Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate
Artikel i vetenskaplig tidskrift, 2022

Background: Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars—in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. Results: The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes (SNG1, FIT3, FZF1 and CBP3) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. Conclusions: The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.


Next generation sequencing


Industrial yeast

Hardwood spent sulfite liquor

Adaptive evolution

Dicarboxylic acids


Vratislav Stovicek

Danmarks Tekniske Universitet (DTU)

Laura Dato

River Stone Biotech ApS

Danmarks Tekniske Universitet (DTU)

Henrik Almqvist

Institutionen för Kemiteknik

Marie Schöpping

Institutionen för Kemiteknik

Chr. Hansen

Chalmers, Biologi och bioteknik, Industriell bioteknik

Ksenia Chekina

Danmarks Tekniske Universitet (DTU)

Lasse Ebdrup Pedersen

Danmarks Tekniske Universitet (DTU)

Anna Koza

Chr. Hansen

Danmarks Tekniske Universitet (DTU)

Diogo Figueira

Biotrend S.A.

Freddy Tjosås

Borregaard ApS

Bruno Sommer Ferreira

Biotrend S.A.

J. Forster

Danmarks Tekniske Universitet (DTU)

G. Liden

Institutionen för Kemiteknik

I. Borodina

Danmarks Tekniske Universitet (DTU)

Biotechnology for Biofuels and Bioproducts

27313654 (eISSN)

Vol. 15 1 22


Kemiska processer


Biokatalys och enzymteknik





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