Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis.

Intrahepatic osteopontin signaling by CREBZF defines a checkpoint for steatosis-to-NASH progression.

BACKGROUND AND AIMS: NASH has emerged as a leading cause of chronic liver disease. However, the mechanisms that govern NASH fibrosis remain largely unknown. CREBZF is a CREB/ATF bZIP transcription factor that causes hepatic steatosis and metabolic defects in obesity. APPROACH AND RESULTS: Here, we show that CREBZF is a key mechanism of liver fibrosis checkpoint that promotes hepatocyte injury and exacerbates diet-induced NASH in mice. CREBZF deficiency attenuated liver injury, fibrosis, and inflammation in diet-induced mouse models of NASH. CREBZF increases HSC activation and fibrosis in a hepatocyte-autonomous manner by stimulating an extracellular matrix protein osteopontin, a key regulator of fibrosis. The inhibition of miR-6964-3p mediates CREBZF-induced production and secretion of osteopontin in hepatocytes. Adeno-associated virus -mediated rescue of osteopontin restored HSC activation, liver fibrosis, and NASH progression in CREBZF-deficient mice. Importantly, expression levels of CREBZF are increased in livers of diet-induced NASH mouse models and humans with NASH. CONCLUSIONS: Osteopontin signaling by CREBZF represents a previously unrecognized intrahepatic mechanism that triggers liver fibrosis and contributes to the severity of NASH.

The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity.

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

Enhanced production of amyrin in Yarrowia lipolytica using a combinatorial protein and metabolic engineering approach.

BACKGROUND: Amyrin is an important triterpenoid and precursor to a wide range of cosmetic, pharmaceutical and nutraceutical products. In this study, we metabolically engineered the oleaginous yeast, Yarrowia lipolytica to produce α- and ß-amyrin on simple sugar and waste cooking oil. RESULTS: We first validated the in vivo enzymatic activity of a multi-functional amyrin synthase (CrMAS) from Catharanthus roseus, by expressing its codon-optimized gene in Y. lipolytica and assayed for amyrins. To increase yield, prevailing genes in the mevalonate pathway, namely HMG1, ERG20, ERG9 and ERG1, were overexpressed singly and in combination to direct flux towards amyrin biosynthesis. By means of a semi-rational protein engineering approach, we augmented the catalytic activity of CrMAS and attained ~ 10-folds higher production level on glucose. When applied together, protein engineering with enhanced precursor supplies resulted in more than 20-folds increase in total amyrins. We also investigated the effects of different fermentation conditions in flask cultures, including temperature, volumetric oxygen mass transfer coefficient and carbon source types. The optimized fermentation condition attained titers of at least 100 mg/L α-amyrin and 20 mg/L ß-amyrin. CONCLUSIONS: The design workflow demonstrated herein is simple and remarkably effective in amplifying triterpenoid biosynthesis in the yeast Y. lipolytica.

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae.

OBJECTIVES: Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus. RESULTS: Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower. CONCLUSIONS: Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

Comparative transcriptome analysis reveals the key regulatory genes for higher alcohol formation by yeast at different α-amino nitrogen concentrations.

Higher alcohols are important flavor substance in alcoholic beverages. The content of α-amino nitrogen (α-AN) in the fermentation system affects the formation of higher alcohols by Saccharomyces cerevisiae. In this study, the effect of α-AN concentration on the higher alcohol productivity of yeast was explored, and the mechanism of this effect was investigated through metabolite and transcription sequence analyses. We screened 12 most likely genes and constructed the recombinant strain to evaluate the effect of each gene on high alcohol formation. Results showed that the AGP1, GDH1, and THR6 genes were important regulators of higher alcohol metabolism in S. cerevisiae. This study provided knowledge about the metabolic pathways of higher alcohols and gave an important reference for the breeding of S. cerevisiae with low-yield higher alcohols to deal with the fermentation system with different α-AN concentrations in the brewing industry.

Increased RNA production in Saccharomyces cerevisiae by simultaneously overexpressing FHL1, IFH1, and SSF2 and deleting HRP1.

