The potency of mitochondria enlargement for mitochondria-mediated terpenoid production in yeast.

Terpenoids are widely used in the food, beverage, cosmetics, and pharmaceutical industries. Microorganisms have been extensively studied for terpenoid production. In yeast, the introduction of the mevalonate (MVA) pathway in organelles in addition to the augmentation of its own MVA pathway have been challenging. Introduction of the MVA pathway into mitochondria is considered a promising approach for terpenoid production because acetyl-CoA, the starting molecule of the MVA pathway, is abundant in mitochondria. However, mitochondria comprise only a small percentage of the entire cell. Therefore, we hypothesized that increasing the total mitochondrial volume per cell would increase terpenoid production. First, we ascertained that the amounts of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the final molecules of the MVA pathway, were 15-fold higher of the strain expressing the MVA pathway in mitochondria than in the wild-type yeast strain. Second, we found that different deletion mutants induced different mitochondrial volumes by measuring the mitochondrial volume in various deletion mutants affecting mitochondrial morphology; for example,Δmdm32 increased mitochondrial volume, and Δfzo1 decreased it. Finally, the effects of mitochondrial volume on amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) were investigated using mutants harboring large or small mitochondria expressing the MVA pathway in mitochondria. Amounts of IPP/DMAPP and terpenoids (squalene or ß-carotene) increased when the mitochondrial volume expanded. Introducing the MVA pathway into mitochondria for terpenoid production in yeast may become more attractive by enlarging the mitochondrial volume. KEY POINTS: • IPP/DMAPP content increased in the strain expressing the MVA pathway in mitochondria • IPP/DMAPP and terpenoid contents are positively correlated with mitochondrial volume • Enlarging the mitochondria may improve mitochondria-mediated terpenoid production.

High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system.

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine-a precursor to phenol-we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Beneficial effect of optimizing the expression balance of the mevalonate pathway introduced into the mitochondria on terpenoid production in Saccharomyces cerevisiae.

Terpenoids are used in various industries, and Saccharomyces cerevisiae is a promising microorganism for terpenoid production. Introducing the mevalonate (MVA) pathway into the mitochondria of a strain with an augmented inherent cytosolic MVA pathway increased terpenoid production but also led to the accumulation of toxic pyrophosphate intermediates that negatively affected terpenoid production. We first engineered the inherent MVA pathway in the cytosol and then introduced the MVA pathway into the mitochondria using several promoter combinations, considering the toxicity of pyrophosphate intermediates. However, the highest titer, 183 mg/L, tends to be only 5% higher than that of the strain that only augmented the inherent MVA pathway (SYCM1; 174 mg/L). Next, we hypothesized that, in addition to the toxicity of pyrophosphate, other compounds in the MVA pathway could affect the squalene titer. Thus, we constructed a combinatorial strain library expressing MVA pathway enzymes in the mitochondria with various promoter combinations. The highest squalene titer (230 mg/L) was 32% higher than that of SYCM1. The promoter set revealed that mitigation of mono- and pyrophosphate compound accumulation was important for mitochondrial usage. This study demonstrated that a combinatorial strain library is useful for discovering the optimal gene expression balance in engineering yeast.

Comprehensive metabolic profiling of Geotrichum candidum and comparison with Saccharomyces cerevisiae.

