Accepted Article

Received Date : 29-Jul-2014 Revised Date : 19-Aug-2014 Accepted Date : 21-Aug-2014 Article type

: Short Communication

Editor

: Jens Nielsen

Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function

Leqian Liu, Peter Otoupal, Anny Pan, Hal S. Alper1,2,*

1

McKetta Department of Chemical Engineering The University of Texas at Austin 200 E Dean Keeton St. Stop C0400 Austin, TX 78712 2

Institute for Cellular and Molecular Biology The University of Texas at Austin 2500 Speedway Avenue Austin, Texas 78712

*Corresponding author: Phone: (512) 471-4417, Fax: (512) 471-7060, E-mail:

[email protected], Mailing Address: 200 E Dean Keeton St., C0400, Austin, TX 78712

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1567-1364.12201 This article is protected by copyright. All rights reserved.

Accepted Article

Running title: PLASMID EXPRESSSION ENGINEERING IN YARROWIA LIPOLYTICA

Abstract Manipulating gene expression using different types of plasmids (centromeric, 2-micron, and autonomously replicating sequence) can expand expression levels and enables a quick characterization of combinations, both of which are highly desired for metabolic engineering applications. However, for the powerful industrial workhorse Yarrowia lipolytica, only a lowcopy CEN plasmid is available. In this study, we engineered the CEN plasmid from Y. lipolytica by fusing different promoters upstream of the centromeric region to regulate its function and expand its range. By doing this, we successfully improved the copy number and gene expression at plasmid level by 80% and enabled a dynamic range of nearly 2.7 fold. This improvement was seen to be independent from both the promoter and gene used in the expression cassette. These results present a better starting point for more potent plasmids in Y. lipolytica.

Key Words: Plasmid, Yarrowia lipolytica, Copy number, Expression level, Metabolic engineering

Yarrowia lipolytica, an unconventional oleaginous yeast, has emerged as a powerful industrial workhouse due to its innate lipogenesis capacity, an increasingly developed genetic toolbox, and highly accurate genome information (Dujon et al., 2004, Bankar et al., 2009, Curran & Alper, 2012, Liu et al., 2013, Liu & Alper, 2014). The attractiveness of this organism is highlighted by

recent efforts in harnessing this strain for value-added chemical production including lipids,

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organic acids (Papanikolaou et al., 2002, Yu et al., 2012, Blazeck et al., 2013, Tai & Stephanopoulos, 2013, Xue et al., 2013, Blazeck et al., 2014) as well as protein production such

as lipase and glycoproteins (Fickers et al., 2011, De Pourcq et al., 2012). The ability to tune gene expression is essential for the success of metabolic engineering efforts. To this end, previous work including endogenous promoter characterization (Juretzek et al., 2000), hybrid promoter engineering (Blazeck et al., 2011, Blazeck et al., 2013), and multi-copy integration (Le Dall et al., 1994) have increased the capacity to engineer this yeast. However, this tuning capacity has not been matched at the plasmid level, a convenience that would enable quick characterization of genetic constructs through a simple transformation and also a tool that can enable fundamental studies of replication and segregation (Ghosh et al., 2006).

In the widely used baker’s yeast Saccharomyces cerevisiae, plasmid-based gene

expression control is enabled by several varieties of plasmids including centromeric (CEN)

plasmids (low copy), 2-micron plasmids (high copy), and autonomously replicating sequence (ARS) plasmids (high copy) (Romanos et al., 1992, Chen et al., 2012, Karim et al., 2013). However, for the case of Y. lipolytica, a suitable 2-micron has not been identified and the CEN and ARS elements cannot be separated (Fournier et al., 1993, Vernis et al., 2001, Yamane et al.,

2008) leaving only low copy CEN-based plasmids. In S. cerevisiae, it has been reported that a CEN plasmid can be converted into an ARS-like plasmid by fusing a promoter upstream of the centromeric region leading to increase in copy number and gene expression (ChlebowiczŚledziewska & Śledziewski, 1985). Here, we sought to invoke a similar strategy for a Y. lipolytica CEN plasmid to both increase copy number and expression level (Figure 1A) (materials and methods in the Supporting Information).

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To accomplish this task, we first selected various endogenous promoters and synthetic (including truncated) variants (P-GPD, P-EXP, P-POT1, P-MET2, P-TEF and truncated P-TEF: P-TEF(272) and P-TEF(136) with 272 bp and 136 bp upstream of the ATG starting site (Juretzek et al., 2000, Blazeck et al., 2011, Blazeck et al., 2013) as well as two cell-cycle dependent promoters (P-CDC2 and P-YOX1) to regulate the centromere function in Y. lipolytica

