Industrial Yeast Research Articles

Single nucleotide polymorphisms associated with wine fermentation and adaptation to nitrogen limitation in wild and domesticated yeast strains

For more than 20 years, Saccharomyces cerevisiae has served as a model organism for genetic studies and molecular biology, as well as a platform for biotechnology (e.g., wine production). One of the important ecological niches of this yeast that has been extensively studied is wine fermentation, a complex microbiological process in which S. cerevisiae faces various stresses such as limited availability of nitrogen. Nitrogen deficiencies in grape juice impair fermentation rate and yeast biomass production, leading to sluggish or stuck fermentations, resulting in considerable economic losses for the wine industry. In the present work, we took advantage of the “1002 Yeast Genomes Project” population, the most complete catalogue of the genetic variation in the species and a powerful resource for genotype-phenotype correlations, to study the adaptation to nitrogen limitation in wild and domesticated yeast strains in the context of wine fermentation. We found that wild and domesticated yeast strains have different adaptations to nitrogen limitation, corroborating their different evolutionary trajectories. Using a combination of state-of-the-art bioinformatic (GWAS) and molecular biology (CRISPR-Cas9) methodologies, we validated that PNP1, RRT5 and PDR12 are implicated in wine fermentation, where RRT5 and PDR12 are also involved in yeast adaptation to nitrogen limitation. In addition, we validated SNPs in these genes leading to differences in fermentative capacities and adaptation to nitrogen limitation. Altogether, the mapped genetic variants have potential applications for the genetic improvement of industrial yeast strains.

The impacts of nicotinamide and inositol on the available cells and product performance of industrial baker's yeasts

A suitable nutrient supply, especially of vitamins, is very significant for the deep display of the inherent genetic properties of microorganisms. Here, using the chemically defined minimal medium (MM) for yeast, nicotinamide and inositol were confirmed to be more beneficial for the performance of two industrial baker's yeasts, a conventional and a high-sugar-tolerant strain. Increasing nicotinamide or inositol to proper levels could enhance the both strains on cell growth and activity and product performance, including trehalose accumulation and leavening performance. The activity of key enzymes (PCK, TPS) and the content of intermediate metabolites (G6P, UDPG) in the trehalose synthesis pathway were promoted by a moderate supply of nicotinamide and inositol. That were also proved that an appropriate amount of niacinamide promoted the transcription of longevity-related genes (PNC1, SIR2), and the proper concentration of inositol altered the phospholipid composition in cells, namely, phosphatidylinositol and phosphatidyl choline. Furthermore, the cell growth and the leavening performance of the both strains were promoted after adjusting inositol to choline to the proper ratio, resulting directly in content changes of phosphatidylinositol and phosphatidyl choline in the cells. While the two strains responded to the different proper ratio of inositol to choline probably due to their specific physiological characteristics. Such beneficial effects of increased nicotinamide levels were confirmed in natural media, molasses and corn starch hydrolyzed sugar media. Meanwhile, such adjustment of inositol to choline ratio could lessen the inhibition of excess inositol on cell growth of the two tested strains in corn starch hydrolyzed sugar media. However, in molasse, such phenomenon was not observed probably since there was higher Ca2+ in it. The results indicated that the effects of nutrient factors, such as vitamins, on cell growth and other properties found out from the simple chemically defined minimal medium were an effective measure to use in improving the recipe of natural media at least for baker's yeast.

A seven-transmembrane methyltransferase catalysing N-terminal histidine methylation of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are oxidative enzymes that help break down lignocellulose, making them highly attractive for improving biomass utilization in industrial biotechnology. The catalytically essential N-terminal histidine (His1) of LPMOs is post-translationally modified by methylation in filamentous fungi to protect them from auto-oxidative inactivation, however, the responsible methyltransferase enzyme is unknown. Using mass-spectrometry-based quantitative proteomics in combination with systematic CRISPR/Cas9 knockout screening in Aspergillus nidulans, we identify the N-terminal histidine methyltransferase (NHMT) encoded by the gene AN4663. Targeted proteomics confirm that NHMT was solely responsible for His1 methylation of LPMOs. NHMT is predicted to encode a unique seven-transmembrane segment anchoring a soluble methyltransferase domain. Co-localization studies show endoplasmic reticulum residence of NHMT and co-expression in the industrial production yeast Komagataella phaffii with LPMOs results in His1 methylation of the LPMOs. This demonstrates the biotechnological potential of recombinant production of proteins and peptides harbouring this specific post-translational modification.