Geotrichum candidum is a dimorphic yeast used in cheese processing. To our knowledge, no major metabolites have been identified to date in G. candidum except for some amino acid and fatty acid metabolites. This has limited research on the commercial use of G. candidum. In this study, we aimed to analyze temporal changes in the intra- and extra-cellular metabolites of G. candidum and Saccharomyces cerevisiae cultured in YM medium as reference. As a result of metabolite analysis, it was observed that G. candidum tends to accumulate　pentose phosphate pathway compounds, which are involved in nucleic acid synthesis, after 48 h of cultivation when compared to S. cerevisiae. In addition, G. candidum accumulated higher amounts of the antioxidant glutathione in the medium than did S. cerevisiae. In addition, G. candidum accumulated large amounts of B vitamins such as pantothenic acid and nicotinic acid in the medium. Finally, we examined the potential of G. candidum as a host for the production of useful compounds such as pantothenic acid. When cultured in medium supplemented with the pantothenic acid precursor ß-alanine, G. candidum produced 12-fold higher amounts of pantothenic acid (30 µM) than that by S. cerevisiae. This study indicates that G. candidum accumulates various useful compounds that are dissimilar to those produced by S. cerevisiae. Furthermore, G. candidum has the potential to produce useful chemicals under appropriate culture conditions.

Metabolomics-based development of bioproduction processes toward industrial-scale production.

Microbial biomanufacturing offers a promising, environment-friendly platform for next-generation chemical production. However, its limited industrial implementation is attributed to the slow production rates of target compounds and the time-intensive engineering of high-yield strains. This review highlights how metabolomics expedites bioproduction development, as demonstrated through case studies of its integration into microbial strain engineering, culture optimization, and model construction. The Design-Build-Test-Learn (DBTL) cycle serves as a standard workflow for strain engineering. Process development, including the optimization of culture conditions and scale-up, is crucial for industrial production. In silico models facilitate the development of strains and processes. Metabolomics is a powerful driver of the DBTL framework, process development, and model construction.

Direct production of 4-hydroxybenzoic acid from cellulose using cellulase-displaying Pichia pastoris.

4-hydroxybenzoic acid (4-HBA) is an industrially important aromatic compound, and there is an urgent need to establish a bioprocess to produce this compound in a sustainable and environmentally friendly manner from renewable feedstocks such as cellulosic biomass. Here, we developed a bioprocess to directly produce 4-HBA from cellulose using a recombinant Pichia pastoris strain that displays heterologous cellulolytic enzymes on its cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system. ß-glucosidase (BGL) from Aspergillus aculeatus, endoglucanase (EG) from Trichoderma reesei, and cellobiohydrolase (CBH) from Talaromyces emersonii were co-displayed on the cell surface of P. pastoris using an appropriate GPI-anchoring domain for each enzyme. The cell-surface cellulase activity was further enhanced using P. pastoris SPI1 promoter- and secretion signal sequences. The resulting strains efficiently hydrolyzed phosphoric acid swollen cellulose (PASC) to glucose. Then, we expressed a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from Providencia rustigianii in the cellulase-displaying strain. This strain produced 975 mg/L of 4-HBA from PASC, which corresponding to 36.8% of the theoretical maximum yield, after 96 h of batch fermentation without the addition of commercial cellulase. This 4-HBA yield was over two times higher than that obtained from glucose (12.3% of the theoretical maximum yield). To our knowledge, this is the first report on the direct production of an aromatic compound from cellulose using cellulase-displaying yeast.

Enhanced production of 3,4-dihydroxybutyrate from xylose by engineered yeast via xylonate re-assimilation under alkaline condition.

To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.

Construction of an l-Tyrosine Chassis in Pichia pastoris Enhances Aromatic Secondary Metabolite Production from Glycerol.

