Deletion of NGG1 in a recombinant Saccharomyces cerevisiae improved xylose utilization and affected transcription of genes related to amino acid metabolism.

Production of biofuels and biochemicals from xylose using yeast cell factory is of great interest for lignocellulosic biorefinery. Our previous studies revealed that a natural yeast isolate Saccharomyces cerevisiae YB-2625 has superior xylose-fermenting ability. Through integrative omics analysis, NGG1, which encodes a transcription regulator as well as a subunit of chromatin modifying histone acetyltransferase complexes was revealed to regulate xylose metabolism. Deletion of NGG1 in S. cerevisiae YRH396h, which is the haploid version of the recombinant yeast using S. cerevisiae YB-2625 as the host strain, improved xylose consumption by 28.6%. Comparative transcriptome analysis revealed that NGG1 deletion down-regulated genes related to mitochondrial function, TCA cycle, ATP biosynthesis, respiration, as well as NADH generation. In addition, the NGG1 deletion mutant also showed transcriptional changes in amino acid biosynthesis genes. Further analysis of intracellular amino acid content confirmed the effect of NGG1 on amino acid accumulation during xylose utilization. Our results indicated that NGG1 is one of the core nodes for coordinated regulation of carbon and nitrogen metabolism in the recombinant S. cerevisiae. This work reveals novel function of Ngg1p in yeast metabolism and provides basis for developing robust yeast strains to produce ethanol and biochemicals using lignocellulosic biomass.

Design, Evolution, and Characterization of a Xylose Biosensor in Escherichia coli Using the XylR/xylO System with an Expanded Operating Range.

Genetically encoded biosensors are extensively utilized in synthetic biology and metabolic engineering. However, reported xylose biosensors are far too sensitive with a limited operating range to be useful for most sensing applications. In this study, we describe directed evolution of Escherichia coli XylR, and construction of biosensors based on XylR and the corresponding operator xylO. The operating range of biosensors containing the mutant XylR was increased by nearly 10-fold comparing with the control. Two individual amino acid mutations (either L73P or N220T) in XylR were sufficient to extend the linear response range to upward of 10 g/L xylose. The evolved biosensors described here are well suited for developing whole-cell biosensors for detecting varying xylose concentrations across an expanded range. As an alternative use of this system, we also demonstrate the utility of XylR and xylO as a xylose inducible system to enable graded gene expression through testing with ß-galactosidase gene and the lycopene synthetic pathway. This evolution strategy identified a less-sensitive biosensor for real applications, thus providing new insights into strategies for expanding operating ranges of other biosensors for synthetic biology applications.

Condition-specific promoter activities in Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae is widely studied for production of biofuels and biochemicals. To improve production efficiency under industrially relevant conditions, coordinated expression of multiple genes by manipulating promoter strengths is an efficient approach. It is known that gene expression is highly dependent on the practically used environmental conditions and is subject to dynamic changes. Therefore, investigating promoter activities of S. cerevisiae under different culture conditions in different time points, especially under stressful conditions is of great importance. RESULTS: In this study, the activities of various promoters in S. cerevisiae under stressful conditions and in the presence of xylose were characterized using yeast enhanced green fluorescent protein (yEGFP) as a reporter. The stresses include toxic levels of acetic acid and furfural, and high temperature, which are related to fermentation of lignocellulosic hydrolysates. In addition to investigating eight native promoters, the synthetic hybrid promoter P3xC-TEF1 was also evaluated. The results revealed that P TDH3 and the synthetic promoter P3xC-TEF1 showed the highest strengths under almost all the conditions. Importantly, these two promoters also exhibited high stabilities throughout the cultivation. However, the strengths of P ADH1 and P PGK1 , which are generally regarded as 'constitutive' promoters, decreased significantly under certain conditions, suggesting that cautions should be taken to use such constitutive promoters to drive gene expression under stressful conditions. Interestingly, P HSP12 and P HSP26 were able to response to both high temperature and acetic acid stress. Moreover, P HSP12 also led to moderate yEGFP expression when xylose was used as the sole carbon source, indicating that this promoter could be used for inducing proper gene expression for xylose utilization. CONCLUSION: The results here revealed dynamic changes of promoter activities in S. cerevisiae throughout batch fermentation in the presence of inhibitors as well as using xylose. These results provide insights in selection of promoters to construct S. cerevisiae strains for efficient bioproduction under practical conditions. Our results also encouraged applications of synthetic promoters with high stability for yeast strain development.