Engineering xylose fermentation in an industrial yeast: continuous cultivation as a tool for selecting improved strains.

Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic feedstock has received much attention as an alternative energy resource for biorefineries toward reducing the demand for fossil resources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae's inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2, and XYL3). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under microaerobic in the hemicellulosic hydrolysate. DPY06 exhibited 35% superior volumetric ethanol productivity compared to its parental strain.

Physiology of Saccharomyces cerevisiae during growth on industrial sugar cane molasses can be reproduced in a tailor-made defined synthetic medium

Fully defined laboratory media have the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. We developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg + K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the physiology of Saccharomyces cerevisiae in different actual molasses-based media. We demonstrate the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. Here we present in detail the development of a fully defined synthetic molasses medium and the physiology of yeast strains in this medium compared to industrial molasses. This tailor-made medium was able to satisfactorily reproduce the physiology of S. cerevisiae in industrial molasses. Thus, we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

Bioethanol production from sugarcane molasses with supplemented nutrients by industrial yeast

Sugarcane molasses, a sugar industry by-product, is being used as a convenient source of carbon for microorganisms to produce bioethanol. The industrial yield of bioethanol derived from Saccharomyces cerevisiae decreases with increasing temperature in the summer season and it is a common issue in the tropical region. It is inevitable to boost the production of bioethanol on an industrial scale to make it commercially profitable. This study has been conducted to analyze and compare the effect of three supplements on boosting alcohol products including thiamine (vitamin B1), pyridoxine (vitamin B6), and D-biotin (vitamin H). Collected molasses were diluted to 20 Brix to make them consumable by yeast, and thiamine, pyridoxine, and biotin were added as 1 mg/l, 1 mg/l, and 0.44 µg/l, respectively. Results depicted that the addition of vitamins impacted very positively on increasing alcohol production, yeast growth, and dry yeast weight where the residual glucose concentration was further decreased. Despite an extended lag phase resulting from higher vitamin doses, there was a significant improvement observed in both alcohol generation and yeast cell growth. This study presents the optimal dosing of supplements in the fermentation broth as a strategy to enhance bioethanol production.

Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase

Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila, termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-β-phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5–2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli-expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries.

High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis.

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3h with differences in the concentration of bioadhered cells ranging from 2.2×102cell/cm2 to 7.6×106cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.

Construction of versatile yeast plasmid vectors transferable by Agrobacterium-mediated transformation and their application to bread-making yeast strains

Agrobacterium-mediated transformation (AMT) potentially has great advantages over other DNA introduction methods: e.g., long DNA and numerous recipient strains can be dealt with at a time merely by co-cultivation with donor Agrobacterium cells. However, AMT was applied only to several laboratory yeast strains, and has never been considered as a standard gene-introduction method for yeast species. To disseminate the AMT method in yeast species, it is necessary to develop versatile AMT plasmid vectors including shuttle type ones, which have been unavailable yet for yeasts. In this study, we constructed a series of AMT plasmid vectors that consist of replicative (shuttle)- and integrative-types and harbor a gene conferring resistance to either G418 or aureobasidin A for application to prototrophic yeast strains. The vectors were successfully applied to five industrial yeast strains belonging to Saccharomyces cerevisiae after a modification of a previous AMT protocol, i.e., simply inputting a smaller number of yeast cells to the co-cultivation than that in the previous protocol. The revised protocol enabled all five yeast strains to generate recombinant colonies not only at high efficiency using replicative-type vectors, but also readily at an efficiency around 10-5 using integrative one. Further modification of the protocol demonstrated AMT for multiple yeast strains at a time with less labor. Therefore, AMT would facilitate molecular genetic approaches to many yeast strains in basic and applied sciences.

