Integrated perspective on microbe-based production of itaconic acid: from metabolic and strain engineering to upstream and downstream strategies

ABSTRACT: The discovery of itaconic acid as a product of citric acid pyrolytic distillation in 1837 opened the possibility of using it as a polymer building block. Itaconic acid, featuring two carboxylic acids and an unsaturated group, can potentially be used as a building block in several chemical syntheses, with a particular emphasis on polymer manufacture. The elucidation of biochemical pathways originating from itaconic acid, first in Aspergillus terreus and, recently, in several species of the Ustilago genus, has intensified and diversified research focused on microbe-based itaconic acid production, including at an industrial scale. These efforts include the engineering of naturally producing species/strains along with the exploration of other species that do not naturally produce itaconic acid but may offer potential benefits. The use of renewable wastes or sugar-enriched residues as substrates to produce itaconic acid, from a circular bioeconomy perspective, is another important aspect of the advancements in microbial itaconic acid production. This review provides an overview of the achievements as well as the challenges concerning the engineering of the producing strains/species, substrate selection, optimisation of bioreactor operation, and downstream itaconic acid purification methods.info:eu-repo/semantics/publishedVersio

Genome-wide identification of Saccharomyces cerevisiae genes required for tolerance to acetic acid

<p>Abstract</p> <p>Background</p> <p>Acetic acid is a byproduct of <it>Saccharomyces cerevisiae </it>alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying <it>S. cerevisiae </it>tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory.</p> <p>Results</p> <p>The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake.</p> <p>Conclusions</p> <p>Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are novel candidate genes for genetic engineering to obtain more robust yeast strains against acetic acid toxicity. Among these genes there are number of transcription factors that are documented regulators of a large percentage of the genes found to exert protection against acetic acid thus being considered interesting targets for subsequent genetic engineering. The increase of potassium concentration in the growth medium was found to improve the expression of maximal tolerance to acetic acid, consistent with the idea that the adequate manipulation of nutrient concentration of industrial growth medium can be an interesting strategy to surpass the deleterious effects of this weak acid in yeast cells.</p

Insights into the transcriptional regulation of poorly characterized alcohol acetyltransferase-encoding genes (HgAATs) shed light into the production of acetate esters in the wine yeast Hanseniaspora guilliermondii

Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.info:eu-repo/semantics/publishedVersio

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates

The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.The authors thank Juan Carlos Parajo and Hector Ruiz for assistance in the pre-treatment of lignocellulose biomass. Research described in this article was financially supported by FEDER and "Fundacao para a Ciencia e a Tecnologia" (FCT) (Contracts PEst-OE/EQB/LA0023/2011, PTDC/BIO/66151/2006, PTDC/AGR-ALI/102608/2008 and ERA-IB/0002/2010 and PhD grant (SFRH/BD/64776/2009) to FP)

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards inhibitory compounds in industrial biomass hydrolysates

The understanding of the determinants of yeast tolerance to inhibitory compounds present in fermentation media at the genetic level is of essential importance for the improvement of second generation bio-ethanol conversion technology. The aim of this study was to systematically identify, at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors derived from lignocellulose biomass pre-treatment. Based on the screening of the EUROSCARF haploid mutant collection, 242 and 216 determinants of yeast resistance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate (SH) were identified, respectively. Twenty-two mutants with deleted genes involved in Oxidative stress response, Lipid Metabolism, Aminoacid metabolism, Vacuolar acidification, Intracellular trafficking and protein sorting, Transcription machinery and RNA processing and Mitochondrial function showed a strong susceptibility phenotype in both WSH and SH, 8 of them being for the first time identified as conferring resistance to lignocellulose-derived inhibitors. The intersection of our WSH and SH datasets and those obtained in previous genome-wide studies on single chemical stress resistance, together with results obtained during comparative fermentative performance analysis [1], provided data for further evaluation of the key genes involved in global adaptation to toxic biomass hydrolysates. This study expands our understanding of the genes and underlying molecular mechanisms that are directly involved in yeast response to the multiple stresses occurring during lignocellulose fermentations under industrially relevant conditions

Genetic adaptive mechanisms mediating response and tolerance to acetic acid stress in the human pathogen Candida glabrata: role of the CgHaa1-dependent signaling pathway

