Glycolate production by a Chlamydomonas reinhardtii mutant lacking carbon-concentrating mechanism.

The green alga Chlamydomonas reinhardtii serves as a model organism for plant and photosynthesis research due to many commonalities in metabolism and to the fast growth rate of C. reinhardtii which accelerates experimental turnaround time. In addition, C. reinhardtii is a focus of research efforts in metabolic engineering and synthetic biology for the potential production of biofuels and value-added chemicals. Here, we report that the C. reinhardtii cia5 mutant, which lacks a functional carbon-concentrating mechanism (CCM), can produce substantial amounts of glycolate, a high-value cosmetic ingredient, when the mutant is cultured under ambient air conditions. In order to reveal the metabolic basis of glycolate accumulation by the cia5 mutant, we investigated the metabolomes of the cia5 mutant and a wild type strain CC-125 (WT) through the global metabolic profiling of intracellular and extracellular fractions using gas chromatography and mass spectrometry. We observed the intracellular and extracellular metabolic profiles of the WT and the cia5 mutant were similar during the mixotrophic phase at 30 h. However, when the cells entered the photoautotrophic phase (i.e., 96 h and 120 h), both the intracellular and extracellular metabolic profiles of cia5 mutant differed significantly when compared to WT. In the cia5 mutant strain, a group of photorespiration pathway intermediates including glycolate, glyoxylate, glycine, and serine accumulated to significantly higher levels compared to WT. In the photorespiration pathway, glycolate is metabolized to glyoxylate and glycine leading to NH3 and CO2 generation during the mitochondrial conversion of glycine to serine. This result provides further evidence that the CIA5 mutation increased the photorespiration rate. Because the cia5 mutant lacks a CCM, and C. reinhardtii might harbor an inefficient or incomplete photorespiration pathway, glycolate may accumulate when the CCM is not functional. We envision that investigating photorespiration controls in C. reinhardtii provides tools for producers to use the cia5 mutant to produce glycolate as well as platform to engineer alternative pathways for glycolate metabolism.

Giant Cell-Rich Solitary Fibrous Tumor in the Nasopharynx: Case Report and Literature Review.

Solitary fibrous tumors (SFTs) can occur in several locations outside the pleura, but rarely in the sinonasal tract, and particularly not in the nasopharynx. Herein, we describe an unusual case of giant cell-rich SFT (GCRSFT) occurring in the nasopharynx. A 64-year-old man experienced dizziness and headache for more than 10 years with no obvious cause. Computed tomography (CT) scan showed a 3.9 cm × 2 cm tumor on the posterior lateral wall of the left nasopharynx, and angiography revealed a hypervascular tumor fed by branches of the left carotid artery. Hence, preoperative embolization was performed, and then the tumor was endoscopically resected. The symptoms were relieved after the resection, and postoperative head CT and video laryngoscopy showed that the tumor was completely resected. We next characterized the specific pathological characteristics of the resected tumor. Histologically, the tumor was characterized by varying cellular proliferation of cytologically bland spindle cells within a collagenous stroma, with prominent interspersed branching vessels. Mitotic activity was low (2/50HPF), and there was no evidence of pleomorphism or tumor necrosis. Moreover, multinucleated giant cells with deep nuclear staining and distributed in pseudovascular spaces were found within the tumor. We ruled out the possibility that our case was giant cell fibroblastoma (GCF) by immunohistochemical analysis, showing that the tumor cells were positive for CD34, CD99, STAT6, and BCL-2, and that the Ki-67 labeling index was 3%, indicating that our case was SFT and not GCF. The patient's condition is generally good after a 14-month follow-up. This report serves to broaden the morphologic spectrum of GCRSFT and will help clinicians and pathologists better understand this entity to prevent misdiagnosis.

Review on D-Allulose: In vivo Metabolism, Catalytic Mechanism, Engineering Strain Construction, Bio-Production Technology.

