Iron bioleaching and polymers accumulation by an extreme acidophilic bacterium.

In many European regions, both local metallic and non-metallic raw materials are poorly exploited due to their low quality and the lack of technologies to increase their economic value. In this context, the development of low cost and eco-friendly approaches, such as bioleaching of metal impurities, is crucial. The acidophilic strain Acidiphilium sp. SJH reduces Fe(III) to Fe(II) by coupling the oxidation of an organic substrate to the reduction of Fe(III) and can therefore be applied in the bioleaching of iron impurities from non-metallic raw materials. In this work, the physiology of Acidiphilium sp. SJH and the reduction of iron impurities from quartz sand and its derivatives have been studied during growth on media supplemented with various carbon sources and under different oxygenation conditions, highlighting that cell physiology and iron reduction are tightly coupled. Although the organism is known to be aerobic, maximum bioleaching performance was obtained by cultures cultivated until the exponential phase of growth under oxygen limitation. Among carbon sources, glucose has been shown to support faster biomass growth, while galactose allowed highest bioleaching. Moreover, Acidiphilium sp. SJH cells can synthesise and accumulate Poly-ß-hydroxybutyrate (PHB) during the process, a polymer with relevant application in biotechnology. In summary, this work gives an insight into the physiology of Acidiphilium sp. SJH, able to use different carbon sources and to synthesise a technologically relevant polymer (PHB), while removing metals from sand without the need to introduce modifications in the process set up.

Valorisation of cotton post-industrial textile waste into lactic acid: chemo-mechanical pretreatment, separate hydrolysis and fermentation using engineered yeast.

BACKGROUND: The textile industry has several negative impacts, mainly because it is based on a linear business model that depletes natural resources and produces excessive amounts of waste. Globally, about 75% of textile waste is disposed of in landfills and only 25% is reused or recycled, while less than 1% is recycled back into new garments. In this study, we explored the valorisation of cotton fabric waste from an apparel textile manufacturing company as valuable biomass to produce lactic acid, a versatile chemical building block. RESULTS: Post-industrial cotton patches were pre-treated with the aim of developing a methodology applicable to the industrial site involved. First, a mechanical shredding machine reduced the fabric into individual fibres of maximum 35 mm in length. Afterwards, an alkaline treatment was performed, using NaOH at different concentrations, including a 16% (w/v) NaOH enriched waste stream from the mercerisation of cotton fabrics. The combination of chemo-mechanical pre-treatment and enzymatic hydrolysis led to the maximum recovery yield of 90.46 ± 3.46%, corresponding to 74.96 ± 2.76 g/L of glucose released, which represents a novel valorisation of two different side products (NaOH enriched wastewater and cotton textile waste) of the textile industry. The Saccharomyces cerevisiae strain CEN.PK m850, engineered for redirecting the natural alcoholic fermentation towards a homolactic fermentation, was then used to valorise the glucose-enriched hydrolysate into lactic acid. Overall, the process produced 53.04 g/L ± 0.34 of L-lactic acid, with a yield of 82.7%, being the first example of second-generation biomass valorised with this yeast strain, to the best of our knowledge. Remarkably, the fermentation performances were comparable with the ones obtained in the control medium. CONCLUSION: This study validates the exploitation of cotton post-industrial waste as a possible feedstock for the production of commodity chemicals in microbial cell-based biorefineries. The presented strategy demonstrates the possibility of implementing a circular bioeconomy approach in manufacturing textile industries.

Impact of ergosterol content on acetic and lactic acids toxicity to Saccharomyces cerevisiae.

