Advanced stimuli-responsive membranes for smart separation.

Membranes have been extensively studied and applied in various fields owing to their high energy efficiency and small environmental impact. Further conferring membranes with stimuli responsiveness can allow them to dynamically tune their pore structure and/or surface properties for efficient separation performance. This review summarizes and discusses important developments and achievements in stimuli-responsive membranes. The most commonly utilized stimuli, including light, pH, temperature, ions, and electric and magnetic fields, are discussed in detail. Special attention is given to stimuli-responsive control of membrane pore structure (pore size and porosity/connectivity) and surface properties (wettability, surface topology, and surface charge), from the perspective of determining the appropriate membrane properties and microstructures. This review also focuses on strategies to prepare stimuli-responsive membranes, including blending, casting, polymerization, self-assembly, and electrospinning. Smart applications for separations are also reviewed as well as a discussion of remaining challenges and future prospects in this exciting field. This review offers critical insights for the membrane and broader materials science communities regarding the on-demand and dynamic control of membrane structures and properties. We hope that this review will inspire the design of novel stimuli-responsive membranes to promote sustainable development and make progress toward commercialization.

Microbial succession and its effect on key aroma components during light-aroma-type Xiaoqu Baijiu brewing process.

Light-aroma-type Baijiu is a Chinese distilled alcoholic beverage produced from fermented sorghum. Microbial composition and dynamics during Baijiu production have a great influence on the flavor and quality of Chinese Baijiu. However, the microbial changes that occur during brewing of Xiaoqu Baijiu are poorly understood. In this study, the microbial composition of light-aroma-type Xiaoqu Baijiu at the saccharification and fermentation stages was investigated to explore microbial dynamics and their effects on aroma components using high-throughput sequencing and gas chromatography-flame ionization detection (GC-FID). Rhizopus, Pichia, Wickerhamomyces, Saccharomyces, Acinetobacter, Lactobacillus, and Weissella constituted the core microbes for Xiaoqu Baijiu production. Microbial succession during brewing could be divided into two phases: at the saccharification and early fermentation stages (F-0d to F-4d), Rhizopus and Acinetobacter were identified as the predominant microbes, accounting for 78.2-90.8% and 53.9-89.5% of the fungal and bacterial communities, respectively, whereas at the middle and late stages of fermentation (F-5d to F-14d), the abundance of Pichia, Wickerhamomyces, Saccharomyces, and Lactobacillus increased. Redundancy analysis (RDA) and Mantel tests indicated that the water, amino acid nitrogen, acid, and reducing sugar contents were significantly correlated with the fungal and bacterial communities in grains (p < 0.05). Pichia, Rhizopus, Saccharomyces, and Wickerhamomyces, especially Saccharomyces, were closely related to the contents of major alcohols, esters and aldehydes, and these microbes had an important functional role in the formation of Xiaoqu Baijiu flavor. This work provides insights into the microbial succession that occurs during brewing of light-aroma-type Xiaoqu Baijiu and the microbial contribution to flavor, which have potential for optimizing production and enhancing the flavor of Baijiu.

High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia pastoris.

As a promising biocatalyst, Yarrowia lipolytica lipase 2 (YlLip2) is limited in its industrial applications due to its low thermostability. In this study, a thermostable YlLip2 mutant was overexpressed in Pichia pastoris and its half-life time was over 30 min at 80 °C. To obtain a higher protein secretion level, the gene dosage of the mutated lip2 gene was optimized and the lipase activity was improved by about 89%. Then, the YlLip2 activity of the obtained strain further increased from 482 to 1465 U/mL via optimizing the shaking flask culture conditions. Subsequently, Hac1p and Vitreoscilla hemoglobin (VHb) were coexpressed with the YlLip2 mutant to reduce the endoplasmic reticulum stress and enhance the oxygen uptake efficiency in the recombinant strains, respectively. Furthermore, high-density fermentations were performed in a 3 L bioreactor and the production of the YlLip2 mutant reached 9080 U/mL. The results demonstrated that the expression level of the thermostable YlLip2 mutant was predominantly enhanced via the combination of these strategies in P. pastoris, which forms a consolidated basis for its large-scale production and future industrial applications.

Efficient Heterologous Production of Rhizopus oryzae Lipase via Optimization of Multiple Expression-Related Helper Proteins.