Rare sugar D-allulose as a substitute sweetener is produced through the isomerization of D-fructose by D-tagatose 3-epimerases (DTEases) or D-allulose 3-epimerases (DAEases). D-Allulose is a kind of low energy monosaccharide sugar naturally existing in some fruits in very small quantities. D-Allulose not only possesses high value as a food ingredient and dietary supplement, but also exhibits a variety of physiological functions serving as improving insulin resistance, antioxidant enhancement, and hypoglycemic controls, and so forth. Thus, D-allulose has an important development value as an alternative to high-energy sugars. This review provided a systematic analysis of D-allulose characters, application, enzymatic characteristics and molecular modification, engineered strain construction, and processing technologies. The existing problems and its proposed solutions for D-allulose production are also discussed. More importantly, a green and recycling process technology for D-allulose production is proposed for low waste formation, low energy consumption, and high sugar yield.

Alkannin represses growth of pancreatic cancer cells based on the down regulation of miR-199a.

Alkannin displays tumor suppressive activity by initiating apoptosis. Here, we corroborated its role in pancreatic carcinoma (PANC-1) cells and addressed the molecular mechanism in which microRNA-199a (miR-199a) and Klotho might be implicated. PANC-1 and MIN6 cells were treated by alkannin and its role was evaluated in cellular viability. Next we assessed the ability of PANC-1 cells to proliferate, migrate, and invade as well as apoptosis process. Besides, proliferating cell nuclear antigen (PCNA), CyclinD1, p53, and caspases were quantified using Western blot. miR-199a was detected by qRT-PCR. miR-199a-silenced or -replenished cells were established to study its function role in Klotho in conjunction with alkannin. Further, Klotho-overexpressed or -silenced cells were constructed to investigate the alteration of mTOR and MEK/ERK pathways. Alkannin repressed the viability of PANC-1 cells instead of MIN6 cells. Alkannin counteracted the growth of PANC-1 cells through inhibiting proliferation, migration, and invasion and facilitating apoptosis, which was evidenced by the modulation on PCNA, CyclinD1, p53, and cleavage of caspases. The silence of miR-199a by alkannin was also involved in the antitumor process. Alkannin enhanced Klotho expression possibly through silencing miR-199a. Besides, mTOR and MEK/ERK signaling were counteracted by Klotho overexpression while facilitated by its silence. Alkannin inhibited the growth of PANC-1 cells via modulating miR-199a-Klotho node. During this process, mTOR and MEK/ERK pathways were blunted.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae.

Bioconversion of xylose-the second most abundant sugar in nature-into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.

Screening for the Biomarkers Associated with Myocardial Infarction by Bioinformatics Analysis.

We aimed to find novel biomarkers associated with myocardial infarction (MI). The array data of GSE62646 were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were analyzed with limma package. Functional enrichment analyses were performed by DAVID v6.7 online tool. The micro-RNA-messenger RNA (miRNA-mRNA) pairs were predicted by miRWalk database, and the protein-mRNA interactions were predicted by StarBase. Then, miRNA-mRNA-protein regulatory network was constructed. Antigen processing and presentation were only the pathway enriched by DEG1 set such as KLRC4 (killer cell lectin-like receptor subfamily C, member 4) and KLRC2 (killer cell lectin-like receptor subfamily C, member 2). KLRC4 and KLRC2 were differentially expressed in MI patients. DLC1 (DLC1 Rho GTPase activating protein, degree = 179) was the most significant node in miRNA-mRNA-protein network. EIF4AIII (eukaryotic translation initiation factor 4A3) and FUS (FUS RNA binding protein) were the key proteins that regulated the most DEGs. KLRC4, KLRC2, and DLC1 were the biomarkers and may play important roles in the progression of MI. Furthermore, EIF4AIII and FUS may also be involved in MI progression.

Deletion of JEN1 and ADY2 reduces lactic acid yield from an engineered Saccharomyces cerevisiae, in xylose medium, expressing a heterologous lactate dehydrogenase.

Microorganisms have evolved to produce specific end products for many reasons, including maintaining redox balance between NAD+ and NADH. The yeast Saccharomyces cerevisiae, for example, produces ethanol as a primary end product from glucose for the regeneration of NAD+. Engineered S. cerevisiae strains have been developed to ferment lignocellulosic sugars, such as xylose, to produce lactic acid by expression of a heterologous lactate dehydrogenase (ldhA from Rhizopus oryzae) without genetic perturbation to the native ethanol pathway. Surprisingly, the engineered yeast strains predominantly produce ethanol from glucose, but produce lactic acid as the major product from xylose. Here, we provide initial evidence that the shift in product formation from ethanol to lactic acid during xylose fermentation is at least partially dependent on the presence of functioning monocarboxylate transporter genes/proteins, including JEN1 and ADY2, which are downregulated and unstable in the presence of glucose, but upregulated/stable on xylose. Future yeast metabolic engineering studies may find the feedstock/carbon selection, such as xylose, an important step toward improving the yield of target end products.