The increased resilience of Candida glabrata to azoles and the continuous emergence of strains resistant to other antifungals demands the development of new therapeutic approaches focused on non-conventional biological targets. Genes contributing to increase C. glabrata competitiveness in the different infection sites are an interesting and unexplored cohort of therapeutic targets. To thrive in the vaginal tract and avoid exclusion C. glabrata cells have evolved dedicated responses rendering them capable of tolerating multiple environmental challenges, including the presence of acetic and lactic acids produced by the commensal microbiota. In this work a cohort of vaginal clinical isolates were phenotyped for their tolerance to acetic acid stress at a low pH as well as for several traits that are known to influence sensitivity to this organic acid, including the structure of the cell envelope and the ability to consume the acid in the presence of glucose. The role played by the ORF CAGL0L09339g, an homologue of the ScHaa1, a critical regulator of acetic acid resistance in S. cerevisiae[1], in C. glabrata response and tolerance to acetic acid stress at pH 4 was also scrutinized using a transcriptomic analysis. The role of CgHaa1 as well as of several of its target genes in mediating virulence of C. glabrata against epithelial vaginal cells was also studied.Funding received by the Institute for Bioengineering and Biosciences from the Portuguese Foundation for Science and Technology (FCT) (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (project no. 007317) is acknowledged. FCT is also acknowledged for funding the Centre of Biological Engineering through contracts FCOMP-01-0124-FEDER- 020243 and PTDC/EBB-EBI/120495/2010info:eu-repo/semantics/publishedVersio

Transcriptomic and chemogenomic analyses unveil the essential role of Com2-regulon in response and tolerance of Saccharomyces cerevisiae to stress induced by sulfur dioxide

During vinification Saccharomyces cerevisiae cells are frequently exposed to high concentrations of sulfur dioxide (SO2) that is used to avoid overgrowth of unwanted bacteria or fungi present in the must. Up to now the characterization of the molecular mechanisms by which S. cerevisiae responds and tolerates SO2 was focused on the role of the sulfite efflux pump Ssu1 and investigation on the involvement of other players has been scarce, especially at a genome-wide level. In this work, we uncovered the essential role of the poorly characterized transcription factor Com2 in tolerance and response of S. cerevisiae to stress induced by SO2 at the enologically relevant pH of 3.5. Transcriptomic analysis revealed that Com2 controls, directly or indirectly, the expression of more than 80% of the genes activated by SO2, a percentage much higher than the one that could be attributed to any other stress-responsive transcription factor. Large-scale phenotyping of the yeast haploid mutant collection led to the identification of 50 Com2-targets contributing to the protection against SO2 including all the genes that compose the sulfate reduction pathway (MET3, MET14, MET16, MET5, MET10) and the majority of the genes required for biosynthesis of lysine (LYS2, LYS21, LYS20, LYS14, LYS4, LYS5, LYS1 and LYS9) or arginine (ARG5,6, ARG4, ARG2, ARG3, ARG7, ARG8, ORT1 and CPA1). Other uncovered determinants of resistance to SO2 (not under the control of Com2) included genes required for function and assembly of the vacuolar proton pump and enzymes of the antioxidant defense, consistent with the observed cytosolic and mitochondrial accumulation of reactive oxygen species in SO2-stressed yeast cells.This work was funded by INNOVINE&WINE, Norte-01-0145-FEDER-000038, co-financed by the European Region-al Development Fund (ERDF) through Norte 2020 and by ERFD through POCI-COMPETE 2020. Support received by FCT-Portuguese Foundationfor Science and Technology(PTDC/EXPL/AGR-TEC/1823/2013 and PTDC/AGR-TEC/3315/2014) and by INTERACT project –“Integrated Research in Environment, Agro-Chain and Technology”, no. NORTE-01-0145-FEDER-000017, in its line of research enti-tled VitalityWine. Supportreceived by Biosystems and In-tegrative Sciences Institute (BioISI; FCT/UID/Multi/04046/2018) and iBB-Institute for Bioengi-neering and Biosciences (UID/BIO/04565/2019) by FCT and from Programa Operacional Regional de Lisboa 2020 (pro-ject no. 007317 and PTDC/AGR-TEC/3315/2014_LISBOA-01-0145-FEDER-016834) is also acknowledged.The authors thank Professor Isabel Sá-Correia for the help and guidance in conducting the chemogenomic analysi

MOMO - multi-objective metabolic mixed integer optimization : application to yeast strain engineering