Bioactive plant-based secondary metabolites such as stilbenoids, flavonoids, and benzylisoquinoline alkaloids (BIAs) are produced from l-tyrosine (l-Tyr) and have a wide variety of commercial applications. Therefore, building a microorganism with high l-Tyr productivity (l-Tyr chassis) is of immense value for large-scale production of various aromatic compounds. The aim of this study was to develop an l-Tyr chassis in the nonconventional yeast Pichia pastoris (Komagataella phaffii) to produce various aromatic secondary metabolites (resveratrol, naringenin, norcoclaurine, and reticuline). Overexpression of feedback-inhibition insensitive variants of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (ARO4K229L) and chorismate mutase (ARO7G141S) enhanced l-Tyr titer from glycerol in P. pastoris. These engineered P. pastoris strains increased the titer of resveratrol, naringenin, and norcoclaurine by 258, 244, and 3400%, respectively, after expressing the corresponding heterologous pathways. The titer of resveratrol and naringenin further increased by 305 and 249%, resulting in yields of 1825 and 1067 mg/L, respectively, in fed-batch fermentation, which is the highest titer from glycerol reported to date. Furthermore, the resveratrol-producing strain accumulated intermediates in the shikimate pathway. l-Tyr-derived aromatic compounds were produced using crude glycerol byproducts from biodiesel fuel (BDF) production. Constructing an l-Tyr chassis is a promising strategy to increase the titer of various aromatic secondary metabolites and P. pastoris is an attractive host for high-yield production of l-Tyr-derived aromatic compounds from glycerol.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus ß-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Optimization of 1,2,4-butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance.

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.

An ion-pair free LC-MS/MS method for quantitative metabolite profiling of microbial bioproduction systems.

Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a 'terminator catalog' by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3'-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.

Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering.

1,2,4-Butanetriol can be used to produce energetic plasticizer as well as several pharmaceutical compounds. Although Saccharomyces cerevisiae has some attractive characters such as high robustness for industrial production of useful chemicals by fermentation, 1,2,4-butanetriol production by S. cerevisiae has not been reported. 1,2,4-butanteriotl is produced by an oxidative xylose metabolic pathway completely different from the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways conventionally used for xylose assimilation in S. cerevisiae. In the present study, S. cerevisiae was engineered to produce 1,2,4-butanetriol by overexpression of xylose dehydrogenase (XylB), xylonate dehydratase (XylD), and 2-ketoacid decarboxylase. Further improvement of the recombinant strain was performed by the screening of optimal 2-ketoacid decarboxylase suitable for 1,2,4-butanetriol production and the enhancement of Fe uptake ability to improve the XylD enzymatic activity. Eventually, 1.7 g/L of 1,2,4-butanetriol was produced from 10 g/L xylose with a molar yield of 24.5%. Furthermore, 1.1 g/L of 1,2,4-butanetriol was successfully produced by direct fermentation of rice straw hydrolysate.

Combined Cell Surface Display of ß-d-Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass.

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d-xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro-organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499-SsXR-AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a ß-d-glucosidase (Aspergillus aculeatus ß-glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5-fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499-XR-ScMAL11-BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.

Sustainable production of glutathione from lignocellulose-derived sugars using engineered Saccharomyces cerevisiae.

Glutathione has diverse physiological functions, and therefore, the demand for it has increased recently. Currently, industrial mass production of glutathione is performed from D-glucose via fermentation by the budding yeast Saccharomyces cerevisiae. However, use of D-glucose often competes with demands for various other industries, leading to high production costs. To affordably produce glutathione, we aimed to produce high amounts of glutathione from D-glucose and D-xylose, which are the main constituents of lignocellulosic biomass pre-treated with acids. Genetically engineered S. cerevisiae strains that can produce high amounts of glutathione and assimilate D-xylose were constructed and cultured in media containing D-xylose. Among these recombinant strains, a S. cerevisiae GCI (XR/XDH/XK) strain over-expressing γ-glutamylcysteine synthetase, glutathione synthetase, D-xylose reductase, xylitol dehydrogenase, and xylulokinase genes successfully consumed D-xylose in the medium and produced the highest amount of glutathione. When strains were grown in media containing D-glucose and D-xylose, the GCI (XR/XDH/XK) strain showed 4.6-fold higher volumetric glutathione production (mg/L-broth), 2.2-fold higher glutathione content (%), and 2.1-fold higher cell growth (g-cell/L-broth) than the vector control strain of YPH499 (Vector). Furthermore, when recombinant S. cerevisiae strains were grown in medium containing fermentation inhibitory materials, the GCI (XR/XDH/XK) strain produced 5.8- and higher volumetric glutathione, 2.6-fold higher intracellular glutathione, and 2.9-fold higher cell growth than the vector control YPH499 (Vector) strain. The gradual sugar consumption by recombinant S. cerevisiae strains in medium containing D-glucose and D-xylose leads to high yields of glutathione. These results indicate the potential for glutathione production from lignocellulosic materials.

