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Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose

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Abstract

Background

Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern.

Results

A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter. Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR. No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains. Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density (low oxygen) cultures consuming xylose more slowly than low-density cultures. The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures. Statistically similar ethanol yields at high and low density (approximately 0.25 g ethanol/ g xylose) suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion.

Conclusion

This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works. Instead, NAD + availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation. While increased aeration could provide increased conversion of NAD + to NADH (and a stoichiometric increase in fermentation flux), this increase would not be expected improve ethanol yield beyond 50% of the theoretical maximum. Based on these findings, future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD + / NADH availability through non-respiratory pathways.

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Data availability

All data from this publication is made available with this manuscript and supplementary materials. Any missing information will be provided by the authors.

Abbreviations

AP:

Antarctic phosphatase

BSA:

Bovine serum albumin

CEN.PK2-1C:

Common lab strain of S. cerevisiae

CTec3:

Novazyme enzyme mixture containing cellulases and hemicellulases

D5A:

Industrial strain of S. cerevisiae isolated from cheese whey

gDCW:

Grams dry cell weight

HTec3:

Novazyme enzyme mixture containing primarily hemicellulases

HPLC:

High-performance liquid chromatography

KanMX:

Sequence inferring kanamycin resistance

LB:

Lysogen broth

NatMX:

Sequence inferring ClonNAT resistance

NEB:

New England Biolabs

NO-PPP:

Non oxidative pentose phosphate pathway

ORP:

Oxidation-reduction potential

O-PP:

Poxidative pentose phosphate pathway

PPP:

Pentose phosphate pathway

pRH290:

Cloning plasmid containing STB5

pRH452:

Cloning plasmid containing PGI1

pRH483:

Cloning plasmid containing NLS-xlR-linker-SSN6 repression protein for TEF-X2-1

pRH506:

Plasmid containing TEF-X2-1

pRH635:

Empty cloning plasmid

pRH759:

Cloning plasmid containing TEF-X2-1pRH759: Cloning plasmid containing TEF-X2-1

pRH760:

Cloning plasmid containing STB5 constitutively expressed under TEF-X2-1

pRH761:

Cloning plasmid containing PGI1 constitutively expressed under TEF-X2-1

pRH762:

Cloning plasmid containing STB5 and PGI1 constitutively expressed under TEF-X2-1

pRH769:

Final plasmid expressing STB5 and PGI1 under xylose inducible control of TEF-X2-1

pRS418:

Empty control plasmid

qRT-PCR:

Quantitative reverse transcriptase polymerase chain reaction

SSF:

Simultaneous saccharification and fermentation

TEF-X2-1:

A novel xylose-inducible promoter construct

XDH:

Xylitol dehydrogenase

XI:

Xylose isomerase

XK:

Xylulokinase

XR:

Xylose reductase

YB-2625:

Industrial strain of strain of S. cerevisiae isolated from bagasse

YP:

Yeast peptone media

YPDYP:

Media with glucose carbon source

YPD + XYP:

Media with glucose and xylose carbon source

YPXYP:

Media with xylose carbon source

YRH1458:

Lab background (CEN.PK2-1C) with integrated XR

XDH:

XK and empty control vector

YRH1460:

Lab background (CEN.PK2-1C) with integrated XR

XDH:

XK and xylose inducible PGI1 and STB5

YRH1461:

Industrial background (YB-2625) with integrated XR

XDH:

XK and empty control vector

YRH1463:

Industrial background (YB-2625) with integrated XR

XDH:

XK and xylose-inducible PGI1 and STB5

YRH1464:

Industrial background (D5A) with integrated XR

XDH:

XK and empty control vector

YRH1466:

Industrial background (D5A) with integrated XR

XDH:

XK and xylose-inducible PGI1 and STB5

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Funding

Partial funding for this study was provided through a multistate hatch grant from Oregon State University Agricultural Experiment Station to the corresponding author.

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Author notes

  1. William Hohenschuh

    Present address: EMD Serono Inc., Rockland, MA, 02370, USA

  2. Ganti S. Murthy

    Present address: Biosciences and Biomedical Engineering, Indian Institute of Technology-Indore, POD 1A704 Khandwa Road, Simrol, Madhya Pradesh, 453552, India

Authors and Affiliations

  1. Biological and Ecological Engineering, Oregon State University, 116 Gilmore Hall, Corvallis, OR, 97331, USA

    William Hohenschuh & Ganti S. Murthy

  2. USDA, ARS, NCAUR, Peoria, IL, 61604, USA

    Ronald E. Hector & Jeffrey A. Mertens

Authors
  1. William Hohenschuh
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  2. Ronald E. Hector
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Contributions

WH performed the research presented here and created the first draft of the manuscript. RH * provided training, guidance, and laboratory support for the strain production work and was involved in revising the manuscript. JM * provided training and laboratory support for the strain analysis and was involved in revising the manuscript. GM acted as the lead investigator and provided significant input on experimental methods during the strain characterization. He was also heavily involved in editing and formatting the manuscript. All authors read and approved the manuscript. *Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Corresponding author

Correspondence to Ganti S. Murthy.

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Hohenschuh, W., Hector, R.E., Mertens, J.A. et al. Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose. Syst Microbiol and Biomanuf 1, 42–57 (2021). https://doi.org/10.1007/s43393-020-00002-y

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  • DOI: https://doi.org/10.1007/s43393-020-00002-y

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