Enhanced pyruvic acid yield in an osmotic stress-resistant mutant of Yarrowia lipolytica

BACKGROUND: Pyruvic acid (PA), a vital α-oxocarboxylic acid, plays an important role in energy and carbon metabolism. The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has considerable potential for the production of PA. An increased NaCl concentration reportedly increases the biomass and PA yield of Y. lipolytica. RESULTS: To increase the yield of PA, the NaCl-tolerant Y. lipolytica A4 mutant was produced using the atmospheric and room temperature plasma method of mutation. The A4 mutant showed growth on medium containing 160 g/L NaCl. The PA yield of the A4 mutant reached 97.2 g/L at 120 h (0.795 g/g glycerol) in a 20-L fermenter with glycerol as the sole carbon source, which was 28.9% higher than that of the parental strain. CONCLUSION: The PA yield from Y. lipolytica can be improved by increasing its NaCl tolerance.

Global Prevalence and Incidence Estimates of Oral Lichen Planus: A Systematic Review and Meta-analysis.

Importance: Integrated information on the global prevalence and incidence of oral lichen planus (OLP) is lacking. Objective: To examine the global prevalence and incidence of OLP in a systematic review and meta-analysis. Data Sources: A systematic review of population-based studies and clinic-based studies reporting the prevalence and incidence of OLP was performed using 3 electronic medical databases (Cochrane Database of Systematic Reviews, Embase, and MEDLINE) from their inception to March 2019. The search terms included "(lichen planus or LP) and (prevalence or incidence or epidemiology)." No language restriction was applied. Study Selection: Observational descriptive studies investigating the prevalence and incidence of OLP were included. Data Extraction and Synthesis: Data were extracted by continent, sex, and other characteristics. The risk of bias was assessed by the Joanna Briggs Institute Critical Appraisal Instrument for Studies Reporting Prevalence Data using random-effects models to synthesize available evidence. Main Outcomes and Measures: The primary outcome was the prevalence (with 95% CIs) of OLP among the overall population and among subgroups. Between-study heterogeneity was assessed using the I2 statistic. Results: Among 46 studies, the overall pooled estimated prevalence of OLP was 0.89% (95% CI, 0.38%-2.05%) among the general population (n = 462 993) and 0.98% (95% CI, 0.67%-1.43%) among clinical patients (n = 191 963). Among the 15 population-based studies, the prevalence of OLP was 0.57% (95% CI, 0.15%-2.18%) in Asia, 1.68% (95% CI, 1.09%-2.58%) in Europe, and 1.39% (95% CI, 0.58%-3.28%) in South America. Among the 31 clinic-based studies, the prevalence was 1.43% (95% CI, 1.12%-1.83%) in Africa, 0.87% (95% CI, 0.61%-1.25%) in Asia, 1.03% (95% CI, 0.51%-2.09%) in Europe, 0.11% (95% CI, 0.07%-0.16%) in North America, and 3.18% (95% CI, 0.97%-9.95%) in South America. The pooled prevalence of OLP by sex was 1.55% (95% CI, 0.83%-2.89%) for women and 1.11% (95% CI, 0.57%-2.14%) for men in the population-based studies and 1.69% (95% CI, 1.05%-2.70%) for women and 1.09% (95% CI, 0.67%-1.77%) for men in the clinic-based studies. In 5 clinic-based studies providing the age distribution of patients with OLP, the prevalence by age was 0.62% (95% CI, 0.33%-1.13%) among patients younger than 40 years and 1.90% (95% CI, 1.16%-3.10%) among patients 40 years and older. Conclusions and Relevance: This study identified the global prevalence and incidence of OLP in terms of its spatial, temporal, and population distribution. The overall estimated pooled prevalence of OLP was 0.89% among the general population and 0.98% among clinical patients. A higher prevalence of OLP was found in non-Asian countries, among women, and among people 40 years and older. The findings should be considered with caution because of the high heterogeneity of the included studies.

Quantitative iTRAQ LC-MS/MS proteomics reveals the cellular response to heterologous protein overexpression and the regulation of HAC1 in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC-MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion. BIOLOGICAL SIGNIFICANCE: Pichia pastoris expression systems have been successfully used for over 20years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris.

Bleach boosting effect of xylanase A from Bacillus halodurans C-125 in ECF bleaching of wheat straw pulp.

Past studies have revealed major difficulties in applications of xylanase in the pulp and paper industry as enzymes isolated from many different species could not tolerate high temperatures or highly alkaline conditions. The thermostable xylanase A from Bacillus halodurans C-125 (C-125 xylanase A) was successfully cloned and expressed in Pichia pastoris with a yield as high as 3361 U/mL in a 2 L reactor. Its thermophilic and basophilic properties (optimal activity at 70 °C and pH 9.0), together with the fact it is cellulase-free, render this enzyme attractive for compatible applications in the pulp and paper industry. The pretreatment of wheat straw pulp with C-125 xylanase A at pH 9.0 and 70 °C for 90 min induced the release of both chromophores (Ab(237), Ab(254), Ab(280)) and hydrophobic compounds (Ab(465)) into the filtrate as well as sugar degradation. Moreover, the addition of 10 U xylanase to 1 g wheat straw pulp (dry weight) as pretreatment improved brightness by 5.2% ISO and decreased the kappa number by 5.0% when followed by hydrogen peroxide bleaching. In addition, compared with two commercial enzymes, Pulpzyme HC and AU-PE89, which are normally incorporated in ECF bleaching of wheat straw pulp, C-125 xylanase A proved to be more effective in enhancing brightness as well as preserving paper strength properties. When evaluating the physical properties of pulp samples, such as tensile index, tearing index, bursting index, and post-color (PC) number, the enzymes involved in pretreating pulps exhibited better or the same performances as chemical treatment. Compared with chemical bleaching, chlorine consumption can be significantly reduced by 10% for xylanase-pretreated wheat straw pulp while maintaining the brightness together with the kappa number at the same level. Scanning electron microscopy revealed significant surface modification of enzyme-pretreated pulp fibers with no marked fiber disruptions.

[Mutational research on the role of lysine 21 in the Pichia stipitis xylose reductase].

The xylose reductase of Pichia stipitis is one of the most important enzymes. It can be used to build up recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing ethanol. Intercellular redox imbalance caused by NADPH preference over NADH for Pichia stipitis xylose reductase (PsXR) has been considered to be one of the main factors for poor ethanol productivity. Some key amino acids of PsXR, which affect the activity or coenzyme preference, were investigated in our previous study. In this study, Lys21 were rational designed for site-directed mutagenesis to alter coenzyme specificity of PsXR from NADPH and NADH into single NADH. The wild gene and mutagenesis genes were ligated into pET28b, and were transferred into E.coli BL21(DE3). After induced by IPTG, the xylose reductases were purified. Purified mutants K21A (Lys21-->Ala), K21R(Lys21-->Arg) were characterized by steady-state kinetic analysis. The results showed that the coenzyme dependence of K21A was completely reversed to NADH.