Overcoming the thermodynamic equilibrium of an isomerization reaction through oxidoreductive reactions for biotransformation.

Isomerases perform biotransformations without cofactors but often cause an undesirable mixture of substrate and product due to unfavorable thermodynamic equilibria. We demonstrate the feasibility of using an engineered yeast strain harboring oxidoreductase reactions to overcome the thermodynamic limit of an isomerization reaction. Specifically, a yeast strain capable of consuming lactose intracellularly is engineered to produce tagatose from lactose through three layers of manipulations. First, GAL1 coding for galactose kinase is deleted to eliminate galactose utilization. Second, heterologous xylose reductase (XR) and galactitol dehydrogenase (GDH) are introduced into the ∆gal1 strain. Third, the expression levels of XR and GDH are adjusted to maximize tagatose production. The resulting engineered yeast produces 37.69 g/L of tagatose from lactose with a tagatose and galactose ratio of 9:1 in the reaction broth. These results suggest that in vivo oxidoreaductase reactions can be employed to replace isomerases in vitro for biotransformation.

Bioprocessing and technoeconomic feasibility analysis of simultaneous production of d-psicose and ethanol using engineered yeast strain KAM-2GD.

The objective of this study was to analyze the processing and technoeconomic feasibility of coproduction of d-psicose and ethanol in a modified dry grind ethanol process. The yeast strain was constructed by expressing d-psicose 3-epimerases (DPE) in Sachharomyces cerevisiae. The strain was capable of converting d-fructose to d-psicose at 55 °C with a conversion efficiency of 26.6%. A comprehensive process model for modified dry grind ethanol plant with 396,000 MT/yr corn processing capacity was developed using SuperPro Designer. Predicted ethanol and d-psicose yields were 390.4 L and 75.3 kg per MT of corn, with total annual production of 154.6 million L and 29,835 MT respectively. The capital investment for the plant was estimated as 150.3 million USD with total operating cost of 85.2 million USD/yr. The unit production cost and minimum selling price of d-psicose with an internal rate of return of 15% were calculated as $0.43/kg and $1.29/kg respectively.

Changes of Serum Procalcitonin (PCT), C-Reactive Protein (CRP), Interleukin-17 (IL-17), Interleukin-6 (IL-6), High Mobility Group Protein-B1 (HMGB1) and D-Dimer in Patients with Severe Acute Pancreatitis Treated with Continuous Renal Replacement Therapy (CRRT) and Its Clinical Significance.

BACKGROUND The aim of this study was to investigate the changes in serum levels of procalcitonin (PCT), C-reactive protein (CRP), interleukin-17 (IL-17), interleukin-6 (IL-6), high mobility group protein-B1 (HMGB1), and D-dimer in severe acute pancreatitis (SAP) patients during treatment with continuous renal replacement therapy (CRRT) and the clinical significance. MATERIAL AND METHODS A total of 92 SAP patients admitted to our hospital from January 2017 to December 2017 were selected and randomly divided into the observation group and the control group using a random number table method, with 46 cases in each group. The control group was given conventional therapy, and the observation group was given CRRT in addition to conventional therapy. RESULTS After 1 week, the total effective rate of treatment in the observation group was significantly higher than that in the control group (P<0.05). In the observation group, each index showed a continuous downward trend at 6, 12, and 24 hours after treatment, and at different time points after treatment, the indexes were significantly lower than those in the control group (P<0.05). CONCLUSIONS CRRT is more effective in the treatment of SAP, and its effects are more obvious in removing a variety of inflammatory factors and reducing the serum levels of PCT, HMGB1, and D-dimer, which is of great clinical significance.

A Mutation in PGM2 Causing Inefficient Galactose Metabolism in the Probiotic Yeast Saccharomyces boulardii.