Ribonucleic acid (RNA) and its degradation products are widely used in the food industry. In this study, we constructed Saccharomyces cerevisiae mutants with FHL1, IFH1, SSF1, and SSF2 overexpression and HRP1 deletion, individually to evaluate the effect on RNA production. The RNA content of recombinant strains W303-1a-FHL1, W303-1a-SSF2, and W303-1a-ΔHRP1 was increased by 14.94%, 24.4%, and 19.36%, respectively, compared with the RNA content of the parent strain. However, W303-1a-IFH1 and W303-1a-SSF1 showed no significant change in RNA production compared with the parent strain. IFH1 and FHL1 encode Ifh1p and Fhl1p, respectively, which combine to form a complex that plays a key role in the transcription of the ribosomal protein (RP) gene. Ssf2p, encoded by SSF2, plays an important role in ribosome biosynthesis and Hrp1p is a negative regulator of cell growth in S. cerevisiae. Subsequently, a high RNA production strain, W112, was constructed by simultaneously overexpressing FHL1, IFH1, and SSF2 and deleting HRP1. The RNA content of W112 was 38.8% higher than the parent strain. The growth performance, RP transcription levels, and rRNA content were also investigated in the recombinant strains. This study provides a new strategy for the construction of S. cerevisiae strains containing large amounts of RNA, and it will make a significant contribution to progress in the nucleic acid industry. KEY POINTS: • Simultaneously overexpressing FHL1, IFH1, and SSF2 and deleting HRP1 can significantly increases RNA production. • The production of RNA increased by 38.8% in Saccharomyces cerevisiae. • The cell size and growth rate of the strains with higher RNA content also increased.

Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.

OBJECTIVE: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass. RESULTS: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0-9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT. CONCLUSIONS: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.

Improve the production of D-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation.

D-Limonene, a cyclized monoterpene, possesses citrus-like olfactory property and multi-physiological functions, which can be used as a bioactive compound and flavor to improve the overall quality of alcoholic beverages. In our previous study, we established an orthogonal pathway of D-limonene synthesis by introducing neryl diphosphate synthase 1 (tNDPS1) and D-limonene synthase (tLS) in Saccharomyces cerevisiae. To further increase D-limonene formation, the metabolic flux of the mevalonate (MVA) pathway was enhanced by overexpressing the key genes tHMGR1, ERG12, IDI1, and IDI1WWW, respectively, or co-overexpressing. The results showed that strengthening the MVA pathway significantly improved D-limonene production, while the best strain yielded 62.31 mg/L D-limonene by co-expressing tHMGR1, ERG12, and IDI1WWW genes in alcoholic beverages. Furthermore, we also studied the effect of enhancing the MVA pathway on the growth and fermentation of engineered yeasts during alcoholic beverage fermentation. Besides, to further resolve the problem of yeast growth inhibition, we separately investigated transporter proteins of the high-yielding D-limonene yeasts and the parental strain under the stress of different D-limonene concentration, suggesting that the transporters of Aus1p, Pdr18p, Pdr5p, Pdr3p, Pdr11p, Pdr15p, Tpo1p, and Ste6p might play a more critical role in alleviating cytotoxicity and improving the tolerance to D-limonene. Finally, we verified the functions of three transporter proteins, finding that the transporter of Aus1p failed to transport D-limonene, and the others (Pdr5p and Pdr15p) could improve the tolerance of yeast to D-limonene. This study provided a valuable platform for other monoterpenes' biosynthesis in yeast during alcoholic beverage fermentation.

Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation.

d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W-N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W-N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 fusion protein, and the best strain yielded 9.8 mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

Identification by comparative transcriptomics of core regulatory genes for higher alcohol production in a top-fermenting yeast at different temperatures in beer fermentation.

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.

Regulating the Golgi apparatus sorting of proteinase A to decrease its excretion in Saccharomyces cerevisiae.

Beer foam stability, a key factor in evaluating overall beer quality, is influenced by proteinase A (PrA). Actin-severing protein cofilin and Golgi apparatus-localized Ca2+ ATPase Pmr1 are involved in protein sorting at the trans-Golgi network (TGN) in yeast Curwin et al. (Mol Biol Cell 23:2327-2338, 2012). To reduce PrA excretion into the beer fermentation broth, we regulated the Golgi apparatus sorting of PrA, thereby facilitating the delivery of more PrA to the vacuoles in the yeast cells. In the present study, the cofilin-coding gene COF1 and the Pmr1-coding gene PMR1 were overexpressed in the parental strain W303-1A and designated as W + COF1 and W + PMR1, respectively. The relative expression levels of COF1 in W + COF1 and PMR1 in W + PMR1 were 5.26- and 19.76-fold higher than those in the parental strain. After increases in the expression levels of cofilin and Pmr1 were confirmed, the PrA activities in the wort broth fermented with W + COF1, W + PMR1, and W303-1A were measured. Results showed that the extracellular PrA activities of W + COF1 and W + PMR1 were decreased by 9.24% and 13.83%, respectively, at the end of the main fermentation compared with that of W303-1A. Meanwhile, no apparent differences were found on the fermentation performance of recombinant and parental strains. The research uncovers an effective strategy for decreasing PrA excretion in Saccharomyces cerevisiae.

