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

Abstract

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.

Valorisation of wine wastes by de novo biosynthesis of resveratrol using a recombinant xylose-consuming industrial Saccharomyces cerevisiae strain

“…In this strain, the 4 genes involved in the non-oxidative PPP reactions ( TAL1, RPE1, RKE1 and TKL1 ) were previously overexpressed. 47 When compared to L323, which is derived from the same Ethanol Red parent strain with the sole expression of the RBP, L326 produced 107.3 mg L −1 of resveratrol from 20 g L −1 of glucose, 22% higher than the 88.0 mg L −1 titre obtained with L323 (Fig. 2).…”

“…42 The strain L323 is described in Costa et al , 27 expressing the resveratrol biosynthetic pathway (RBP), consisting of the genes AtPAL2, At4CL2, AtC4H and VvVST1 . The strain L326 was developed by insertion of the RBP into a previously engineered xylose-consuming strain developed by Stovicek et al 38 The strain L543 was developed by (over)expression of the genes AtATR2 and ScCYB5 in the strain L326. A detailed description of the genotype of all the strains is provided in Table S4 †…”

“…Several studies focused on the overexpression of XK, either the native S. cerevisiae XKS1 27,28,34 or the Scheffersomyces stipitis XYL3, 35 as well as the non-oxidative PPP genes (RPE1, RKI1, TKL1, TAL1) to enhance xylose assimilation. 27,28,33,[36][37][38] Besides its central role in xylose metabolism, the PPP is also vital to producing several valuable compounds like polyols, biofuels or phenylpropanoids. The PPP is tightly related to several metabolic steps of glycolysis, either by the assimilation of glucose-6-phosphate in the oxidative part of the PPP or the reversible reactions of the non-oxidative PPP to form fructose-6-phosphate or glyceraldehyde-3-phosphate.…”

“…Several studies focused on the overexpression of XK, either the native S. cerevisiae XKS1 27,28,34 or the Scheffersomyces stipitis XYL3 , 35 as well as the non-oxidative PPP genes ( RPE1 , RKI1 , TKL1 , TAL1 ) to enhance xylose assimilation. 27,28,33,36–38…”

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts

“…Since early in human civilization, it has been extensively used in the production of fermented food and beverages, which nowadays include bread, chocolate, wine, beer, cider, sake, spirits (rum, vodka, whisky, brandy), and other alcoholic beverages arising from the fermentation of fruits, honey, and tea ( Walker and Stewart, 2016 ; Parapouli et al, 2020 ). Nowadays, many enzymes, pharmaceuticals, nutraceuticals, and other added-value bioproducts can be produced in engineered yeast cell factories ( Borodina and Nielsen, 2014 ; Runguphan and Keasling, 2014 ; Kwak and Jin, 2017 ; Nielsen, 2019 ; Fathi et al, 2021 ; Wang et al, 2021 ; Arnesen et al, 2022 ; Stovicek et al, 2022 ). The implementation of a sustainable circular bioeconomy requires the development of Advanced Yeast Biorefineries to produce biofuels (e.g., bioethanol and biodiesel), chemicals, materials, and other bioproducts from organic residues from agriculture, forestry, and industry residues ( Madhavan et al, 2017 ; Mukherjee et al, 2017 ; Yamakawa et al, 2018 ; Panahi et al, 2019 ; Saini and Sharma, 2021 ; Usmani et al, 2021 ; Raj et al, 2022 ).…”