[en] The genetic stability and metabolic robustness of production strains is one of the key criteria for the production of bio-based products by microbial fermentation on an industrial scale. These criteria were here explored in an industrial ethanol-producer strain of Saccharomyces cerevisiae able to co-ferment D-xylose and L-arabinose with glucose through the chromosomal integration of several copies of pivotal genes for the use of these pentose (C5) sugars. Using batch sequential cultures in a controlled bioreactor that mimics long-term fermentation in an industrial setting, this strain was found to exhibit significant fluctuations in D-xylose and L-arabinose consumption as early as the 50th generation and beyond. These fluctuations seem not related to the few low-consumption C5 sugar clones that appeared throughout the sequential batch cultures at a frequency lower than 1.5% and that were due to the reduction in the number of copies of transgenes coding for C5 sugar assimilation enzymes. Also, subpopulations enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work highlighted the existence of genetic and metabolic instabilities in an industrial yeast which, although modest in our conditions, could be more deleterious in harsher industrial conditions, leading to reduced production performance.
Disciplines :
Biotechnology
Author, co-author :
Duperray, Maëlle; Toulouse Biotechnology Institute, INSA/University of Toulouse, CNRS, INRAE, Toulouse, France
Delvenne, Mathéo ; Université de Liège - ULiège > TERRA Research Centre
François, Jean Marie; Toulouse Biotechnology Institute, INSA/University of Toulouse, CNRS, INRAE, Toulouse, France ; Toulouse White Biotechnology, INSA, INRAE, CNRS, Toulouse, France
Delvigne, Frank ; Université de Liège - ULiège > TERRA Research Centre > Microbial technologies
Capp, Jean-Pascal; Toulouse Biotechnology Institute, INSA/University of Toulouse, CNRS, INRAE, Toulouse, France
Language :
English
Title :
Genomic and metabolic instability during long-term fermentation of an industrial Saccharomyces cerevisiae strain engineered for C5 sugar utilization.
Capp J. P. (2010). Noise-driven heterogeneity in the rate of genetic-variant generation as a basis for evolvability. Genetics 185, 395–404. 10.1534/genetics.110.118190
Capp J. P. (2021). Interplay between genetic, epigenetic, and gene expression variability: considering complexity in evolvability. Evol. Appl. 14, 893–901. 10.1111/eva.13204
D'ambrosio V. Dore E. Di Blasi R. Van Den Broek M. Sudarsan S. Horst J. T. et al. (2020). Regulatory control circuits for stabilizing long-term anabolic product formation in yeast. Metab. Eng. 61, 369–380. 10.1016/j.ymben.2020.07.006
Delvigne F. Zune Q. Lara A. R. Al-Soud W. Sorensen S. J. (2014). Metabolic variability in bioprocessing: implications of microbial phenotypic heterogeneity. Trends Biotechnol. 32, 608–616. 10.1016/j.tibtech.2014.10.002
Demeke M. M. Dietz H. Li Y. Foulquié-Moreno M. R. Mutturi S. Deprez S. et al. (2013). Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering. Biotechnol. Biofuels 6, 89.
De Mol M. L. Marcoen V. Maryns I. Snoeck N. Beauprez J. J. De Maeseneire S. L. et al. (2023). Evaluation of long-term fermentation performance with engineered Saccharomyces cerevisiae strains. Fermentation 9, 721. 10.3390/fermentation9080721
Dietz H. (2013). Konstruktion und Charakterisierung von pentosefermentierenden Industriehefestämmen. PhD. Johann Wolfgang Goethe-Universität Frankfurt am Main.