Organic acid stress often represents a major hurdle in industrial bio-based microbial processes. Organic acids can be released from lignocellulosic feedstocks pretreatment and can also be desirable products obtained by microbial fermentation with applications in different industrial sectors. Yeasts are prominent cell factories. However, the presence of organic acids can compromise yeast metabolism, impairing fermentation performances and limiting the economic feasibility of the processes. Plasma membrane remodeling is deeply involved in yeast tolerance to organic acids, but the detailed mechanisms and potentials of this phenomenon remain largely to be studied and exploited. We investigated the impact of ergosterol on Saccharomyces cerevisiae tolerance against organic acid stress by coupling in vitro and in vivo assays. In the in vitro assay, synthetic lipid vesicles were prepared containing different concentrations of ergosterol. We observed changes in organic acids diffusion through the membrane as a function of ergosterol content. Then, we extended our approach in vivo, engineering S. cerevisiae with the aim of changing the ergosterol content of cells. We focused on ECM22, an important transcription factor, involved in the regulation of ergosterol biosynthesis. The overexpression of ECM22 was sufficient to increase ergosterol levels in S. cerevisiae, resulting in an enhanced tolerance toward lactic acid stress. In this work we propose an in vitro approach, using synthetic lipid vesicles, as a complementary method to be used when studying the impact of the plasma membrane lipid composition on the diffusion of organic acids.

pCEC-red: a new vector for easier and faster CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

CRISPR-Cas9 technology is widely used for precise and specific editing of Saccharomyces cerevisiae genome to obtain marker-free engineered hosts. Targeted double-strand breaks are controlled by a guide RNA (gRNA), a chimeric RNA containing a structural segment for Cas9 binding and a 20-mer guide sequence that hybridises to the genomic DNA target. Introducing the 20-mer guide sequence into gRNA expression vectors often requires complex, time-consuming, and/or expensive cloning procedures. We present a new plasmid for CRISPR-Cas9 genome editing in S. cerevisiae, pCEC-red. This tool allows to (i) transform yeast with both Cas9 and gRNA expression cassettes in a single plasmid and (ii) insert the 20-mer sequence in the plasmid with high efficiency, thanks to Golden Gate Assembly and (iii) a red chromoprotein-based screening to speed up the selection of correct plasmids. We tested genome-editing efficiency of pCEC-red by targeting the ADE2 gene. We chose three different 20-mer targets and designed two types of repair fragments to test pCEC-red for precision editing and for large DNA region replacement procedures. We obtained high efficiencies (∼90%) for both engineering procedures, suggesting that the pCEC system can be used for fast and reliable marker-free genome editing.

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

L-lactate production in engineered Saccharomyces cerevisiae using a multistage multiobjective automated design framework.

The design of alternative biodegradable polymers has the potential of severely reducing the environmental impact, cost and production time currently associated with the petrochemical industry. In fact, growing demand for renewable feedstock has recently brought to the fore synthetic biology and metabolic engineering. These two interdependent research areas focus on the study of microbial conversion of organic acids, with the aim of replacing their petrochemical-derived equivalents with more sustainable and efficient processes. The particular case of Lactic acid (LA) production has been the subject of extensive research because of its role as an essential component for developing an eco-friendly biodegradable plastic-widely used in industrial biotechnological applications. Because of its resistance to acidic environments, among the many LA-producing microbes, Saccharomyces cerevisiae has been the main focus of research into related biocatalysts. In this study, we present an extensive in silico investigation of S. cerevisiae cell metabolism (modeled with Flux Balance Analysis) with the overall aim of maximizing its LA production yield. We focus on the yeast 8.3 steady-state metabolic model and analyze it under the impact of different engineering strategies including: gene knock-in, gene knock-out, gene regulation and medium optimization; as well as a comparison between results in aerobic and anaerobic conditions. We designed ad-hoc constrained multiobjective evolutionary algorithms to automate the engineering process and developed a specific postprocessing methodology to analyze the genetic manipulation results obtained. The in silico results reported in this paper empirically show that our method is able to automatically select a small number of promising genetic and metabolic manipulations, deriving competitive strains that promise to impact microorganisms design in the production of sustainable chemicals.

Enzymatic Modification of Cellulose To Unlock Its Exploitation in Advanced Materials.