Production and sensory analysis of grape flavoured beer by co-fermentation of an industrial and a genetically modified laboratory yeast strain

The so-called “craft beer revolution” has increased the demand for new styles of beers, often with new ingredients like flavour extracts. In recent years, synthetic biology has realized the production of a plethora of plant secondary metabolites in microbial hosts, which could provide an alternative source for these compounds. In this study, we selected a in situ flavour production approach for grape flavour addition. We used an O-methyl anthranilate (OmANT) producing laboratory Saccharomyces cerevisiae strain in co-fermentations with an industrial beer yeast strain WLP644. The laboratory strain provided an ease of genetic manipulation and the desirable properties of the WLP644 strain were not modified in this approach. In shake flasks, a 10:90 ratio of the yeasts produced grape flavoured beer with the yeast produced flavour compound in a range normally used for flavoured beverages. Hopped and unhopped beers were analysed by VTT’s trained sensory panel and with olfactory GC–MS. OmANT was successfully detected from the beers as a floral odour and flavour. Moreover, no off-flavours were detected and aroma profiles outside the grape flavour were rather similar. These results indicate that the co-fermentation principle is a suitable approach to change the flavour profiles of beers with a simple yeast strain drop-in approach.

Mimicked Mixing-Induced Heterogeneities of Industrial Bioreactors Stimulate Long-Lasting Adaption Programs in Ethanol-Producing Yeasts.

Commercial-scale bioreactors create an unnatural environment for microbes from an evolutionary point of view. Mixing insufficiencies expose individual cells to fluctuating nutrient concentrations on a second-to-minute scale while transcriptional and translational capacities limit the microbial adaptation time from minutes to hours. This mismatch carries the risk of inadequate adaptation effects, especially considering that nutrients are available at optimal concentrations on average. Consequently, industrial bioprocesses that strive to maintain microbes in a phenotypic sweet spot, during lab-scale development, might suffer performance losses when said adaptive misconfigurations arise during scale-up. Here, we investigated the influence of fluctuating glucose availability on the gene-expression profile in the industrial yeast Ethanol Red™. The stimulus-response experiment introduced 2 min glucose depletion phases to cells growing under glucose limitation in a chemostat. Even though Ethanol Red™ displayed robust growth and productivity, a single 2 min depletion of glucose transiently triggered the environmental stress response. Furthermore, a new growth phenotype with an increased ribosome portfolio emerged after complete adaptation to recurring glucose shortages. The results of this study serve a twofold purpose. First, it highlights the necessity to consider the large-scale environment already at the experimental development stage, even when process-related stressors are moderate. Second, it allowed the deduction of strain engineering guidelines to optimize the genetic background of large-scale production hosts.

PEP4-Allele Modification Provides an Industrial Brewing Yeast with Malate Stress Tolerance

Acid stress is a challenging condition that yeast cells must overcome during fermentation. Enhancing the inherent tolerance of industrial Saccharomyces cerevisiae to organic acid stresses is crucial for increasing fermentation efficiency and reducing economic costs. In a previous study, we constructed a Saccharomyces cerevisiae strain SWY85S with improved tolerance to citric acid stress by modifying the second PEP4-allele. Malic acid is a dominant organic acid in grapefruit, which forms the acidic constituents of wine fermentation mash and finished products. We investigated the malic acid stress tolerance of the strain SWY85S in comparison with that of a strain with one PEP4-allele disrupted and the wild-type strain in this study. Our results revealed that the strain SWY85S demonstrated greater tolerance of malic acid stress, regardless of whether it was cultured with adequate nutrient supplies or under amino acid starvation. Moreover, the strain SWY85S performed remarkably in converting glucose to ethanol during fermentation under malic acid stress. This study provides insights into the role of a vacuolar PEP4-allele coding product in response to environmental stress and the physiological mechanism of yeast to withstand organic acid stress.

Microbial Communities of Flor Velums and the Genetic Stability of Flor Yeasts Used for a Long Time for the Industrial Production of Sherry-like Wines

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for the production of sherry-like wines by biological wine aging. We sequenced the genome of the industrial flor yeast strain I-329 from a collection of microorganisms for winemaking “Magarach” and the metagenomes of two flor velums based on this strain and continuously maintained for several decades. The winery uses two processes for the production of sherry-like wine: batch aging and a continuous process similar to the criaderas–solera system. The 18S rRNA gene profiling and sequencing of metagenomes of flor velums revealed the presence of the yeasts Pichia membranifaciens and Malassezia restricta in minor amounts along with the dominant S. cerevisiae I-329 flor yeast. Bacteria Oenococcus oeni and Lentilactobacillus hilgardii together accounted for approximately 20% of the velum microbiota in the case of a batch process, but less than 1% in the velum used in the continuous process. Collection strain I-329 was triploid for all chromosomes except diploid chromosomes I and III, while the copy numbers of all chromosomes were equal in industrial velums. A comparative analysis of the genome of strain I-329 maintained in the collection and metagenomes of industrial velums revealed only several dozens of single nucleotide polymorphisms, which indicates a long-term genetic stability of this flor yeast strain under the harsh conditions of biological wine aging.