BACKGROUND: In this paper, we explore the concept of multi-objective optimization in the field of metabolic engineering when both continuous and integer decision variables are involved in the model. In particular, we propose a multi-objective model that may be used to suggest reaction deletions that maximize and/or minimize several functions simultaneously. The applications may include, among others, the concurrent maximization of a bioproduct and of biomass, or maximization of a bioproduct while minimizing the formation of a given by-product, two common requirements in microbial metabolic engineering. RESULTS: Production of ethanol by the widely used cell factory Saccharomyces cerevisiae was adopted as a case study to demonstrate the usefulness of the proposed approach in identifying genetic manipulations that improve productivity and yield of this economically highly relevant bioproduct. We did an in vivo validation and we could show that some of the predicted deletions exhibit increased ethanol levels in comparison with the wild-type strain. CONCLUSIONS: The multi-objective programming framework we developed, called MOMO, is open-source and uses POLYSCIP (Available at http://polyscip.zib.de/). as underlying multi-objective solver. MOMO is available at http://momo-sysbio.gforge.inria.fr

Identification of Saccharomyces cerevisiae genes involved in the resistance to multiple stresses during Very-High-Gravity and lignocellulosic biomass industrial fermentations

Most of the current processes for bioethanol production are based on the use of Very-High-Gravity (VHG) technology and the processing of lignocellulosic biomass, limited by the high osmotic pressure and ethanol concentration in the fermentation medium, and by inhibitors resulting from biomass pre-treatments, respectively. Aiming the optimization of strains for industrial bioethanol production an integrated approach was undertaken to identify genes required for simultaneous yeast resistance to different fermentation-related stresses. The integration of previous chemogenomics data was used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to different inhibitors present during lignocellulosic fermentations. The expression of BUD31 and HPR1 lead to the increase of both ethanol yield and fermentation rate, while PHO85, VRP1 and YGL024w expression is required for maximal ethanol production in VHG fermentations. Five genes, ERG2, PRS3, RAV1, RPB4 and VMA8 were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or for maximal fermentation rate of this substrate [1]. Moreover, the yeast disruptome was screened for strains with increased susceptibility to inhibitory compounds present in an industrial lignocellulosic hydrolysate obtained from wheat straw. With this genome-wide analysis, 42 determinants of resistance to inhibitors were identified showing a high susceptibility phenotype compared to the parental strain. The identified genes stand as preferential targets for genetic engineering manipulation to generate more robust and efficient industrial strains

Identification of candidate genes for yeast engineering to improve bioethanol production in very high gravity and lignocellulosic biomass industrial fermentations

<p>Abstract</p> <p>Background</p> <p>The optimization of industrial bioethanol production will depend on the rational design and manipulation of industrial strains to improve their robustness against the many stress factors affecting their performance during very high gravity (VHG) or lignocellulosic fermentations. In this study, a set of <it>Saccharomyces cerevisiae </it>genes found, through genome-wide screenings, to confer resistance to the simultaneous presence of different relevant stresses were identified as required for maximal fermentation performance under industrial conditions.</p> <p>Results</p> <p>Chemogenomics data were used to identify eight genes whose expression confers simultaneous resistance to high concentrations of glucose, acetic acid and ethanol, chemical stresses relevant for VHG fermentations; and eleven genes conferring simultaneous resistance to stresses relevant during lignocellulosic fermentations. These eleven genes were identified based on two different sets: one with five genes granting simultaneous resistance to ethanol, acetic acid and furfural, and the other with six genes providing simultaneous resistance to ethanol, acetic acid and vanillin. The expression of <it>Bud31 </it>and <it>Hpr1 </it>was found to lead to the increase of both ethanol yield and fermentation rate, while <it>Pho85</it>, <it>Vrp1 </it>and <it>Ygl024w </it>expression is required for maximal ethanol production in VHG fermentations. Five genes, <it>Erg2</it>, <it>Prs3</it>, <it>Rav1</it>, <it>Rpb4 </it>and <it>Vma8</it>, were found to contribute to the maintenance of cell viability in wheat straw hydrolysate and/or the maximal fermentation rate of this substrate.</p> <p>Conclusions</p> <p>The identified genes stand as preferential targets for genetic engineering manipulation in order to generate more robust industrial strains, able to cope with the most significant fermentation stresses and, thus, to increase ethanol production rate and final ethanol titers.</p