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

ABSTRACT In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the production of a Klebsiella pneumoniae carbapenemase (KPC). KPC-producing isolates often exhibit a range of carbapenem MICs. To better understand the factors that contribute to overall carbapenem resistance, we analyzed 27 KPC-producing K. pneumoniae isolates with different levels of carbapenem resistance, 11 with low-level (i.e., meropenem or imipenem MIC ≤ 4 μg/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 μg/ml), and 14 with high-level (i.e., imipenem or meropenem MIC ≥ 16 μg/ml) carbapenem resistance, that were received from throughout the United States. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates either contained an increased bla KPC gene copy number (n = 3) or had deletions directly upstream of the bla KPC gene (n = 7). Four additional isolates lacked elevated KPC production but had high-level carbapenem resistance. Porin sequencing analysis identified 22 isolates potentially lacking a functional OmpK35 and three isolates potentially lacking a functional OmpK36. The highest carbapenem MICs were found in two isolates that lacked both functioning porins and produced elevated amounts of KPC. The 11 isolates with low-level carbapenem resistance contained neither an upstream deletion nor increased bla KPC copy number. These results suggest that both bla KPC copy number and deletions in the upstream genetic environment affect the level of KPC production and may contribute to high-level carbapenem resistance in KPC-producing K. pneumoniae, particularly when coupled with OmpK36 porin loss.

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Erratum to: The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris

Applied Microbiology and Biotechnology ◽

10.1007/s00253-012-4383-7 ◽

2012 ◽

Vol 96 (3) ◽

pp. 851-851

Author(s):

Qi Shen ◽

Min Wu ◽

Hai-Bin Wang ◽

Hua Naranmandura ◽

Shu-Qing Chen

Keyword(s):

Pichia Pastoris ◽

Fusion Proteins ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy

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The effect of gene copy number and co-expression of chaperone on production of albumin fusion proteins in Pichia pastoris

Applied Microbiology and Biotechnology ◽

10.1007/s00253-012-4337-0 ◽

2012 ◽

Vol 96 (3) ◽

pp. 763-772 ◽

Cited By ~ 36

Author(s):

Qi Shen ◽

Ming Wu ◽

Hai-Bin Wang ◽

Hua Naranmandura ◽

Shu-Qing Chen

Keyword(s):

Pichia Pastoris ◽

Fusion Proteins ◽

Copy Number ◽

Gene Copy Number ◽

Gene Copy

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Effect of gene copy number on production yield of recombinant ergopeptine hydrolase ErgA in Pichia pastoris

New Biotechnology ◽

10.1016/j.nbt.2014.05.928 ◽

2014 ◽

Vol 31 ◽

pp. S188

Author(s):

Julia Panhölzl ◽

Julia Lindenberger ◽

Patricia Menczik ◽

Markus Aleschko ◽

Irene Hahn ◽

...

Keyword(s):

Pichia Pastoris ◽

Copy Number ◽

Gene Copy Number ◽

Production Yield ◽

Gene Copy

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

International Journal of Molecular Sciences ◽

10.3390/ijms19113372 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3372 ◽

Cited By ~ 3

Author(s):

Liangcheng Jiao ◽

Qinghua Zhou ◽

Zhixin Su ◽

Yunjun Yan

Keyword(s):

Large Scale ◽

Rhizopus Oryzae ◽

Feeding Strategy ◽

Gene Copy Number ◽

Gene Copy ◽

Scale Production ◽

Rhizopus Oryzae Lipase ◽

Positive Effects ◽

Multiple Copies ◽

Multiple Expression

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.

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