Nowadays natural biopolymers have a wide variety of uses in various industrial applications, such as food, adhesives and composite materials. Among them, cellulose has attracted the interest of researchers due to its properties: high strength and flexibility, biocompatibility and nontoxicity. Despite that, in many cases its practical use is limited because of poor solubility and/or an unsuitable hydrophilic/hydrophobic balance. In this context, enzymatic modification appears as a powerful strategy to overcome these problems through selective, green and environmentally friendly processes. This minireview discusses the different methods developed for the enzymatic modification of cellulose, emphasizing the type of reaction, the enzymes used (laccases, esterases, lipases, hexokinases, etc.), and the properties and applications of the cellulose derivatives obtained. Considering that cellulose is the most abundant natural polymer on Earth and can be derived from residual lignocellulosic biomass, the impact of its use in bio-based process following the logic of the circular economy is relevant.

Insights on life cycle and cell identity regulatory circuits for unlocking genetic improvement in Zygosaccharomyces and Kluyveromyces yeasts.

Evolution has provided a vast diversity of yeasts that play fundamental roles in nature and society. This diversity is not limited to genotypically homogeneous species with natural interspecies hybrids and allodiploids that blur species boundaries frequently isolated. Thus, life cycle and the nature of breeding systems have profound effects on genome variation, shaping heterozygosity, genotype diversity and ploidy level. The apparent enrichment of hybrids in industry-related environments suggests that hybridization provides an adaptive route against stressors and creates interest in developing new hybrids for biotechnological uses. For example, in the Saccharomyces genus where regulatory circuits controlling cell identity, mating competence and meiosis commitment have been extensively studied, this body of knowledge is being used to combine interesting traits into synthetic F1 hybrids, to bypass F1 hybrid sterility and to dissect complex phenotypes by bulk segregant analysis. Although these aspects are less known in other industrially promising yeasts, advances in whole-genome sequencing and analysis are changing this and new insights are being gained, especially in the food-associated genera Zygosaccharomyces and Kluyveromyces. We discuss this new knowledge and highlight how deciphering cell identity circuits in these lineages will contribute significantly to identify the genetic determinants underpinning complex phenotypes and open new avenues for breeding programmes.

Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch.

Many interspecies hybrids have been discovered in yeasts, but most of these hybrids are asexual and can replicate only mitotically. Whole-genome duplication has been proposed as a mechanism by which interspecies hybrids can regain fertility, restoring their ability to perform meiosis and sporulate. Here, we show that this process occurred naturally during the evolution of Zygosaccharomyces parabailii, an interspecies hybrid that was formed by mating between 2 parents that differed by 7% in genome sequence and by many interchromosomal rearrangements. Surprisingly, Z. parabailii has a full sexual cycle and is genetically haploid. It goes through mating-type switching and autodiploidization, followed by immediate sporulation. We identified the key evolutionary event that enabled Z. parabailii to regain fertility, which was breakage of 1 of the 2 homeologous copies of the mating-type (MAT) locus in the hybrid, resulting in a chromosomal rearrangement and irreparable damage to 1 MAT locus. This rearrangement was caused by HO endonuclease, which normally functions in mating-type switching. With 1 copy of MAT inactivated, the interspecies hybrid now behaves as a haploid. Our results provide the first demonstration that MAT locus damage is a naturally occurring evolutionary mechanism for whole-genome duplication and restoration of fertility to interspecies hybrids. The events that occurred in Z. parabailii strongly resemble those postulated to have caused ancient whole-genome duplication in an ancestor of Saccharomyces cerevisiae.

Conversion of sugar beet residues into lipids by Lipomyces starkeyi for biodiesel production.