Effect of environmental stresses during fermentation on brewing yeast and exploration on the novel flocculation-associated function of RIM15 gene

Flocculation of brewer's yeast is an environment-friendly and cost-effective way to separate yeast cells from fermentation broth for subsequent production. Diverse genetic background and complex fermentation environment cause difficulty to explore flocculation mechanism and regulate yeast flocculation. In this study, comparative transcriptome analysis was carried out between an industrial brewing yeast and its flocculation-enhanced mutant strain, unveiling the differentially-expressed genes were enriched in response to stresses. The expression level of Lg-FLO1 was the highest among all FLO genes. Environmental stresses of fermentation were simulated to stimulated yeast cells and it was found that nitrogen and amino acid starvation promoted the process of flocculation. It is the first time to reveal the nutrient-responsive gene RIM15 has a novel genetic function regulating flocculation. The study provides novel direction and strategies to manage yeast flocculation and achieve effective cell utilization in fermentation.

Optimization of Probiotic Lactobacilli Production for In-Feed Supplementation to Feedlot Cattle

The selection of probiotic bacteria based on their beneficial characteristics does not necessarily mean they can be later scaled up and used for technological applications and formula design. Three probiotic strains—Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085, and Limosolactobacillus mucosae CRL2069, originally isolated from feedlot cattle feces—have demonstrated beneficial characteristics when used as in-feed probiotics. Therefore, the current study was conducted to develop a low-cost culture medium to optimize growth conditions to enhance biomass production. The study also sought to identify appropriate cryoprotective agents to sustain high functional cell numbers after freeze drying. A central composite design was applied to determine the optimal medium composition. This yielded a simplified, low-cost effective medium containing 3% molasses and industrial yeast extracts (0.5 to 2.5%) as carbon and nitrogen sources, which were added to a basal medium for each strain. Established production conditions at 37 °C, without agitation, and pH-controlled for the CRL2085 and CRL2069 strains, and free pH for the CRL2074 strain, allowed us to obtain biomass yields of 12.95, 18.20, and 12.25 g, respectively, at 24-h incubation, compared with the MRS medium. In addition, the cryoprotective effect of the selected agents was demonstrated to be strain-dependent. Thus, the highest viability (109–1010 CFU/g), stability during 30-d storage, and survival rate (88–99%) were achieved when 10% MSG (monosodium glutamate), sucrose + fructose + trehalose + WPC (whey protein concentrate) + 10% MSG, and 1.2% WPC + 10% trehalose, were used for freeze drying CRL2074, CRL2085, and CRL2069, respectively. Moreover, the probiotic strains retained their probiotic functionality when hydrophobic characteristics were evaluated. These results highlight the need to perform strain-specific evaluation of the critical factors involved in the large-scale production of probiotic lactobacilli to sustain viability and stability after the freeze drying and storage processes.

Deletion of QDR genes in a bioethanol-producing yeast strain reduces propagation of contaminating lactic acid bacteria

Bacterial contaminations in yeast fermentation tanks are a recurring problem for the bioethanol production industry. Lactic acid bacteria (LAB), particularly of the genus Lactobacillus, are the most common contaminants. Their proliferation can reduce fermentation efficiency or even impose premature shutdown for cleaning. We have previously reported that laboratory yeast strains naturally excrete amino acids via transporters of the Drug: H+ Antiporter-1 (DHA1) family. This excretion allows yeast to cross-feed LAB, which are most often unable to grow without an external amino acid supply. Whether industrial yeast strains used in bioethanol production likewise promote LAB proliferation through cross-feeding has not been investigated. In this study, we first show that the yeast strain Ethanol Red used in ethanol production supports growth of Lactobacillus fermentum in an amino-acid-free synthetic medium. This effect was markedly reduced upon homozygous deletion of the QDR3 gene encoding a DHA1-family amino acid exporter. We further show that cultivation of Ethanol Red in a nonsterile sugarcane-molasses-based medium is associated with an increase in lactic acid due to LAB growth. When Ethanol Red lacked the QDR1, QDR2, and QDR3 genes, this lactic acid production was not observed and ethanol production was not significantly reduced. Our results indicate that Ethanol Red cultivated in synthetic or molasses medium sustains LAB proliferation in a manner that depends on its ability to excrete amino acids via Qdr transporters. They further suggest that using mutant industrial yeast derivatives lacking DHA1-family amino acid exporters may be a way to reduce the risk of bacterial contaminations during fermentation.

