Alginate lyase is a promising biocatalyst because of its application in saccharification of alginate for the production of biochemicals and renewable biofuels. This study described the isolation of a new alginate metabolizing bacterium, Flavobacterium sp. S20, from sludge samples and the characterization of its alginate lyase Alg2A. The alginate lyase gene, alg2A, was obtained by constructing and screening the genomic library of the strain S20 and overexpressed in Escherichia coli. Substrate specificity assays indicated Alg2A preferred poly-α-l-guluronate as a substrate over poly-β-d-mannuronate. In the saccharification process of a high content (10 %, w/v) of sodium alginate, the recombinant alginate lyase Alg2A yielded 152 of mM the reducing sugars after 69 h of reaction, and the amounts of oligosaccharides with a different degree of polymerization (DP) generated by Alg2A gradually accumulated without significant variation in the distribution of oligosaccharide compositions. These results indicated that Alg2A possessed high enzymatic capability for saccharifying the alginate, which could be used in saccharifying the alginate biomass prior to the main fermentation process for biofuels. In addition, Alg2A had a different endolytic reaction mode from both the two commercial alginate lyases and other alginate lyases from polysaccharide lyase family 7 owing to high yields of penta-, hex-, and hepta-saccharides in the hydrolysis products of Alg2A. Thus, Alg2A could be a good tool for the large-scale preparation of alginate oligosaccharides with high DP.

Taking into account its thermotolerance, high growth rate, and broad substrate spectrum, Kluyveromyces marxianus can be considered an ideal consolidated bioprocessing (CBP). A major obstacle to ethanol production using K. marxianus is the low production of lignocellulolytic enzymes, which reduces the cellulose hydrolysis and ethanol production. Thus, further improvement of enzyme expression and secretion is essential. To improve the expression of lignocellulolytic enzymes, the inulinase promoter and signal sequence from K. marxianus was optimized through mutagenesis. A T(-361)A mutation inside the promoter, a deletion of AT-rich region inside 5'UTR (UTR∆A), and a P10L substitution in the signal sequence increased the secretory expression of the feruloyl esterase Est1E by up to sixfold. T(-361)A and UTR∆A increased the mRNA expression, while the P10L substitution extended the hydrophobic core of signal sequence and promoted secretion of mature protein. P10L and T(-361)A mutations increased secretory expressions of other types of lignocellulolytic enzymes by up to threefold, including endo-1,4-β-glucanase RuCelA, endo-1,4-β-endoxylanase Xyn-CDBFV, and endo-1,4-β-mannanase MAN330. During the fed-batch fermentation of strains carrying optimized modules, the peak activities of RuCelA, Xyn-CDBFV, MAN330, and Est1E reached 24 U/mL, 25,600 U/mL, 10,200 U/mL, and 1220 U/mL, respectively. Importantly, higher yield of enzymes by optimized promoter and signal sequence were achieved in all tested carbon sources, including the major end products of lignocellulose saccharification and fermentation, with growth on xylose resulting in the highest production. The engineered promoter and signal sequence presented increased secretory expressions of different lignocellulolytic enzymes in K. marxianus by means of various carbon resources. Activities of lignocellulolytic enzymes in fed-batch fermentation were the highest activities reported for K. marxianus so far. Our engineered modules are valuable in producing lignocellulolytic enzymes by K. marxianus and in constructing efficient CBP strains for cellulosic ethanol production.

Background: Porcine Parvovirus (PPV) is a Parvovirinae virus that can cause embryonic and fetal loss and death and mummification in affected fetal pigs. Unlike conventional vaccines, virus-like particles (VLPs) inherit the natural structure of their authentic virions and highly immunostimulatory that can induce strong humoral immune and T cell responses with no risk of pathogenicity. The production of PPV VLPs is still a challenge based on traditional expression platforms due to their low yields and high culture costs. Kluyveromyces marxianus is a safe and fast-growing eukaryote that can get high biomass with low-cost cultures. In this study, we investigated the expression and downstream processes of PPV VLPs in K. marxianus, and the potential for effective stand-alone vaccines. Results: After optimization according to the codon bias of K. marxianus, the VP2 protein from Kresse strain was highly expressed. In a 5 L fermentator, the yield of PPV VLPs reached 2.5 g/L, quantified by HPLC, using a defined mineral medium after 48 h fermentation. Two strategies were established to purify intracellular PPV VLPs: (i) Using the cation exchange chromatography coupled with Sephacryl® S-500 HR chromatography to purify VLPs from the supernatants of pH adjusted cell lysates. (ii) Using anion exchange chromatography followed by cross-flow diafiltration to recover the VLPs precipitated in pH adjusted cell lysates. The purity of PPV VLPs reached about 95%, and total recovery was more than 60%. Vaccination of mice with the purified PPV VLPs induced high titers of specific IgG antibodies in sera, and showed hemagglutination inhibitions on both swine and guinea pig erythrocytes. Spleen lymphocyte proliferation and cytokines detection suggested the PPV VLPs produced by K. marxianus provoked the cellular immune and humoral immunity responses in mice. Conclusions: This is the highest production of recombinant PPV VLPs achieved to date. The superiorities, Generally Recognized As Safe (GRAS), high production, short lead time, and low cost, make K. marxianus a greatly competitive platform for bioproduction of PPV VLPs vaccine.