BACKGROUND: Lipids from oleaginous yeasts emerged as a sustainable alternative to vegetable oils and animal fat to produce biodiesel, the biodegradable and environmentally friendly counterpart of petro-diesel fuel. To develop economically viable microbial processes, the use of residual feedstocks as growth and production substrates is required. RESULTS: In this work we investigated sugar beet pulp (SBP) and molasses, the main residues of sugar beet processing, as sustainable substrates for the growth and lipid accumulation by the oleaginous yeast Lipomyces starkeyi. We observed that in hydrolysed SBP the yeast cultures reached a limited biomass, cellular lipid content, lipid production and yield (2.5 g/L, 19.2%, 0.5 g/L and 0.08 g/g, respectively). To increase the initial sugar availability, cells were grown in SBP blended with molasses. Under batch cultivation, the cellular lipid content was more than doubled (47.2%) in the presence of 6% molasses. Under pulsed-feeding cultivation, final biomass, cellular lipid content, lipid production and lipid yield were further improved, reaching respectively 20.5 g/L, 49.2%, 9.7 g/L and 0.178 g/g. Finally, we observed that SBP can be used instead of ammonium sulphate to fulfil yeasts nitrogen requirement in molasses-based media for microbial oil production. CONCLUSIONS: This study demonstrates for the first time that SBP and molasses can be blended to create a feedstock for the sustainable production of lipids by L. starkeyi. The data obtained pave the way to further improve lipid production by designing a fed-batch process in bioreactor.

The Saccharomyces cerevisiae poly (A) binding protein (Pab1): Master regulator of mRNA metabolism and cell physiology.

Pab1, the major poly (A) binding protein of the yeast Saccharomyces cerevisiae, is involved in many intracellular functions associated with mRNA metabolism, such as mRNA nuclear export, deadenylation, translation initiation and termination. Pab1 consists of four RNA recognition motifs (RRM), a proline-rich domain (P) and a carboxy-terminal (C) domain. Due to its modular structure, Pab1 can simultaneously interact with poly (A) tails and different proteins that regulate mRNA turnover and translation. Furthermore, Pab1 also influences cell physiology under stressful conditions by affecting the formation of quinary assemblies and stress granules, as well as by stabilizing specific mRNAs to allow translation re-initiation after stress. The main goal of this review is to correlate the structural complexity of this protein with the multiplicity of its functions.

Copper homeostasis as a target to improve Saccharomyces cerevisiae tolerance to oxidative stress.

The yeast Saccharomyces cerevisiae is widely used as a cell factory for the biotechnological production of various industrial products. During these processes, yeasts meet different kinds of stressors that often cause oxidative stress and thus impair cell growth. Therefore, the development of robust strains is indispensable to improve production, yield and productivity of fermentative processes. Copper plays a key role in the response to oxidative stress, as cofactor of the cytosolic superoxide dismutase (Sod1) and being contained in metallochaperone and metallothioneines with antioxidant properties. In this work, we observed a higher naturally copper internalization in a robust S. cerevisiae strain engineered to produce the antioxidant l-ascorbic acid (L-AA), compared with the wild type strain. Therefore, we investigated the effect of the alteration of copper homeostasis on cellular stress tolerance. CTR1 and FRE1 genes, codifying for a plasma membrane high-affinity copper transporter and for a cell-surface ferric/cupric reductase, respectively, were overexpressed in both wild type and L-AA cells. Remarkably, we found that the sole FRE1 overexpression was sufficient to increase copper internalization leading to an enhanced stress tolerance toward H2O2 exposure, in both strains under investigation. These findings reveal copper homeostasis as a target for the development of robust cell factories.

Transcriptional Response to Lactic Acid Stress in the Hybrid Yeast Zygosaccharomyces parabailii.