Increase ethyl acetate production in Saccharomyces cerevisiae by genetic engineering of ethyl acetate metabolic pathway.

Ethyl acetate has attracted much attention as an important chemical raw material and a flavor component of alcoholic beverages. In this study, the biosynthetic pathway for the production of ethyl acetate in Chinese liquor yeast was unblocked. In addition to engineering Saccharomyces cerevisiae to increased intracellular CoA and acetyl-CoA levels, we also increased the combining efficiency of acetyl-CoA to ethanol. The genes encoding phosphopantothenate-cysteine ligase, acetyl-CoA synthetase, and alcohol acetyltransferase were overexpressed by inserting the strong promoter PGK1p and the terminator PGK1t, respectively, and then combine them. Our results finally showed that the ethyl acetate levels of all engineering strains were improved. The final engineering strain CLy12a-ATF1-ACS2-CAB2 had a significant increase in ethyl acetate yield, reaching 610.26 (± 14.28) mg/L, and the yield of higher alcohols was significantly decreased. It is proved that the modification of ethyl acetate metabolic pathway is extremely important for the production of ethyl acetate from Saccharomyces cerevisiae.

Modulating acetate ester and higher alcohol production in Saccharomyces cerevisiae through the cofactor engineering.

Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In Saccharomyces cerevisiae cells, the syntheses of esters and higher alcohols are considerably influenced by intracellular CoA levels catalyzed by pantothenate kinase. In this work, we examined the effects of cofactor CoA and acetyl-CoA synthesis on the metabolism of esters and higher alcohols. Strains 12α-BAP2 and 12α+ATF1 where generated by deleting and overexpressing BAP2 (encoded branched-chain amino acid permease) and ATF1 (encoded alcohol acetyl transferases), respectively, in the parent 12α strains. Then, 12α-BAP2+CAB1 and 12α-BAP2+CAB3 strains were obtained by overexpressing CAB1 (encoded pantothenate kinase Cab1) and CAB3 (encoded pantothenate kinase Cab3) in the 12α-BAP2 strain, and 12α-BAP2+CAB1+ATF1 and 12α-BAP2+CAB3+ATF1 were generated by overexpressing ATF1 in the pantothenate kinase overexpression strains. The acetate ester level in 12α-BAP2 was slightly changed relative to that in the control strain 12α, whereas the acetate ester levels in 12α-BAP2+CAB1, 12α-BAP2+CAB3, 12α-BAP2+CAB1+ATF1, and 12α-BAP2+CAB3+ATF1 were distinctly increased (44-118% for ethyl acetate and 18-57% for isoamyl acetate). The levels of n-propanol, methyl-1-butanol, isopentanol, isobutanol, and phenethylol levels were changed and varied among the six engineered strains. The levels of acetate esters and higher alcohols can be modulated by changing the CoA and acetyl-CoA levels. The method proposed in this work supplies a practical means of breeding yeast strains by modulating acetate ester and higher alcohol production.

Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans.

BACKGROUND: Aureobasidium pullulans is a yeast-like fungus that can ferment xylose to generate high-value-added products, such as pullulan, heavy oil, and melanin. The combinatorial expression of two xylose reductase (XR) genes and two xylitol dehydrogenase (XDH) genes from Spathaspora passalidarum and the heterologous expression of the Piromyces sp. xylose isomerase (XI) gene were induced in A. pullulans to increase the consumption capability of A. pullulans on xylose. RESULTS: The overexpression of XYL1.2 (encoding XR) and XYL2.2 (encoding XDH) was the most beneficial for xylose utilization, resulting in a 17.76% increase in consumed xylose compared with the parent strain, whereas the introduction of the Piromyces sp. XI pathway failed to enhance xylose utilization efficiency. Mutants with superior xylose fermentation performance exhibited increased intracellular reducing equivalents. The fermentation performance of all recombinant strains was not affected when glucose or sucrose was utilized as the carbon source. The strain with overexpression of XYL1.2 and XYL2.2 exhibited excellent fermentation performance with mimicked hydrolysate, and pullulan production increased by 97.72% compared with that of the parent strain. CONCLUSIONS: The present work indicates that the P4 mutant (using the XR/XDH pathway) with overexpressed XYL1.2 and XYL2.2 exhibited the best xylose fermentation performance. The P4 strain showed the highest intracellular reducing equivalents and XR and XDH activity, with consequently improved pullulan productivity and reduced melanin production. This valuable development in aerobic fermentation by the P4 strain may provide guidance for the biotransformation of xylose to high-value products by A. pullulans through genetic approach.

An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway.