Association of improved oxidative stress tolerance and alleviation of glucose repression with superior xylose-utilization capability by a natural isolate of Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae wild strains generally have poor xylose-utilization capability, which is a major barrier for efficient bioconversion of lignocellulosic biomass. Laboratory adaption is commonly used to enhance xylose utilization of recombinant S. cerevisiae. Apparently, yeast cells could remodel the metabolic network for xylose metabolism. However, it still remains unclear why natural isolates of S. cerevisiae poorly utilize xylose. Here, we analyzed a unique S. cerevisiae natural isolate YB-2625 which has superior xylose metabolism capability in the presence of mixed-sugar. Comparative transcriptomic analysis was performed using S. cerevisiae YB-2625 grown in a mixture of glucose and xylose, and the model yeast strain S288C served as a control. Global gene transcription was compared at both the early mixed-sugar utilization stage and the latter xylose-utilization stage. RESULTS: Genes involved in endogenous xylose-assimilation (XYL2 and XKS1), gluconeogenesis, and TCA cycle showed higher transcription levels in S. cerevisiae YB-2625 at the xylose-utilization stage, when compared to the reference strain. On the other hand, transcription factor encoding genes involved in regulation of glucose repression (MIG1, MIG2, and MIG3) as well as HXK2 displayed decreased transcriptional levels in YB-2625, suggesting the alleviation of glucose repression of S. cerevisiae YB-2625. Notably, genes encoding antioxidant enzymes (CTT1, CTA1, SOD2, and PRX1) showed higher transcription levels in S. cerevisiae YB-2625 in the xylose-utilization stage than that of the reference strain. Consistently, catalase activity of YB-2625 was 1.9-fold higher than that of S. cerevisiae S288C during the xylose-utilization stage. As a result, intracellular reactive oxygen species levels of S. cerevisiae YB-2625 were 43.3 and 58.6% lower than that of S288C at both sugar utilization stages. Overexpression of CTT1 and PRX1 in the recombinant strain S. cerevisiae YRH396 deriving from S. cerevisiae YB-2625 increased cell growth when xylose was used as the sole carbon source, leading to 13.5 and 18.1%, respectively, more xylose consumption. CONCLUSIONS: Enhanced oxidative stress tolerance and relief of glucose repression are proposed to be two major mechanisms for superior xylose utilization by S. cerevisiae YB-2625. The present study provides insights into the innate regulatory mechanisms underlying xylose utilization in wild-type S. cerevisiae, which benefits the rapid development of robust yeast strains for lignocellulosic biorefineries.

Mixture of Peanut Skin Extract and Fish Oil Improves Memory in Mice via Modulation of Anti-Oxidative Stress and Regulation of BDNF/ERK/CREB Signaling Pathways.

Long-term use of fish oil (FO) is known to induce oxidative stress and increase the risk of Alzheimer's disease in humans. In the present study, peanut skin extract (PSE), which has strong antioxidant capacity, was mixed with FO to reduce its side effects while maintaining its beneficial properties. Twelve-week Institute of Cancer Research (ICR) mice were used to conduct animal behavior tests in order to evaluate the memory-enhancing ability of the mixture of peanut skin extract and fish oil (MPF). MPF significantly increased alternations in the Y-maze and cognitive index in the novel object recognition test. MPF also improved performance in the water maze test. We further sought to understand the mechanisms underlying these effects. A significant decrease in superoxide dismutase (SOD) activity and an increase in malonyldialdehyde (MDA) in plasma were observed in the FO group. The MPF group showed reduced MDA level and increased SOD activity in the plasma, cortex and hippocampus. Furthermore, the gene expression levels of brain-derived neurotrophic factor (BDNF) and cAMP responsive element-binding protein (CREB) in the hippocampus were increased in the MPF group, while phosphorylation of protein kinase B (AKT), extracellular signal-regulated kinase (ERK) and CREB in the hippocampus were enhanced. MPF improves memory in mice via modulation of anti-oxidative stress and activation of BDNF/ERK/CREB signaling pathways.

Response of risperidone treatment may be associated with polymorphisms of HTT gene in Chinese schizophrenia patients.

Serotonin transporter (5-HTT) is a key component of the serotonergic neurotransmitter system. Few studies have focused on polymorphisms of the serotonin transporter and antipsychotic response and, in particular, there have so far been no published studies on the association between the serotonin transporter and response to risperidone. This study examined the relationship between two polymorphisms of the serotonin transporter and the efficacy of risperidone treatment in 129 patients with schizophrenia. Our results revealed that patients with l allele of HTTRLP showed a greater improvement than those without l allele on the overall brief psychiatric rating scale (BPRS) (P=0.025). But no such relationship was found for the HTTVNTR. In haplotype analysis, the frequency of L-12 haplotype showed a significant difference between the responder group and the non-responder group (P=0.005). Our study has, for the first time, produced evidence that the potential for therapy in patients with schizophrenia is related to the HTTRLP polymorphism in the HTT gene and haplotype L-12 may help to predict risperidone treatment efficiency.