Lactic acid has a wide range of applications starting from its undissociated form, and its production using cell factories requires stress-tolerant microbial hosts. The interspecies hybrid yeast Zygosaccharomyces parabailii has great potential to be exploited as a novel host for lactic acid production, due to high organic acid tolerance at low pH and a fermentative metabolism with a high growth rate. Here we used mRNA sequencing (RNA-seq) to analyze Z. parabailii's transcriptional response to lactic acid added exogenously, and we explore the biological mechanisms involved in tolerance. Z. parabailii contains two homeologous copies of most genes. Under lactic acid stress, the two genes in each homeolog pair tend to diverge in expression to a significantly greater extent than under control conditions, indicating that stress tolerance is facilitated by interactions between the two gene sets in the hybrid. Lactic acid induces downregulation of genes related to cell wall and plasma membrane functions, possibly altering the rate of diffusion of lactic acid into cells. Genes related to iron transport and redox processes were upregulated, suggesting an important role for respiratory functions and oxidative stress defense. We found differences in the expression profiles of genes putatively regulated by Haa1 and Aft1/Aft2, previously described as lactic acid responsive in Saccharomyces cerevisiae Furthermore, formate dehydrogenase (FDH) genes form a lactic acid-responsive gene family that has been specifically amplified in Z. parabailii in comparison to other closely related species. Our study provides a useful starting point for the engineering of Z. parabailii as a host for lactic acid production.IMPORTANCE Hybrid yeasts are important in biotechnology because of their tolerance to harsh industrial conditions. The molecular mechanisms of tolerance can be studied by analyzing differential gene expression under conditions of interest and relating gene expression patterns to protein functions. However, hybrid organisms present a challenge to the standard use of mRNA sequencing (RNA-seq) to study transcriptional responses to stress, because their genomes contain two similar copies of almost every gene. Here we used stringent mapping methods and a high-quality genome sequence to study the transcriptional response to lactic acid stress in Zygosaccharomyces parabailii ATCC 60483, a natural interspecies hybrid yeast that contains two complete subgenomes that are approximately 7% divergent in sequence. Beyond the insights we gained into lactic acid tolerance in this study, the methods we developed will be broadly applicable to other yeast hybrid strains.

The spoilage yeast Zygosaccharomyces bailii: Foe or friend?

Zygosaccharomyces bailii is a non-Saccharomyces budding yeast known as one of the most aggressive food spoilage microorganisms, often isolated as a contaminant during wine fermentation, as well as from many acidic, high-sugar and canned foods. The spoilage ability relies on the yeast's unique feature of tolerating the most common preservatives such as sulphite, dimethyl dicarbonate, acetic acid and sorbic acid. Therefore, many studies have focused on the description of this peculiar tolerance with the aim of developing preventative measures against Z. bailii food spoilage. These studies demonstrated the involvement of diverse molecular and physiological mechanisms in the yeast resistance, comprising detoxification of preservatives, adaptation of the cytoplasmic pH and modulation of the cell wall/membrane composition. At the same time, the described traits unveiled Z. bailii as a novel potential workhorse for industrial bioprocesses. Here we present the yeast Z. bailii starting from important aspects of its robustness and concluding with the exploitation of its potential in biotechnology. Overall, the article describes Z. bailii from different perspectives, converging in presenting it as one of the most interesting species of the Saccharomycotina subphylum. Copyright © 2017 John Wiley & Sons, Ltd.

The recruitment of the Saccharomyces cerevisiae poly(A)-binding protein into stress granules: new insights into the contribution of the different protein domains.

The Saccharomyces cerevisiae poly(A)-binding protein Pab1 is a modular protein composed of four RNA recognition motifs (RRM), a proline-rich domain (P) and a C-terminus. Thanks to this modularity, Pab1 is involved in different interactions that regulate many aspects of mRNA metabolism, including the assembly of stress granules. In this work, we analyzed the contribution of each domain for the recruitment of the protein within stress granules by comparing the intracellular distribution of synthetic Pab1-GFP variants, lacking one or more domains, with the localization of the endogenous mCherry-tagged Pab1. Glucose starvation and heat shock were used to trigger the formation of stress granules. We found that Pab1 association into these aggregates relies mainly on RRMs, whose number is important for an efficient recruitment of the protein. Interestingly, although the P and C domains do not directly participate in Pab1 association to stress granules, their presence strengthens or decreases, respectively, the distribution of synthetic Pab1 lacking at least one RRM into these aggregates. In addition to describing the contribution of domains in determining Pab1 association within stress granules, the outcomes of this study suggest the modularity of Pab1 as an attractive platform for synthetic biology approaches aimed at rewiring mRNA metabolism.