Fruit Beer with the Bisucciu Sardinian Apricot Cultivar (Prunus armeniaca L.): A Technological and Analytical Approach

The aim of this work was to test native microbial strains and fruits for brewing, with a multidisciplinary approach for a sustainable production linked to the territory. Pediococcus acidilactici B5 and Hanseniaspora uvarum L2 strains were isolated from apricot Bisucciu fruits, a Sardinian local variety (Prunus armeniaca L.), and P. acidilactici B5 was used to ferment a sterile apricot Bisucciu puree, which was then added to a malt wort. The H. uvarum L2 strain and the industrial yeast Saccharomyces cerevisiae US-05 were used sequentially to ferment a portion of this wort (M2); a control was carried out with an industrial yeast, S. cerevisiae T-58 (T58). Beer standard quality parameters were studied and a sensorial analysis performed in the beers obtained from the two fermentations. Intermediate and end molecular products were characterized by proton Nuclear Magnetic Resonance (1H NMR) for glucidic, organic acids and amino acids and by Gas Chromatography–Mass Spectrometry (SPME/GC/MS) for volatile profiles. M2 and T58 samples showed differences in color, foam stability and in the carbohydrates, acids and amino acids profiles. The highest concentrations of ethyl acetate were found in M2, whereas a high concentration of 3-methylbutan-1-ol characterized T58. Sensory analysis highlighted differences in flavor, astringency and balance between the two beers studied.

The Metabolism of Respiring Carbon Sources by Dekkera bruxellensis and Its Relation with the Production of Acetate.

Dekkera bruxellensis has been studied for several aspects of its metabolism over the past years, which has expanded our comprehension on its importance to industrial fermentation processes and uncovered its industrial relevance. Acetate is a metabolite often found in D. bruxellensis aerobic cultivations, whereas its production is linked to decreased ethanol yields. In a previous work, we aimed to understand how acetate metabolism affected the fermentation capacity of D. bruxellensis. In the present work, we evaluated the role of acetate metabolism in respiring cells using ammonium or nitrate as nitrogen sources. Our results showed that galactose is a strictly respiratory sugar and that a relevant part of its carbon is lost, while the remaining is metabolised through the Pdh bypass pathway before being assimilated into biomass. When this pathway was blocked, yeast growth was reduced while more carbon was assimilated to the biomass. In nitrate, more acetate was produced as expected, which increased carbon assimilation, although less galactose was uptaken from the medium. This scenario was not affected by the Pdh bypass inhibition. The confirmation that acetate production was crucial for carbon assimilation was brought by cultivations in pyruvate. All physiological data were connected to the expression patterns of PFK1, PDC1, ADH1, ALD3, ALD5 and ATP1 genes. Other respiring carbon sources could only be properly used by the cells when some external acetate was supplied. Therefore, the results reported herein helped in providing valuable contributions to the understanding of the oxidative metabolism in this potential industrial yeast.

Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

Bioconversion of methanol to 3-hydroxypropionate by engineering Ogataea polymorpha

Methanol bioconversion toward chemical production can be helpful for carbon neutrality. Here metabolic engineering an industrial methylotrophic yeast Ogataea polymorpha was conducted for overproduction of 3-hydroxypropionate (3-HP), an important platform chemical, which can be used for production of special chemicals including acrylamide, acrylic acid, 1,3-propanediol, as well as biodegradable plastics. We developed several metabolic engineering strategies to optimize the 3-HP biosynthetic pathway and enhance the supply of precursors acetyl-CoA and malonyl-CoA, and cofactor NADPH, which enabled the 3-HP production of 1.45 g/L under batch fermentation and 7.10 g/L under fed-batch fermentation at shake flask scale, with a yield of 0.14 g/g methanol. This was, to our knowledge, the highest 3-HP titer from methanol and even one-carbon sources. This study demonstrated the potential of O. polymorpha as a cell factory for chemical production from methanol.