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

https://doi.org/10.1016/j.pep.2018.02.005Get rights and content

Highlights

  • An efficient method was developed to construct multi-copy genes biobricks

  • A 5-copy-ROL-gene recombinant with the highest activity was screened.

  • ROL co-expressed with two ERAD-related proteins further enhanced expression level.

  • The fermented conditions of the derivative recombinants were totally optimized.

Abstract

Rhizopus oryzae lipase (ROL) is an important industrial enzyme limited in application due to its low production in native strains. Here, we used a new combined strategy to overexpress ROL in Pichia pastoris. An efficient method based on bio-brick was developed to construct a series of vectors harboring different copy numbers of ROL gene cassettes, which were then transformed into P. pastoris GS115 to generate a strain with specific copy numbers of ROL. An optimized gene-dosage recombinant strain of GS115/pAOα-5ROL 11# harboring five copies of ROL was screened, revealing production of the highest activity (2700 U/mL), which was 8-fold higher than that of the strain harboring one copy. The activity of GS115/pAOα-5ROL 11# was then enhanced to 3080 U/mL in a shaking flask under optimized culture conditions. Subsequently, the endoplasmic reticulum-associated protein-degradation-related genes Ubc1 or/and Hrd1 were co-expressed with ROL to further increase ROL expression. The activities of the recombinant strains, GS115/5ROL-Ubc1 22#, -Hrd1 15#, and -Hrd1-Ubc1 1#, were 4000 U/mL, 4200 U/mL, and 4750 U/mL, which was 29.9%, 36.4%, and 54.2% higher, respectively, than that observed in GS115/pAOα-5ROL 11#. Using the combined strategy, ROL expression was improved 15.8-fold, with maximum GS115/5ROL-Hrd1-Ubc1 1# activity reaching 33,900 U/mL via a sorbitol/methanol co-feeding strategy in a 3-L fermenter and resulting in a 1.65-, 1.26-, and 1.14-fold enhancement relative to the activities observed in strains GS115/pAOα-5ROL 11#, GS115/5ROL-Ubc1 22#, and GS115/5ROL-Hrd1 15#, respectively. These results indicated that heterologous overexpression of ROL in P. pastoris using this combined strategy is feasible for large-scale industrialization.

Introduction

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are among the most important industrial enzymes involved in catalytic hydrolysis, trans-esterification, and ester-synthesis reactions at the special interface between the insoluble-substrate phase and the aqueous phase [[1], [2], [3], [4]]. Benefiting from these unique attributes, lipases have received additional attention for use in various industrial fields, such as detergents, food, pharmaceutical, biosensor, and biodiesel production [3,5,6].

Lipase from Rhizopus oryzae hydrolyzes the sn-1 and sn-3 positions of ester bonds, thereby showing strong 1,3-regiospecificity and is widely used in biodiesel production [[7], [8], [9]]. The native structure of R. oryzae lipase (ROL) is synthesized as a pre-pro-protein containing a 26-amino-acid signal sequence, a 97-amino-acid prosequence, and a 269-amino-acid mature lipase region (mROL) [10]. Renaturation experiments in vitro showed that the ROL prosequence (proROL) is critical for lipase activity, and that the signal peptide can inhibit correct folding [10]. Additionally, experiments revealed that the prosequence is important for proper folding of ROL and aids in ROL secretion [9,11].

To meet the demand of industrial applications, proROL, comprising the prosequence and mROL regions, has been expressed in Pichia pastoris [[12], [13], [14], [15]]. To improve ROL production, different strategies have been attempted. ROL gene expression was enhanced by increasing ROL gene-copy number [16] or using a sorbitol/methanol co-feeding method to reduce metabolic burden [17]. A 7-fold improvement in ROL-expression level was achieved in P. pastoris by intracellular constitutive co-expression of the Saccharomyces cerevisiae unfolded-protein response (UPR) transcription factor Hac1 along with knockout of the Gas1 gene [18]. ROL was also highly expressed with co-expression of endoplasmic reticulum (ER) oxidoreductase 1 (Ero1) and protein disulfide isomerase (Pdi). Moreover, ROL activity was further increased to 12,019 U/mL by optimizing NH4+ concentration in a 7-L bioreactor [19]. However, ROL expression still requires significant improvement to meet the huge demand of industries. Therefore, new strategies are urgently needed to increase ROL expression.

The methylotrophic yeast P. pastoris is widely used for recombinant heterologous-protein expression [[20], [21], [22]] due to its advantages, including easy growth to high cell density with low nutritional requirements, ease of molecular genetic manipulation, strict regulation of its alcohol oxidase 1 (AOX1) promoter by methanol, and the ability to perform complex eukaryotic posttranslational modifications [23,24]. Among the common strategies used to increase gene expression in P. pastoris are screening strains containing multiple copies of the target gene [25,26]. Several methods are used to increase gene-copy number, including sequential transformations of the gene using a different selectable marker [27], posttransformational vector amplification [28], and construction of plasmids harboring multi-copy gene cassettes [29]. However, excessive increases in gene-copy number might result in massive accumulation of unassembled proteins in the ER, resulting in cellular stress and activation of the UPR [30], which can relieve ER stress by activating downstream genes, such as Hac1, Bip, and Pdi, to aid protein folding and accelerate secretion [30]. Additionally, the UPR can alleviate cellular stress via ER-associated protein degradation (ERAD) [31].