This study is dedicated to efficiently produce Rhizopus oryzae lipase (ROL) by optimizing the expression of multiple expression-related helper proteins in Pichia pastoris. A series of engineered strains harboring different copy numbers of the ROL gene and different copies of the chaperone Pdi gene were first constructed to examine the influence of Pdi gene copy number on ROL production. The results showed that multiple copies of Pdi gene did not significantly improve ROL expression. Then, the effect of the co-overexpression of 10 expression-related helper proteins on ROL secretion was investigated by screening 20 colonies of each transformants. The data from shaking-flask fermentation suggested that Ssa4, Bmh2, Sso2, Pdi, Bip, Hac1, and VHb had positive effects on ROL expression. Subsequently, Ssa4, Bmh2, and Sso2, which all participate in vesicular trafficking and strongly promote ROL expression, were combined to further improve ROL production level. ROL activity of the screened strain GS115/5ROL-Ssa4-Sso2-Bmh2 4# attained 5230 U/mL. Furthermore, when the helper proteins Pdi, Bip, Hac1, and VHb were individually co-expressed with ROL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2 4#, lipase activity increased to 5650 U/mL in the strain GS115/5ROL-Ssa4-Sso2-Bmh2-VHb 9#. Additionally, the maximum ROL activity of 41,700 U/mL was achieved in a 3 L bioreactor for high-density fermentation via a sorbitol⁻methanol co-feeding strategy, reaching almost twofold the value of the initial strain GS115/pAOα-5ROL 11#. Thus, the strategies in this study significantly increased ROL expression level, which is of great potential for the large-scale production of ROL in P. pastoris.

High-level extracellular production of Rhizopus oryzae lipase in Pichia pastoris via a strategy combining optimization of gene-copy number with co-expression of ERAD-related proteins.

Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy. The activity of GS115/pAOα-5ROL 11# was then enhanced to 3080 U/mL in a shaking flask under optimized culture conditions. Subsequently, the endoplasmic reticulum-associated protein-degradation-related genes Ubc1 or/and Hrd1 were co-expressed with ROL to further increase ROL expression. The activities of the recombinant strains, GS115/5ROL-Ubc1 22#, -Hrd1 15#, and -Hrd1-Ubc1 1#, were 4000 U/mL, 4200 U/mL, and 4750 U/mL, which was 29.9%, 36.4%, and 54.2% higher, respectively, than that observed in GS115/pAOα-5ROL 11#. Using the combined strategy, ROL expression was improved 15.8-fold, with maximum GS115/5ROL-Hrd1-Ubc1 1# activity reaching 33,900 U/mL via a sorbitol/methanol co-feeding strategy in a 3-L fermenter and resulting in a 1.65-, 1.26-, and 1.14-fold enhancement relative to the activities observed in strains GS115/pAOα-5ROL 11#, GS115/5ROL-Ubc1 22#, and GS115/5ROL-Hrd1 15#, respectively. These results indicated that heterologous overexpression of ROL in P. pastoris using this combined strategy is feasible for large-scale industrialization.

Clustered, regularly interspaced short palindromic repeat (CRISPR) diversity and virulence factor distribution in avian Escherichia coli.

In order to investigate the diverse characteristics of clustered, regularly interspaced short palindromic repeat (CRISPR) arrays and the distribution of virulence factor genes in avian Escherichia coli, 80 E. coli isolates obtained from chickens with avian pathogenic E. coli (APEC) or avian fecal commensal E. coli (AFEC) were identified. Using the multiplex polymerase chain reaction (PCR), five genes were subjected to phylogenetic typing and examined for CRISPR arrays to study genetic relatedness among the strains. The strains were further analyzed for CRISPR loci and virulence factor genes to determine a possible association between their CRISPR elements and their potential virulence. The strains were divided into five phylogenetic groups: A, B1, B2, D and E. It was confirmed that two types of CRISPR arrays, CRISPR1 and CRISPR2, which contain up to 246 distinct spacers, were amplified in most of the strains. Further classification of the isolates was achieved by sorting them into nine CRISPR clusters based on their spacer profiles, which indicates a candidate typing method for E. coli. Several significant differences in invasion-associated gene distribution were found between the APEC isolates and the AFEC isolates. Our results identified the distribution of 11 virulence genes and CRISPR diversity in 80 strains. It was demonstrated that, with the exception of iucD and aslA, there was no sharp demarcation in the gene distribution between the pathogenic (APEC) and commensal (AFEC) strains, while the total number of indicated CRISPR spacers may have a positive correlation with the potential pathogenicity of the E. coli isolates.