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by Genetically Engineered Filamentous Fungs
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
T. Vichitsoonthonkul, Y. W. Chu, H. S. Sodhi, G. Saunders
Numerous plasmids for fungal transformation have been constructed [37]. The basic components of these plasmids are a gene that can be used for selection of fungal transformants, and a bacterial plasmid sequence that can be used for selection and propagation of the plasmid in E. coli. Many different selective markers have been used in different fungal species (Table 1). Selective markers can be divided into at least three functional groups: genes that complement preexisting mutations and lead to prototrophic growth (auxotrophic markers; see Table la); genes that provide a new function and lead to a recognizable phenotype, for example, drug resistance or growth on a previously nonutilizable nutrient source (drug resistance or added-functional markers; see Table lb); and DNA fragments that give rise to selectable mutations when integrated into the genome of the recipient strain at specific locations (mutagenic markers [58]). Each type of marker has specific uses and, occasionally, combinations of markers can be used to good advantage. A vector to be used as an expression cassette should possess a strong constitutive or regulatable fungal promoter, an efficient translation start, a signal peptide-coding sequence when secretion is the goal, cloning sites for the heterologous gene, and sequences for transcriptional termination and polyadenylation. Two systems, one based on the A. nidulans alcohol dehydrogenase I promoter (aleA) and its positive regulator (alcR) developed at Allelix Inc. [59] and the A. niger var. awamori system, using the homologous glucoamylase promoter, have been developed at Genencor, Inc. [60].
Engineering Trichoderma reesei for the hyperproduction of cellulose induced protein 1 (Cip1) on a sophorose-containing inducer to efficiently saccharify alkali-pretreated corn stover
Published in Preparative Biochemistry & Biotechnology, 2023
Jianghong Li, Yudian Chen, Yushan Gao, Yi Mo, Tingting Long, Bo Yao, Yonghao Li
The T. reesei gene expression vector pPTPDC1 containing both the PDC1 promoter and terminator was constructed in our laboratory, and BsiWI restriction endonuclease sites were added between the promoter and the terminator. pPTPDC1 had previously been used to successfully express the exogenous aabgl1 gene in T. reesei, increasing the β-glucosidase activity of T. reesei by 70-fold.[20] Therefore, after pPTPDC1 was linearized by BsiWI, the Trcip1 gene was ligated between the PDC1 promoter and terminator using the seamless cloning technique to successfully construct a vector overexpressing the Trcip1 gene (pPTPDC1-cip1) (Figure 1A). The T-DNA sequence containing the Trcip1 gene expression cassette and hygromycin B selection marker was integrated into T. reesei Rut C30, and the genomes of the two transformants were extracted. Then, with BsiWI-F and cip1-R as primers, the integration of the Trcip1 gene expression cassette into the genome of T. reesei was demonstrated using PCR (Figure 1B). As verified by DNA sequencing, the expression cassette had no base mutations and could therefore be used for subsequent studies (Figure 1C). The two positive transformants were named T. reesei OE-cip1-1 and OE-cip1-2.
Production of codon-optimized Human papillomavirus type 52 L1 virus-like particles in Pichia pastoris BG10 expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Kartika Sari Dewi, Sheila Chairunnisa, Sri Swasthikawati, Dian Fitria Agustiyanti, Apon Zainal Mustopa, Wien Kusharyoto, Ratih Asmana Ningrum
For Pichia transformation, pD902_HPV52L1 was linearized using SacI restriction enzyme in AOX1 promoter region to facilitate plasmid integration to P. pastoris genome. Figure 5a showed the difference between linear and circular pD902_HPV52L1 in agarose gel electrophoresis. The transformant colonies grown on a YPDS plate medium containing 100 µg/mL zeocin were then selected and re-grow on a YPD plate supplemented with zeocin with various concentration levels. During the growth phase, the copy number of the Sh ble gene will increase to adapt to the higher concentration of zeocin. The study conducted by Sunga et al.[31] revealed that the cell would amplify the entire expression cassette using this method, including the gene of interest. Therefore, the colonies that grew on the highest concentration were suspected of having a multicopy of the l1 gene.
Optimizing secretory expression of recombinant human interferon gamma from Kluyveromyces lactis
Published in Preparative Biochemistry & Biotechnology, 2018
Rajat Pandey, Venkata Dasu Veeranki
While transforming K. lactis with heterologous gene, it is possible for up to 10 copies of the expression cassette to tandemly insert into its genome, and in such cases K. lactis strains having multiple integrations may secrete higher amount of target protein. K. lactis genome contains a fungal acetamidase gene (amdS) which helps in selection of transformants containing integrated expression cassette. K. lactis transformants were grown on a nitrogen-free minimal medium supplemented with 5 mM acetamide. Only those K. lactis transformants which had an integrated IFNG expression cassette (KLIFNG) in turn expressed amdS can break down acetamide to produce nitrogen required for their growth. An advantage of screening of K. lactis transformants with acetamide is that it enriches those cells which have multiple expression cassettes integrated in their genome.[24] Interestingly, the trasnsformants in the first PCR experiment found to contain pVVDRP01 expression cassette were also found positive in the second PCR experiment as they contain multiple copies of pVVDRP01 expression cassette. Only exception was transformant 4 which was positive in first but failed in second PCR experiment. This may be due to PCR error or absence of multiple copies of pVVDRP01 expression cassette. Transformant 5 also showed 2.3 kb band confirming the presence of multiple copies of pVVDRP01 expression cassette but rejected for further analysis due to multiple bands present in the PCR profile which was consistent with other negative transformants.