The probiotic yeast Saccharomyces boulardii has been extensively studied for the prevention and treatment of diarrheal diseases, and it is now commercially available in some countries. S. boulardii displays notable phenotypic characteristics, such as a high optimal growth temperature, high tolerance against acidic conditions, and the inability to form ascospores, which differentiate S. boulardii from Saccharomyces cerevisiae The majority of prior studies stated that S. boulardii exhibits sluggish or halted galactose utilization. Nonetheless, the molecular mechanisms underlying inefficient galactose uptake have yet to be elucidated. When the galactose utilization of a widely used S. boulardii strain, ATCC MYA-796, was examined under various culture conditions, the S. boulardii strain could consume galactose, but at a much lower rate than that of S. cerevisiae While all GAL genes were present in the S. boulardii genome, according to analysis of genomic sequencing data in a previous study, a point mutation (G1278A) in PGM2, which codes for phosphoglucomutase, was identified in the genome of the S. boulardii strain. As the point mutation resulted in the truncation of the Pgm2 protein, which is known to play a pivotal role in galactose utilization, we hypothesized that the truncated Pgm2 might be associated with inefficient galactose metabolism. Indeed, complementation of S. cerevisiaePGM2 in S. boulardii restored galactose utilization. After reverting the point mutation to a full-length PGM2 in S. boulardii by Cas9-based genome editing, the growth rates of wild-type (with a truncated PGM2 gene) and mutant (with a full-length PGM2) strains with glucose or galactose as the carbon source were examined. As expected, the mutant (with a full-length PGM2) was able to ferment galactose faster than the wild-type strain. Interestingly, the mutant showed a lower growth rate than that of the wild-type strain on glucose at 37°C. Also, the wild-type strain was enriched in the mixed culture of wild-type and mutant strains on glucose at 37°C, suggesting that the truncated PGM2 might offer better growth on glucose at a higher temperature in return for inefficient galactose utilization. Our results suggest that the point mutation in PGM2 might be involved in multiple phenotypes with different effects.IMPORTANCESaccharomyces boulardii is a probiotic yeast strain capable of preventing and treating diarrheal diseases. However, the genetics and metabolism of this yeast are largely unexplored. In particular, molecular mechanisms underlying the inefficient galactose metabolism of S. boulardii remain unknown. Our study reports that a point mutation in PGM2, which codes for phosphoglucomutase, is responsible for inferior galactose utilization by S. boulardii After correction of the mutated PGM2 via genome editing, the resulting strain was able to use galactose faster than a parental strain. While the PGM2 mutation made the yeast use galactose slowly, investigation of the genomic sequencing data of other S. boulardii strains revealed that the PGM2 mutation is evolutionarily conserved. Interestingly, the PGM2 mutation was beneficial for growth at a higher temperature on glucose. We speculate that the PGM2 mutation was enriched due to selection of S. boulardii in the natural habitat (sugar-rich fruits in tropical areas).

Glucose repression can be alleviated by reducing glucose phosphorylation rate in Saccharomyces cerevisiae.

Microorganisms commonly exhibit preferential glucose consumption and diauxic growth when cultured in mixtures of glucose and other sugars. Although various genetic perturbations have alleviated the effects of glucose repression on consumption of specific sugars, a broadly applicable mechanism remains unknown. Here, we report that a reduction in the rate of glucose phosphorylation alleviates the effects of glucose repression in Saccharomyces cerevisiae. Through adaptive evolution under a mixture of xylose and the glucose analog 2-deoxyglucose, we isolated a mutant strain capable of simultaneously consuming glucose and xylose. Genome sequencing of the evolved mutant followed by CRISPR/Cas9-based reverse engineering revealed that mutations in the glucose phosphorylating enzymes (Hxk1, Hxk2, Glk1) were sufficient to confer simultaneous glucose and xylose utilization. We then found that varying hexokinase expression with an inducible promoter led to the simultaneous utilization of glucose and xylose. Interestingly, no mutations in sugar transporters occurred during the evolution, and no specific transporter played an indispensable role in simultaneous sugar utilization. Additionally, we demonstrated that slowing glucose consumption also enabled simultaneous utilization of glucose and galactose. These results suggest that the rate of intracellular glucose phosphorylation is a decisive factor for metabolic regulations of mixed sugars.

Engineering and Evolution of Saccharomyces cerevisiae to Produce Biofuels and Chemicals.