BACKGROUND: Microbial biofuel production provides a promising sustainable alternative to fossil fuels. 1-Butanol is recognized as an advanced biofuel and is gaining attention as an ideal green replacement for gasoline. In this proof-of-principle study, the oleaginous yeast Yarrowia lipolytica was first engineered with a heterologous CoA-dependent pathway and an endogenous pathway, respectively. RESULTS: The co-overexpression of two heterologous genes ETR1 and EutE resulted in the production of 1-butanol at a concentration of 65 µg/L. Through the overexpression of multiple 1-butanol pathway genes, the titer was increased to 92 µg/L. Cofactor engineering through endogenous overexpression of a glyceraldehyde-3-phosphate dehydrogenase and a malate dehydrogenase further led to titer improvements to 121 µg/L and 110 µg/L, respectively. In addition, the presence of an endogenous 1-butanol production pathway and a gene involved in the regulation of 1-butanol production was successfully identified in Y. lipolytica. The highest titer of 123.0 mg/L was obtained through this endogenous route by combining a pathway gene overexpression strategy. CONCLUSIONS: This study represents the first report on 1-butanol biosynthesis in Y. lipolytica. The results obtained in this work lay the foundation for future engineering of the pathways to optimize 1-butanol production in Y. lipolytica.

Genetic engineering to alter carbon flux for various higher alcohol productions by Saccharomyces cerevisiae for Chinese Baijiu fermentation.

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded ß-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.

Overexpression of SNF4 and deletions of REG1- and REG2-enhanced maltose metabolism and leavening ability of baker's yeast in lean dough.

Maltose metabolism of baker's yeast (Saccharomyces cerevisiae) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker's yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1/REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant ΔREG1ΔREG2 + SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker's yeast strains had excellent potential applications.

Optimization of sodium percarbonate pretreatment for improving 2,3-butanediol production from corncob.

Sodium percarbonate (SP), a kind of alkaline strong oxidant, was applied to corncob pretreatment. The optimized pretreatment conditions were at 4% (w/v) SP concentration with solid-to-liquid (SLR) ratio of 1:10 treating for 4 hr at 60°C. This pretreatment resulted in 91.06% of cellulose and 84.08% of hemicellulose recoveries with 34.09% of lignin removal in corncob. The reducing sugar yield from SP-pretreated corncob was 0.56 g/g after 72 hr of enzymatic hydrolysis, 1.75-folds higher than that from raw corncob. 2,3-butanediol production by Enterobacer cloacae in simultaneous saccharification fermentation was 29.18 g/L using SP-pretreated corncob as a substrate, which was 11.12 times of that using raw corncob. Scanning electron microscope, X-ray diffraction, and Fourier transform infrared spectra analysis indicated that physical characteristics, crystallinity, and structure of corncob had changed obviously after SP pretreatment. This simple and novel pretreatment method was effective for delignification and carbohydrate retention in microbial production of 2,3-butanediol from lignocellulose biomass.

Effect of ß-mannanase domain from Trichoderma reesei on its biochemical characters and synergistic hydrolysis of sugarcane bagasse.

BACKGROUND: ß-mannanase is a key enzyme for hydrolyzing mannan, a major constituent of hemicellulose, which is the second most abundant polysaccharide in nature. Different structural domains greatly affect its biochemical characters and catalytic efficiency. However, the effects of linker and carbohydrate-binding module (CBM) on ß-mannanase from Trichoderma reesei (Man1) have not yet been fully described. The present study aimed to determine the influence of different domains on the expression efficiency, biochemical characteristics and hemicellulosic deconstruction of Man1. RESULTS: The expression efficiency was improved after truncating CBM. Activities of Man1 and Man1ΔCBM (CBM) in the culture supernatant after 168 h of induction were 34.5 and 42.9 IU mL-1 , although a value of only 0.36 IU mL-1 was detected for Man1ΔLCBM (lacking CBM and linker). Man1 showed higher thermostability than Man1ΔCBM at low temperature, whereas Man1ΔCBM had a higher specificity for galactomannan (Km = 2.5 mg mL-1 ) than Man1 (Km = 4.0 mg mL-1 ). Both Man1 and Man1ΔCBM could synergistically improve the hydrolysis of cellulose, galactomannan and pretreated sugarcane bagasse, with a 10-30% improvement of the reducing sugar yield. CONCLUSION: Linker and CBM domains were vital for mannanase activity and expression efficiency. CBM affected the thermostability and adsorption ability of Man1. The results obtained in the present study should help guide the rational design and directional modification of Man with respect to improving its catalytic efficiency. © 2017 Society of Chemical Industry.