(all promoters were amplified with 50bp from the coding region). Previous studies in S. cerevisiae suggested CDC2 and YOX1 exhibit cell-cycle dependent expression pattern and

express G1-phase and mid G1 to early S phase respectively (Cho et al., 1998, Spellman et al., 1998, Wittenberg & Reed, 2005). Whereas the promoter fused to the CEN region was altered in each plasmid, each construct contained a strong hybrid promoter (P-UAS1B16-Leum) upstream of hrGFP, a green fluorescence protein. Through the use of flow cytometry, we found that altering the promoter upstream of the centromere region in Y. lipolytica leads to similar phenotype found in S. cerevisiae. As a result, it was possible to alter the expression level via regulated centromere function (Figure 1B). For the case of P-TEF(272), P-CDC2, P-POT1 and P-EXP based engineered plasmid, fluorescence was increased by at least 50% compared to the control plasmid construct and up to 80% for the case of P-TEF(272). Interestingly, the P-MET2 promoter upstream of the CEN region resulted in a decreased expression by 30%. Collectively, a nearly 2.7 fold dynamic range was achievable through this strategy. Next, we replaced the hrGFP gene with a β-galactosidase reporter gene for two of the

plasmids (containing the promoters P-POT1 and P-CDC2 upstream of CEN) to test the generalizability of this approach. Both plasmids showed a similar improvement of roughly 50% in the β-galactosidase activity as seen with the hrGFP (Figure 1C). In addition, the increase in expression is preserved when a lower strength native promoter (P-EXP) is used to drive the

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expression of β-galactosidase, thus indicating that the approach is independent of reporter protein and promoter driving the expression cassette (Figure 1D). To confirm that the increased protein expression level was the result of increases in

plasmid copy number, we employed a relative copy number assay based on RT-PCR using a total genomic DNA extract (Moriya et al., 2006, Karim et al., 2013). The copy number in the

augmented plasmids was found to be increased by roughly 80% compared to the initial plasmid construct (Figure 1C). Thus, these engineering efforts successfully increased the copy number as

well as the expression level from Y. lipolytica plasmids.

One commonly observed feature of these Y. lipolytica plasmids is a bimodal distribution

of fluorescence signal (Blazeck et al., 2011). The improvement in expression seen in this work was the cumulative result of both an increase in the signal as well as a mild increase of the size of the positive subpopulation (Figure 1E). In an effort to further understand this bimodal phenotype, we separated the positive and negative subpopulations through cell sorting and cultivated the sorted populations. Whereas the positive population reverted back to the bimodal distribution, the negative population was unable to be grown in selective growth conditions. This data suggests a biased plasmid segregation occurs in Y. lipolytica CEN-based plasmids which can be attributed to the nature of the plasmid as well as the size (about 9.5kb). In summary, we reported the improvement in expression level and copy number at the

plasmid level in Y. lipolytica through altering its centromere function. These efforts resulted in over 80% improvement of plasmid copy number and expression and also enabled a dynamic range of nearly 2.7 fold. This improvement was seen to be independent of both the promoter and gene in the expression cassette. Although the improvement is obtained here is lower in

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magnitude compared with common expression tuning strategies such as hybrid promoter engineering in this yeast (8-fold improvement over typically used endogenous promoters (Blazeck et al., 2011)), the method presented here can applied in conjunction with promoter engineering strategies, thus leading to a synergistic increase in net expression level. As a result, we demonstrate an improved set of plasmids for use in Y. lipolytica.

Acknowledgements This work was funded by the Office of Naval Research Young Investigator Program and the Welch Foundation under grant F-1753. We would like to thank Masayoshi Matsuoka for the gift of plasmid pSl16-Cen1-1(227).

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DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission. Journal of Bioscience and Bioengineering 105: 571-578. Yu Z, Du G, Zhou J & Chen J (2012) Enhanced α-ketoglutaric acid production in Yarrowia

lipolytica WSH-Z06 by an improved integrated fed-batch strategy. Bioresource Technology 114: 597-602. Figure legends Figure 1 Improving expression level and copy number of plasmid in Yarrowia lipolytica with regulated centromere function. (A) A schematic of the engineered plasmids with regulated centromere function. (B) Promoters, including P-MET2, P-GPD, P-TEF, P-TEF(136), P-YOX1, P-EXP, PPOT1, P-CDC2, P-TEF(272), were used to regulate centromere function. Mean fluorescence

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data collected by flow cytometry analysis was used to compare expression at a time point of 48 h in minimal medium. (C) Relative plasmid copy number and β-galactosidase activity with PPOT1 and P-CDC2 based engineered plasmid and control plasmid with P-UAS1B16-Leum driving expression of β-galactosidase. The data was collected by RT-PCR and Gal-Screen™ βGalactosidase Reporter Gene Assay System for Yeast or Mammalian Cells (life technologies) at a time point of 48 h in minimal medium. (D) Relative β-galactosidase activity with P-POT1 and P-CDC2 based engineered plasmids and control plasmid with promoter P-EXP driving expression of β-galactosidase. The data was collected by Gal-Screen™ β-Galactosidase Reporter Gene Assay System for Yeast or Mammalian Cells (life technologies) at a time point of 48 h in minimal medium. (E) Histogram of fluorescence signal showing the bimodal distribution with PPOT1 (blue), P-EXP (red) based engineered plasmids with the control plasmid (yellow). The data was collected by flow cytometry analysis to compare P-POT1, P-EXP based engineered plasmids with the control plasmid at a time point of 48h in minimal medium: P-POT1 based plasmid contains 46.1%, P-EXP based plasmid contains 46.2% and the control plasmid contains 36.6% positive subpopulations. The graph was made using FlowJo software (Tree Star Inc., Ashland, OR). In all graphs, error bars represent standard deviations from biological replicates.

This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function.

Manipulating gene expression using different types of plasmids (centromeric, 2-micron, and autonomously replicating sequence) can expand expression le...
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