Temperature-induced lipocalin (TIL): a shield against stress-inducing environmental shocks in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a well-established workhorse, either for recombinant or natural products, thanks to its natural traits and easily editable metabolism. However, during a bio-based industrial process it meets multiple stresses generated by operative conditions such as non-optimal temperature, pH, oxygenation and product accumulation. The development of tolerant strains is therefore indispensable for the improvement of production, yield and productivity of fermentative processes. In this regard, plants as resilient organisms are a generous source for fishing genes and/or metabolites that can help the cell factory to counteract environmental constraints. Plants possess proteins named temperature-induced lipocalins, TIL, whose levels in the cells correlates with the tolerance to sudden temperature changes and with the scavenging of reactive oxygen species. In this work, the gene encoding for the Arabidopsis thaliana TIL protein was for the first time expressed in S. cerevisiae. The recombinant strain was compared and analysed against the parental counterpart under heat shock, freezing, exposure to organic acid and oxidative agents. In all the tested conditions, TIL expression conferred a higher tolerance to the stress imposed, making this strain a promising candidate for the development of robust cell factories able to overtake the major impairments of industrial processes.

De novo biosynthesis of trans-cinnamic acid derivatives in Saccharomyces cerevisiae.

The production of natural aroma compounds is an expanding field within the branch of white biotechnology. Three aromatic compounds of interest are cinnamaldehyde, the typical cinnamon aroma that has applications in agriculture and medical sciences, as well as cinnamyl alcohol and hydrocinnamyl alcohol, which have applications in the cosmetic industry. Current production methods, which rely on extraction from plant materials or chemical synthesis, are associated with drawbacks regarding scalability, production time, and environmental impact. These considerations make the development of a sustainable microbial-based production highly desirable. Through steps of rational metabolic engineering, we engineered the yeast Saccharomyces cerevisiae as a microbial host to produce trans-cinnamic acid derivatives cinnamaldehyde, cinnamyl alcohol, and hydrocinnamyl alcohol, from externally added trans-cinnamic acid or de novo from glucose as a carbon source. We show that the desired products can be de novo synthesized in S. cerevisiae via the heterologous overexpression of the genes encoding phenylalanine ammonia lyase 2 from Arabidopsis thaliana (AtPAL2), aryl carboxylic acid reductase (acar) from Nocardia sp., and phosphopantetheinyl transferase (entD) from Escherichia coli, together with endogenous alcohol dehydrogenases. This study provides a proof of concept and a strain that can be further optimized for production of high-value aromatic compounds.

Assessing physio-macromolecular effects of lactic acid on Zygosaccharomyces bailii cells during microaerobic fermentation.

The ability of Zygosaccharomyces bailii to grow at low pH and in the presence of considerable amounts of weak organic acids, at lethal condition for Saccharomyces cerevisiae, increased the interest in the biotechnological potential of the yeast. To understand the mechanism of tolerance and growth effect of weak acids on Z. bailii, we evaluated the physiological and macromolecular changes of the yeast exposed to sub lethal concentrations of lactic acid. Lactic acid represents one of the important commodity chemical which can be produced by microbial fermentation. We assessed physiological effect of lactic acid by bioreactor fermentation using synthetic media at low pH in the presence of lactic acid. Samples collected from bioreactors were stained with propidium iodide (PI) which revealed that, despite lactic acid negatively influence the growth rate, the number of PI positive cells is similar to that of the control. Moreover, we have performed Fourier Transform Infra-Red (FTIR) microspectroscopy analysis on intact cells of the same samples. This technique has been never applied before to study Z. bailii under this condition. The analyses revealed lactic acid induced macromolecular changes in the overall cellular protein secondary structures, and alterations of cell wall and membrane physico-chemical properties.

Protein aggregation and membrane lipid modifications under lactic acid stress in wild type and OPI1 deleted Saccharomyces cerevisiae strains.