To mitigate global climate change caused partly by the use of fossil fuels, the production of fuels and chemicals from renewable biomass has been attempted. The conversion of various sugars from renewable biomass into biofuels by engineered baker's yeast (Saccharomyces cerevisiae) is one major direction which has grown dramatically in recent years. As well as shifting away from fossil fuels, the production of commodity chemicals by engineered S. cerevisiae has also increased significantly. The traditional approaches of biochemical and metabolic engineering to develop economic bioconversion processes in laboratory and industrial settings have been accelerated by rapid advancements in the areas of yeast genomics, synthetic biology, and systems biology. Together, these innovations have resulted in rapid and efficient manipulation of S. cerevisiae to expand fermentable substrates and diversify value-added products. Here, we discuss recent and major advances in rational (relying on prior experimentally-derived knowledge) and combinatorial (relying on high-throughput screening and genomics) approaches to engineer S. cerevisiae for producing ethanol, butanol, 2,3-butanediol, fatty acid ethyl esters, isoprenoids, organic acids, rare sugars, antioxidants, and sugar alcohols from glucose, xylose, cellobiose, galactose, acetate, alginate, mannitol, arabinose, and lactose.

Enhanced isoprenoid production from xylose by engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae has limited capabilities for producing fuels and chemicals derived from acetyl-CoA, such as isoprenoids, due to a rigid flux partition toward ethanol during glucose metabolism. Despite numerous efforts, xylose fermentation by engineered yeast harboring heterologous xylose metabolic pathways was not as efficient as glucose fermentation for producing ethanol. Therefore, we hypothesized that xylose metabolism by engineered yeast might be a better fit for producing non-ethanol metabolites. We indeed found that engineered S. cerevisiae on xylose showed higher expression levels of the enzymes involved in ethanol assimilation and cytosolic acetyl-CoA synthesis than on glucose. When genetic perturbations necessary for overproducing squalene and amorphadiene were introduced into engineered S. cerevisiae capable of fermenting xylose, we observed higher titers and yields of isoprenoids under xylose than glucose conditions. Specifically, co-overexpression of a truncated HMG1 (tHMG1) and ERG10 led to substantially higher squalene accumulation under xylose than glucose conditions. In contrast to glucose utilization producing massive amounts of ethanol regardless of aeration, xylose utilization allowed much less amounts of ethanol accumulation, indicating ethanol is simultaneously re-assimilated with xylose consumption and utilized for the biosynthesis of cytosolic acetyl-CoA. In addition, xylose utilization by engineered yeast with overexpression of tHMG1, ERG10, and ADS coding for amorphadiene synthase, and the down-regulation of ERG9 resulted in enhanced amorphadiene production as compared to glucose utilization. These results suggest that the problem of the rigid flux partition toward ethanol production in yeast during the production of isoprenoids and other acetyl-CoA derived chemicals can be bypassed by using xylose instead of glucose as a carbon source. Biotechnol. Bioeng. 2017;114: 2581-2591. © 2017 Wiley Periodicals, Inc.

Metabolic engineering of a haploid strain derived from a triploid industrial yeast for producing cellulosic ethanol.

Many desired phenotypes for producing cellulosic biofuels are often observed in industrial Saccharomyces cerevisiae strains. However, many industrial yeast strains are polyploid and have low spore viability, making it difficult to use these strains for metabolic engineering applications. We selected the polyploid industrial strain S. cerevisiae ATCC 4124 exhibiting rapid glucose fermentation capability, high ethanol productivity, strong heat and inhibitor tolerance in order to construct an optimal yeast strain for producing cellulosic ethanol. Here, we focused on developing a general approach and high-throughput screening method to isolate stable haploid segregants derived from a polyploid parent, such as triploid ATCC 4124 with a poor spore viability. Specifically, we deleted the HO genes, performed random sporulation, and screened the resulting segregants based on growth rate, mating type, and ploidy. Only one stable haploid derivative (4124-S60) was isolated, while 14 other segregants with a stable mating type were aneuploid. The 4124-S60 strain inherited only a subset of desirable traits present in the parent strain, same as other aneuploids, suggesting that glucose fermentation and specific ethanol productivity are likely to be genetically complex traits and/or they might depend on ploidy. Nonetheless, the 4124-60 strain did inherit the ability to tolerate fermentation inhibitors. When additional genetic perturbations known to improve xylose fermentation were introduced into the 4124-60 strain, the resulting engineered strain (IIK1) was able to ferment a Miscanthus hydrolysate better than a previously engineered laboratory strain (SR8), built by making the same genetic changes. However, the IIK1 strain showed higher glycerol and xylitol yields than the SR8 strain. In order to decrease glycerol and xylitol production, an NADH-dependent acetate reduction pathway was introduced into the IIK1 strain. By consuming 2.4g/L of acetate, the resulting strain (IIK1A) exhibited a 14% higher ethanol yield and 46% lower byproduct yield than the IIK1 strain from anaerobic fermentation of the Miscanthus hydrolysate. Our results demonstrate that industrial yeast strains can be engineered via haploid isolation. The isolated haploid strain (4124-S60) can be used for metabolic engineering to produce fuels and chemicals.