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain.

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. ß-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S. cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48 h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72 h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MSE). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1.

Improvement of Xylose Fermentation Ability under Heat and Acid Co-Stress in Saccharomyces cerevisiae Using Genome Shuffling Technique.

Xylose-assimilating yeasts with tolerance to both fermentation inhibitors (such as weak organic acids) and high temperature are required for cost-effective simultaneous saccharification and cofermentation (SSCF) of lignocellulosic materials. Here, we demonstrate the construction of a novel xylose-utilizing Saccharomyces cerevisiae strain with improved fermentation ability under heat and acid co-stress using the drug resistance marker-aided genome shuffling technique. The mutagenized genome pools derived from xylose-utilizing diploid yeasts with thermotolerance or acid tolerance were shuffled by sporulation and mating. The shuffled strains were then subjected to screening under co-stress conditions of heat and acids, and the hybrid strain Hyb-8 was isolated. The hybrid strain displayed enhanced xylose fermentation ability in comparison to both parental strains under co-stress conditions of heat and acids. Hyb-8 consumed 33.1 ± 0.6 g/L xylose and produced 11.1 ± 0.4 g/L ethanol after 72 h of fermentation at 38°C with 20 mM acetic acid and 15 mM formic acid. We also performed transcriptomic analysis of the hybrid strain and its parental strains to screen for key genes for multiple stress tolerances. We found that 13 genes, including 5 associated with cellular transition metal ion homeostasis, were significantly upregulated in Hyb-8 compared to levels in both parental strains under co-stress conditions. The hybrid strain Hyb-8 has strong potential for cost-effective SSCF of lignocellulosic materials. Moreover, the transcriptome data gathered in this study will be useful for understanding the mechanisms of multiple tolerance to high temperature and acids in yeast and facilitate the development of robust yeast strains for SSCF.

Enhanced cell-surface display and secretory production of cellulolytic enzymes with Saccharomyces cerevisiae Sed1 signal peptide.

Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus ß-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Disruption of PHO13 improves ethanol production via the xylose isomerase pathway.

Xylose is the second most abundant sugar in lignocellulosic materials and can be converted to ethanol by recombinant Saccharomyces cerevisiae yeast strains expressing heterologous genes involved in xylose assimilation pathways. Recent research demonstrated that disruption of the alkaline phosphatase gene, PHO13, enhances ethanol production from xylose by a strain expressing the xylose reductase (XR) and xylitol dehydrogenase (XDH) genes; however, the yield of ethanol is poor. In this study, PHO13 was disrupted in a recombinant strain harboring multiple copies of the xylose isomerase (XI) gene derived from Orpinomyces sp., coupled with overexpression of the endogenous xylulokinase (XK) gene and disruption of GRE3, which encodes aldose reductase. The resulting YΔGP/XK/XI strain consumed 2.08 g/L/h of xylose and produced 0.88 g/L/h of volumetric ethanol, for an 86.8 % theoretical ethanol yield, and only YΔGP/XK/XI demonstrated increase in cell concentration. Transcriptome analysis indicated that expression of genes involved in the pentose phosphate pathway (GND1, SOL3, TAL1, RKI1, and TKL1) and TCA cycle and respiratory chain (NDE1, ACO1, ACO2, SDH2, IDH1, IDH2, ATP7, ATP19, SDH4, SDH3, CMC2, and ATP15) was upregulated in the YΔGP/XK/XI strain. And the expression levels of 125 cell cycle genes were changed by deletion of PHO13.

Cell surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate.

Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with ß-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.