BACKGROUND: Lactic acid is a versatile chemical platform with many different industrial applications. Yeasts have been demonstrated as attractive alternative to natural lactic acid producers since they can grow at low pH, allowing the direct purification of the product in the desired acidic form. However, when very high concentrations of organic acids are reached, the major limitation for a viable production is the toxic effect of the product. The accumulation in the cytosol of H(+) and of the weak organic counter-anions triggers a cellular reprogramming. Here, the effects of lactic acid exposure on Saccharomyces cerevisiae have been evaluated by Fourier transform infrared (FTIR) microspectroscopy. In addition to -omic techniques, describing these responses in terms of systems and networks, FTIR microspectroscopy allows a rapid acquisition of the cellular biochemical fingerprint, providing information on the major classes of macromolecules. RESULTS: FTIR analyses on Saccharomyces cerevisiae cells under lactic acid stress at low pH revealed some still uncharacterized traits: (1) a direct correlation between lactic acid exposure and a rearrangement in lipid hydrocarbon tails, together with a decrease in the signals of phosphatidylcholine (PC), one of the main components of cell membrane; (2) a rearrangement in the cell wall carbohydrates, including glucans and mannans (3) a significant yet transient protein aggregation, possibly responsible for the observed transient decrease of the growth rate. When repeated on the isogenic strain deleted in OPI1, encoding for a transcriptional repressor of genes involved in PC biosynthesis, FTIR analysis revealed that not only the PC levels were affected but also the cell membrane/wall composition and the accumulation of protein aggregates, resulting in higher growth rate in the presence of the stressing agent. CONCLUSIONS: This work revealed novel effects evoked by lactic acid on cell membrane/wall composition and protein aggregation in S. cerevisiae cells. We consequently demonstrated that the targeted deletion of OPI1 resulted in improved lactic acid tolerance. Considering that stress response involves many and different cellular networks and regulations, most of which are still not implemented in modelling, these findings constitute valuable issues for interpreting cellular rewiring and for tailoring ameliorated cell factories for lactic acid production.

Assessing an effective feeding strategy to optimize crude glycerol utilization as sustainable carbon source for lipid accumulation in oleaginous yeasts.

BACKGROUND: Microbial lipids can represent a valuable alternative feedstock for biodiesel production in the context of a viable bio-based economy. This production can be driven by cultivating some oleaginous microorganisms on crude-glycerol, a 10% (w/w) by-product produced during the transesterification process from oils into biodiesel. Despite attractive, the perspective is still economically unsustainable, mainly because impurities in crude glycerol can negatively affect microbial performances. In this view, the selection of the best cell factory, together with the development of a robust and effective production process are primary requirements. RESULTS: The present work compared crude versus pure glycerol as carbon sources for lipid production by three different oleaginous yeasts: Rhodosporidium toruloides (DSM 4444), Lipomyces starkeyi (DSM 70295) and Cryptococcus curvatus (DSM 70022). An efficient yet simple feeding strategy for avoiding the lag phase caused by growth on crude glycerol was developed, leading to high biomass and lipid production for all the tested yeasts. Flow-cytometry and fourier transform infrared (FTIR) microspectroscopy, supported by principal component analysis (PCA), were used as non-invasive and quick techniques to monitor, compare and analyze the lipid production over time. Gas chromatography (GC) analysis completed the quali-quantitative description. Under these operative conditions, the highest lipid content (up to 60.9% wt/wt) was measured in R. toruloides, while L. starkeyi showed the fastest glycerol consumption rate (1.05 g L(-1) h(-1)). Being productivity the most industrially relevant feature to be pursued, under the presented optimized conditions R. toruloides showed the best lipid productivity (0.13 and 0.15 g L(-1) h(-1) on pure and crude glycerol, respectively). CONCLUSIONS: Here we demonstrated that the development of an efficient feeding strategy is sufficient in preventing the inhibitory effect of crude glycerol, and robust enough to ensure high lipid accumulation by three different oleaginous yeasts. Single cell and in situ analyses allowed depicting and comparing the transition between growth and lipid accumulation occurring differently for the three different yeasts. These data provide novel information that can be exploited for screening the best cell factory, moving towards a sustainable microbial biodiesel production.