Enhanced xylose fermentation by engineered yeast expressing NADH oxidase through high cell density inoculums.

Accumulation of reduced byproducts such as glycerol and xylitol during xylose fermentation by engineered Saccharomyces cerevisiae hampers the economic production of biofuels and chemicals from cellulosic hydrolysates. In particular, engineered S. cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD+-linked xylitol dehydrogenase (XDH) produces substantial amounts of the reduced byproducts under anaerobic conditions due to the cofactor difference of XR and XDH. While the additional expression of a water-forming NADH oxidase (NoxE) from Lactococcus lactis in engineered S. cerevisiae with the XR/XDH pathway led to reduced glycerol and xylitol production and increased ethanol yields from xylose, volumetric ethanol productivities by the engineered yeast decreased because of growth defects from the overexpression of noxE. In this study, we introduced noxE into an engineered yeast strain (SR8) exhibiting near-optimal xylose fermentation capacity. To overcome the growth defect caused by the overexpression of noxE, we used a high cell density inoculum for xylose fermentation by the SR8 expressing noxE. The resulting strain, SR8N, not only showed a higher ethanol yield and lower byproduct yields, but also exhibited a high ethanol productivity during xylose fermentation. As noxE overexpression elicits a negligible growth defect on glucose conditions, the beneficial effects of noxE overexpression were substantial when a mixture of glucose and xylose was used. Consumption of glucose led to rapid cell growth and therefore enhanced the subsequent xylose fermentation. As a result, the SR8N strain produced more ethanol and fewer byproducts from a mixture of glucose and xylose than the parental SR8 strain without noxE overexpression. Our results suggest that the growth defects from noxE overexpression can be overcome in the case of fermenting lignocellulose-derived sugars such as glucose and xylose.

Short communication: Conversion of lactose and whey into lactic acid by engineered yeast.

Lactose is often considered an unwanted and wasted byproduct, particularly lactose trapped in acid whey from yogurt production. But using specialized microbial fermentation, the surplus wasted acid whey could be converted into value-added chemicals. The baker's yeast Saccharomyces cerevisiae, which is commonly used for industrial fermentation, cannot natively ferment lactose. The present study describes how an engineered S. cerevisiae yeast was constructed to produce lactic acid from purified lactose, whey, or dairy milk. Lactic acid is an excellent proof-of-concept chemical to produce from lactose, because lactic acid has many food, pharmaceutical, and industrial uses, and over 250,000 t are produced for industrial use annually. To ferment the milk sugar lactose, a cellodextrin transporter (CDT-1, which also transports lactose) and a ß-glucosidase (GH1-1, which also acts as a ß-galactosidase) from Neurospora crassa were expressed in a S. cerevisiae strain. A heterologous lactate dehydrogenase (encoded by ldhA) from the fungus Rhizopus oryzae was integrated into the CDT-1/GH1-1-expressing strain of S. cerevisiae. As a result, the engineered strain was able to produce lactic acid from purified lactose, whey, and store-bought milk. A lactic acid yield of 0.358g/g of lactose was achieved from whey fermentation, providing an initial proof of concept for the production of value-added chemicals from excess industrial whey using engineered yeast.

Recycling Carbon Dioxide during Xylose Fermentation by Engineered Saccharomyces cerevisiae.

