Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris

Abstract

Background The microbial transglutaminase (MTG) is inactive when only the mature sequence is expressed in Pichia pastoris. Although co-expression of MTG and its N-terminal pro-peptide can obtain the active MTG, the enzyme activity was still low. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number recombinants of P. pastoris are achievable only by cloning of gene concatemers, so methods for rapid and reliable multicopy strains are therefore desirable. Results The coexpression strains harboring different copies mtg were obtained successfully by stepwise increasing Zeocin concentration based on the rDNA sequence of P. pastoris . The genome of coexpression strains with the highest enzyme activity was analyzed by real-time fluorescence quantitative PCR, and three copies of mtg gene ( mtg- 3c) was calculated according to the standard curve of gap and mtg genes ( gap is regarded as the single-copy reference gene). The maximum enzyme activity of mtg- 3c was up to 1.41 U/mL after being inducted for 72 h in 1 L flask under optimal culture conditions, and two protein bands were observed at the expected molecular weights (40 kDa and 5 kDa) by Western blot. Furthermore, among the strains detected, compared with mtg -2c, mtg -6c or mtg -8c, mtg -3c is the highest expression level and enzyme activity, implying that mtg -3c is the most suitable for co-expression pro-peptide and MTG. Conclusions This study provides an effective strategy for improving the expression level of active MTG by directional increasing of mtg copies in P. pastoris. Electronic supplementary material The online version of this article (10.1186/s12896-019-0542-6) contains supplementary material, which is available to authorized users.

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

“…That is a result of the negative effects of a high concentration of target protein on the growth and metabolism of the strain [40][41][42]. Song et al (2019) reported that a co-expressing strain (pro/rDNA-mtg) with three copies of mtg genes (mtg-3c) exhibited higher transglutaminase activity than mtg-2c, mtg-6c, or mtg-8c [25]. Dagar et al (2018) reported that the expression of human interleukin-3 protein increased with the addition of up to 8 copies of the expression cassettes, then drastically decreased thereafter [26].…”

“…Currently recombinant K. phaffii strains with high copy numbers can only be obtained by increasing gene concatemers, so alternative methods for generating multicopy strains rapidly and reliably are highly desirable [25]. Additionally, in order to study the effects of different promoters on protein expression levels, it is necessary to accurately and quantitatively analyze the copy number of foreign genes inserted into the recombinant yeast.…”

“…In the process of strain propagation, reduced copy number can indicate plasmid loss. Real-time quantification PCR (qPCR) has been used to detect the copy numbers of many foreign genes including transglutaminase [25], human interleukin-3 [26], and lipase [27] in recombinant yeasts, but to date it has not been used in fib gene copy number estimation.…”

Magnetic nanoparticle mediated-gene delivery for simpler and more effective transformation of Pichia pastoris

“…This situation might be attributed to occur multi-copy integration of GFP gene into the genomes of yeast cells forming 4 th colony (Fig. 7 E) 42,43 . Comparing with LiCl or electroporation methods (Table 2), the suggested magnetotransformation system achieved an efficient transformation and resulted in sufficient numbers of transformants.…”

Effect of differential expression of pro-region on the transglutaminase productivity in pichia pastoris

“…Multi-copy integration is anticipated to occur by single homologous recombination during integration, resulting in tandem integrated copies of the vector 20 .The copy number of genes for TG expression is an important factor. In pichia pastoris, TG with 3 copy numbers recorded the best activity of 1.41U/ml 21 .Therefore, the co-expression strain was selected on YPD with two concentration of G418 (contains 100 ug/ml, 150ug/ml) to ensure the successful transformant and appropriate copy number selection for TG expression. Strategy for multi-site integration of pro-region via rDNA and selection of an appropriate copy of TG produced 3.9u/ml in the shake flask, indicating the role of pro-region in proper folding of more enzyme extracellularly (Fig 3a and 3b).…”