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African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens—KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L— induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV.

Although, yeast Saccharomyces cerevisiae is expected to be used as a host for lactic acid production, improvement of yeast lactic acid tolerance is required for efficient non-neutralizing fermentation. In this study, we optimized the expression levels of various transcription factors to improve the lactic acid tolerance of yeast by a previously developed cocktail δ-integration strategy. By optimizing the expression levels of various transcription factors, the maximum d-lactic acid production and yield under non-neutralizing conditions were improved by 1.2. and 1.6 times, respectively. Furthermore, overexpression of PDR3, which is known as a transcription factor involved in multi-drug resistance, effectively improved lactic acid tolerance in yeast. In addition, we clarified for the first time that high expression of PDR3 contributes to the improvement of lactic acid tolerance. PDR3 is considered to be an excellent target gene for studies on yeast stress tolerance and further researches are desired in the future.

In biotechnological industry, increased expression cassette stability and copy number serve as important means of maintaining consistently high production levels of heterologous proteins in Saccharomyces cerevisiae. In this study, we combined δ sequences for site-specific integration with TPI1 gene from Schizosaccharomyces pombe (POT1) as a selection marker to realize high-copy integration and stable expression of heterologous proteins in S. cerevisiae. With the newly developed POT1 platform, a 32-copy integration of enhanced green fluorescent protein (EGFP) expression cassette was obtained in a single round and was stably maintained after 100 generations of growth in a rich complex medium. Talaromyces emersonii cellobiohydrolase I gene was synthesized with S. cerevisiae codon bias and expressed under the control of TPI1 promoter and terminator via POT1-mediated δ-integration; the highest specific activity yielded 238 mU g-1 of dry cell weight when p-nitrophenyl-β-D-cellobioside was used as substrate, whereas the highest activity in cellulose hydrolysis reached 67% Avicel conversion. POT1-mediated δ-integration produces high protein levels over a wide dynamic range and enables broad applications in metabolic engineering and synthetic biology.

It is of utmost importance to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production. In this study, two xylose isomerase-based industrial S. cerevisiae strains, O7 and P5, were constructed by δ-integration of the xylose isomerase (XI) gene xylA from the fungus Orpinomyces sp. and from the bacterium Prevotella ruminicola , respectively. The xylose consumption of the strains O7 and P5 at 48-h fermentation was 17.71 and 26.10 g/L, respectively, in synthetic medium with xylose as the sole sugar source. Adaptive evolution further improved the xylose fermentation capacity of the two strains to 51.0 and 28.9% in average, respectively. The transcriptomes of these two strains before and after evolution were analyzed using RNA-Seq. The expression levels of the genes involved in cell integrity, non-optimal sugar utilization, and stress response to environment were significantly up-regulated after evolution and did not depend on the origin of xylA ; the expression levels of the genes involved in transmembrane transport, rRNA processing, cytoplasmic translation, and other processes were down-regulated. The expression of genes involved in central carbon metabolism was fine-tuned after the evolution. The analysis of transcription factors (TFs) indicated that most of the genes with significant differential expression were regulated by the TFs related to cell division, DNA damage response, or non-optimal carbon source utilization. The results of this study could provide valuable references for the construction of efficient xylose-fermenting XI strains.

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining δ-integration and polyploidization through cell fusion. Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase/α-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The α-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.

δ-Integration can improve the expression stability and increase the copy number of exogenous genes in Saccharomyces cerevisiae. Generally, δ-integration vectors employ auxotrophic markers, such as LEU2-d, HIS3, TRP1, and URA3, to screen transformants. In this study, two cellulase genes (β-glucosidase and endoglucanase) were integrated into the S. cerevisiae W303-1A chromosome as reporters, by δ-integration with the aforementioned auxotrophic markers. Eight strains, L-BGL, H-BGL, T-BGL, U-BGL, L-EG, H-EG, T-EG, and U-EG, were selected and tested. After 5 days growth, cellulase activities (U ml⁻¹) were 3, 2.3, 1.8, 1.5, 29, 12, 8, and 1.5, respectively. The results showed that auxotrophic markers influenced the expression of cellulase genes.

Introducing large numbers of target genes into the chromosome of Saccharomyces cerevisiae via δ-sequence-mediated integration is a good strategy for exploring the effects of gene dosage on expression and secretion of heterologous proteins. The expression of exogenous genes might be further improved through meiosis in an isogenic triploid. Here, a stable strain A-8 was screened from 35 sexual spore colonies obtained from an isogenic triploid integratively expressing bgl1 from Aspergillus aculeatus. The corresponding β-glucosidase activity in this strain was increased by ~120 % compared with the parent strain BGL-a. Measurement of doubling time, flow cytometry, and mating experiments further confirmed that A-8 was a spore-forming strain obtained from a triploid parent. Thus, combining δ-integration and meiosis in an isogenic triploid is a promising approach for improving the expression of exogenous proteins in S. cerevisiae.