Global climate change caused by the emission of anthropogenic greenhouse gases (GHGs) is a grand challenge to humanity. To alleviate the trend, the consumption of fossil fuels needs to be largely reduced and alternative energy technologies capable of controlling GHG emissions are anticipated. In this study, we introduced a synthetic reductive pentose phosphate pathway (rPPP) into a xylose-fermenting Saccharomyces cerevisiae strain SR8 to achieve simultaneous lignocellulosic bioethanol production and carbon dioxide recycling. Specifically, ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum and phosphoribulokinase from Spinacia oleracea were introduced into the SR8 strain. The resulting strain with the synthetic rPPP was able to exhibit a higher yield of ethanol and lower yields of byproducts (xylitol and glycerol) than a control strain. In addition, the reduced release of carbon dioxide by the engineered strain was observed during xylose fermentation, suggesting that the carbon dioxide generated by pyruvate decarboxylase was partially reassimilated through the synthetic rPPP. These results demonstrated that recycling of carbon dioxide from the ethanol fermentation pathway in yeast can be achieved during lignocellulosic bioethanol production through a synthetic carbon conservative metabolic pathway. This strategy has a great potential to alleviate GHG emissions during the production of second-generation ethanol.

Lactose fermentation by engineered Saccharomyces cerevisiae capable of fermenting cellobiose.

Lactose is an inevitable byproduct of the dairy industry. In addition to cheese manufacturing, the growing Greek yogurt industry generates excess acid whey, which contains lactose. Therefore, rapid and efficient conversion of lactose to fuels and chemicals would be useful for recycling the otherwise harmful acid whey. Saccharomyces cerevisiae, a popular metabolic engineering host, cannot natively utilize lactose. However, we discovered that an engineered S. cerevisiae strain (EJ2) capable of fermenting cellobiose can also ferment lactose. This finding suggests that a cellobiose transporter (CDT-1) can transport lactose and a ß-glucosidase (GH1-1) can hydrolyze lactose by acting as a ß-galactosidase. While the lactose fermentation by the EJ2 strain was much slower than the cellobiose fermentation, a faster lactose-fermenting strain (EJ2e8) was obtained through serial subcultures on lactose. The EJ2e8 strain fermented lactose with a consumption rate of 2.16g/Lh. The improved lactose fermentation by the EJ2e8 strain was due to the increased copy number of cdt-1 and gh1-1 genes. Looking ahead, the EJ2e8 strain could be exploited for the production of other non-ethanol fuels and chemicals from lactose through further metabolic engineering.

Optimization of an acetate reduction pathway for producing cellulosic ethanol by engineered yeast.

Xylose fermentation by engineered Saccharomyces cerevisiae expressing NADPH-linked xylose reductase (XR) and NAD+ -linked xylitol dehydrogenase (XDH) suffers from redox imbalance due to cofactor difference between XR and XDH, especially under anaerobic conditions. We have demonstrated that coupling of an NADH-dependent acetate reduction pathway with surplus NADH producing xylose metabolism enabled not only efficient xylose fermentation, but also in situ detoxification of acetate in cellulosic hydrolysate through simultaneous co-utilization of xylose and acetate. In this study, we report the highest ethanol yield from xylose (0.463 g ethanol/g xylose) by engineered yeast with XR and XDH through optimization of the acetate reduction pathway. Specifically, we constructed engineered yeast strains exhibiting various levels of the acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) activities. Engineered strains exhibiting higher activities of AADH and ACS consumed more acetate and produced more ethanol from a mixture of 20 g/L of glucose, 80 g/L of xylose, and 8 g/L of acetate. In addition, we performed environmental and genetic perturbations to further improve the acetate consumption. Glucose-pulse feeding to continuously provide ATPs under anaerobic conditions did not affect acetate consumption. Promoter truncation of GPD1 and gene deletion of GPD2 coding for glycerol-3-phosphate dehydrogenase to produce surplus NADH also did not lead to improved acetate consumption. When a cellulosic hydrolysate was used, the optimized yeast strain (SR8A6S3) produced 18.4% more ethanol and 41.3% less glycerol and xylitol with consumption of 4.1 g/L of acetate than a control strain without the acetate reduction pathway. These results suggest that the major limiting factor for enhanced acetate reduction during the xylose fermentation might be the low activities of AADH and ACS, and that the redox imbalance problem of XR/XDH pathway can be exploited for in situ detoxification of acetic acid in cellulosic hydrolysate and increasing ethanol productivity and yield. Biotechnol. Bioeng. 2016;113: 2587-2596. © 2016 Wiley Periodicals, Inc.