ERAD can eliminate misfolded proteins from the ER to reduce cellular stress [32] by promoting retro-translocation of unassembled proteins to the cytosol by a quality control system, followed by their degradation by the ubiquitin-proteasome system [31,32]. Some co-factors, such as Hrd1, a polytopic E3 ubiquitin ligase, are required for ERAD for the degradation of incorrectly folded proteins in the ER [33,34]. Additionally, the ubiquitin-conjugating enzyme Ubc1 is responsible for selective degradation of proteins [35] and is also involves in ERAD. These findings show that the UPR and ERAD coordinate to alleviate the ER stress [32].

There are several reported strategies focusing on increasing the protein-folding capacity to realize highly effective secretion involving ROL overexpression, such as co-overexpression of the UPR activator Hac1 [36], enhancing expression of the ER chaperone Bip [37] and/or Pdi [38], increasing expression of Ero1 [39], or knocking out ERAD-related genes to strengthen the folding pathway of the UPR [40]. Although such strategies were widely used to increase gene-expression levels in P. pastoris, the efficiency of disposing of misfolded proteins in ERAD might still represent a bottleneck of efficient protein expression. A previous report showed that co-expression of ERAD-related genes successfully enhances glucose oxidase expression [41]. However, few studies have explored the specific role of ERAD.

Previous work indicated that ROL overexpression under the AOX1 promoter in P. pastoris activates the UPR [42], suggesting the existence of high levels of unfolded ROL protein. As mentioned, increasing the expression of ERAD-related proteins to enhance the efficiency of removing misfolded proteins might contribute to reducing ER stress, resulting in improved expression levels. Therefore, the screening of strain(s) harboring proper copy numbers of target genes and increasing ERAD efficiency were undertaken in this study to enhance ROL expression.

In this study, an efficient method was designed to construct vectors harboring different gene-copy numbers of ROL-expression cassettes to provide an efficient method for optimizing ROL gene dosage in P. pastoris. Additionally, to reduce ER stress, the ERAD-related genes Ubc1 and/or Hrd1 were for the first time co-overexpressed along with the ROL gene to improve ROL expression. Furthermore, high-density fermentation was performed for the derivative recombinant engineering strains in a 3-L bioreactor via sorbitol/methanol co-feeding method to achieve the maximum expression level of ROL.

Section snippets

Strains, plasmids, and media

P. pastoris GS115 (his4), Escherichia coli Top10 cells, and the vectors pPICZA, pPICZαA, pPIC3.5K, and pAO815 were purchased from Invitrogen (Carlsbad, CA, USA). R. oryzae (CCTCC AF 93144) was purchased from the China Center for Type Culture Collection (Hubei, China) and used for ROL gene cloning. The pMD19-T simple vector, PrimeStar HS DNA polymerase, restriction endonucleases, and DNA ligation kit were commercially obtained from TaKaRa (Dalian, China) and used for gene manipulation and

Construction of expression vectors

Several vectors with different ROL gene-copy numbers were constructed to precisely optimize the gene dosage and effectively improve ROL expression. For this purpose, a one-copy ROL-expression plasmid pAOα-ROL used as the original vector was constructed (Fig. 1). Subsequently, the vectors pAOα-nROL (n = 2, 3, 4, …8) harboring different copy numbers of the ROL gene were generated (Fig. 2a). The plasmids were further verified by restriction analysis with BglII and BamHI (Data not shown). It is

Discussion

ROL, an important industry lipase, is widely used for esterification of docosahexaenoic acid and for preparation of biodiesel production [8,9]. Currently, lipase-catalyzed transesterification for biodiesel production is an attractive option. ROL immobilized on the anion-exchange resin Amberlite IRA-93 was used for biodiesel biosynthesis from soybean oil, reaching a 90.5% yield [47], and liquid ROL was employed as an effective catalyst to catalyze biodiesel production from rapeseed oil

Conclusions

A new strategy based on the bio-brick method was developed to construct multi-copy vectors in vitro. Strains containing one to eight copies of ROL were generated to investigate the effects of gene dosage on ROL expression. The results showed ROL copy number greatly influenced protein level. Subsequent optimization of the cultivation conditions of the derivative dominant strains in shake flasks and co-expression of the ERAD-related genes Ubc1 and Hrd1 along with ROL to further enhanced ROL

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (Nos: 31070089, 31170078, and J1103514), the National High Technology Research and Development Program of China (No: 2011AA02A204), the National Natural Science Foundation of Hubei Province (No. 2015CFA085), and the Fundamental Research Funds for HUST (No: 2014NY007). The authors would like to acknowledge the Analytical and Testing Center of HUST for their valuable assistance in protein and real-time qPCR

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      Similarly, the expression of recombinant human interleukin-3 (hIL-3) increased with copy number where a maximum protein concentration of 310 mg/L was achieved in case of an eight copy recombinant Pichia X-33 clone [10]. A recombinant strain of P. pastoris GS115 carrying five copies of the Rhizopus oryzae lipase (ROL) gene led to an eight-fold higher activity of 2700 U/mL compared to the strain harboring only one copy of the gene [11]. Thus, gene dosage seems to be a viable strategy to increase production titers during heterologous gene expression in P. pastoris.

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