. <I>cerevisiae</I> has been engineered with a minimal set of genes that should overcome this problem; however, additional genetic changes are required for optimal fermentative conversion of xylose into biofuel. Despite extensive knowledge of the regulatory networks controlling glucose metabolism, less is known about the regulation of xylose metabolism and how to rewire these networks for effective biofuel production. Here we report genetic mutations that enabled the conversion of xylose into bioethanol by a previously ineffective yeast strain. By comparing altered protein and metabolite abundance within yeast cells containing these mutations, we determined that the mutations synergistically alter metabolic pathways to improve the rate of xylose conversion. One change in a gene with well-characterized aerobic mitochondrial functions was found to play an unexpected role in anaerobic conversion of xylose into ethanol. The results of this work will allow others to rapidly generate yeast strains for the conversion of xylose into biofuels and other products.</P></▼2>

2016

Public Library of Science

cc-by

1553-7390

1553-7404

12

10

pp.e1006372

10.1371/journal.pgen.1006372

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART87573347

https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1006372&type=printable