Francois J. M. Alkim C. Morin N. (2020). Engineering microbial pathways for production of bio-based chemicals from lignocellulosic sugars: current status and perspectives. Biotechnol. Biofuels 13, 118. 10.1186/s13068-020-01744-6
Garcia Sanchez R. Karhumaa K. Fonseca C. Sànchez Nogué V. Almeida J. R. Larsson C. U. et al. (2010). Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering. Biotechnol. Biofuels 3, 13. 10.1186/1754-6834-3-13
Generoso W. C. Brinek M. Dietz H. Oreb M. Boles E. (2017). Secretion of 2,3-dihydroxyisovalerate as a limiting factor for isobutanol production in Saccharomyces cerevisiae. FEMS Yeast Res. 17. 10.1093/femsyr/fox029
Hastings P. J. Lupski J. R. Rosenberg S. M. Ira G. (2009). Mechanisms of change in gene copy number. Nat. Rev. Genet. 10, 551–564. 10.1038/nrg2593
Heieck K. Arnold N. D. Bruck T. B. (2023). Metabolic stress constrains microbial L-cysteine production in Escherichia coli by accelerating transposition through mobile genetic elements. Microb. Cell Fact. 22, 10. 10.1186/s12934-023-02021-5
Jacobus A. P. Gross J. Evans J. H. Ceccato-Antonini S. R. Gombert A. K. (2021). Saccharomyces cerevisiae strains used industrially for bioethanol production. Essays Biochem. 65, 147–161. 10.1042/ebc20200160
Laughery M. F. Hunter T. Brown A. Hoopes J. Ostbye T. Shumaker T. et al. (2015). New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae. Yeast 32, 711–720. 10.1002/yea.3098
Lee S. W. Rugbjerg P. Sommer M. O. A. (2021). Exploring selective pressure trade-offs for synthetic addiction to extend metabolite productive lifetimes in yeast. ACS Synth. Biol. 10, 2842–2849. 10.1021/acssynbio.1c00240
Liu J. Francois J. M. Capp J. P. (2019). Gene expression noise produces cell-to-cell heterogeneity in eukaryotic homologous recombination rate. Front. Genet. 10, 475. 10.3389/fgene.2019.00475
Lv Y. Gu Y. Xu J. Zhou J. Xu P. (2020). Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield. Metab. Eng. 61, 79–88. 10.1016/j.ymben.2020.05.005
Mu X. Zhang F. (2023). Diverse mechanisms of bioproduction heterogeneity in fermentation and their control strategies. J. Industrial Microbiol. Biotechnol. 50, kuad033. 10.1093/jimb/kuad033
Olsson L. Rugbjerg P. Torello Pianale L. Trivellin C. (2022). Robustness: linking strain design to viable bioprocesses. Trends Biotechnol. 40, 918–931. 10.1016/j.tibtech.2022.01.004
Rugbjerg P. Myling-Petersen N. Porse A. Sarup-Lytzen K. Sommer M. O. A. (2018). Diverse genetic error modes constrain large-scale bio-based production. Nat. Commun. 9, 787. 10.1038/s41467-018-03232-w
Rugbjerg P. Sommer M. O. A. (2019). Overcoming genetic heterogeneity in industrial fermentations. Nat. Biotechnol. 37, 869–876. 10.1038/s41587-019-0171-6
Ryan O. W. Poddar S. Cate J. H. (2016). CRISPR-Cas9 genome engineering in Saccharomyces cerevisiae cells. Cold Spring Harb. Protoc. 2016, prot086827. 10.1101/pdb.prot086827
Schreiber F. Ackermann M. (2020). Environmental drivers of metabolic heterogeneity in clonal microbial populations. Curr. Opin. Biotechnol. 62, 202–211. 10.1016/j.copbio.2019.11.018
Shirvani R. Barshan-tashnizi M. Shahali M. (2020). An investigation into gene copy number determination in transgenic yeast; the importance of selecting a reliable real-time PCR standard. Biologicals 65, 10–17. 10.1016/j.biologicals.2020.04.001
Solis-Escalante D. Kuijpers N. G. Bongaerts N. Bolat I. Bosman L. Pronk J. T. et al. (2013). amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae. FEMS Yeast Res. 13, 126–139. 10.1111/1567-1364.12024
Takhaveev V. Heinemann M. (2018). Metabolic heterogeneity in clonal microbial populations. Curr. Opin. Microbiol. 45, 30–38. 10.1016/j.mib.2018.02.004
Uphoff S. Lord N. D. Okumus B. Potvin-Trottier L. Sherratt D. J. Paulsson J. (2016). Stochastic activation of a DNA damage response causes cell-to-cell mutation rate variation. Science 351, 1094–1097. 10.1126/science.aac9786
Verduyn C. Postma E. Scheffers W. A. Van Dijken J. P. (1992). Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8, 501–517. 10.1002/yea.320080703
Xiao Y. Bowen C. H. Liu D. Zhang F. (2016). Exploiting nongenetic cell-to-cell variation for enhanced biosynthesis. Nat. Chem. Biol. 12, 339–344. 10.1038/nchembio.2046
Zhang H. Zeidler A. F. Song W. Puccia C. M. Malc E. Greenwell P. W. et al. (2013). Gene copy-number variation in haploid and diploid strains of the yeast Saccharomyces cerevisiae. Genetics 193, 785–801. 10.1534/genetics.112.146522
