DEVELOPMENT OF SYNTHETIC BIOLOGICAL TOOLS -SYNTHETIC INDUCIBLE PROMOTER

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Comparison of erythritol-inducible promoters on CalB gene expression and protein production

The expression levels and enzymatic activities of CalB were analyzed for the four promoters in the bioreactor (Figure 3.8, the detailed condition of fermentation and analysis are described in Appendix E – Park et al. Microbial Cell Factories, 2019). A good correlation between promoter strength and lipase activity were observed. A similar expression was obtained with pEYD1 and pTEF, while 2.5- and 2.7-fold higher expression of CalB than pTEF was shown with pEYK1-3AB and pHU8EYK (Figure 3.8 (A)). The specific lipase activities of CalB with pEYK1-3AB and pHU8EYK were 2.8- and 2.5-fold higher than that obtained with pTEF, respectively (Figure 3.8 (B)). The enzymatic productivities obtained with pEYK1-3AB and pHU8EYK were 1.7- and 1.6-fold higher than the lipase productivity obtained from the previous study with LIP2 promoter [Fickers et al. 2005].
Figure 3.8. Expression levels (A) and enzymatic activities (B) of CalB gene under the control of promoters pTEF (strain JMY7536), pEYKA3B (strain JMY7539), pHu8EYK (strain JMY7544), and pEYD1 (strain JMY7548).
Cells were grown at 28 °C in YNBGE medium, in 2Mag mini bioreactors. Gene expression levels were analyzed from the cells grown for 24h, and the expression levels were normalized to that the one of actin. The specific lipase activities were analyzed after 24 h and 48 h of cultivation.

CalB production in bioreactor

From the activity analysis with different promoters, pEYK1-3AB was selected to test process conditions. The strain JMY7989, a prototroph derivative of strain JMY7539 was grown for 48 h in YNBG2E medium in DASGIP bioreactor, with pH and pO2 regulation (more details of fermentation condition are described in Appendix E – Park et al. Microbial Cell Factories, 2019). The exponential growth phase lasted for 12 h with a specific growth rate of 0.29 ± 0.00 h−1 and final biomass of 6.96 ± 0.04 g L−1 (Figure 3.9 (A), Table 3.5). Within the first 24 h, the main carbon source (glycerol) had been entirely consumed, and the inducer (erythritol) assimilated by the cells. Lipase activity reached its highest titer (28,024 ± 743 U mL−1) after 24 h of culture (Figure 3.9 (A)). Then, it decreased slightly after until the end of the culture (20,150 ± 1,060 U mL−1). Analysis of culture supernatant by SDS-PAGE clearly highlighted that CalB is the only secreted protein in those conditions (Figure 3.9 (B)). During the enzyme production phase (between 3.5 and 24 h of culture), the lipase volumetric productivity was of 1,357 ± 34 U mL−1 h−1 (Table 3.5). Culture of strain JMY7990 (pTEF-CalB prototroph) in the same experimental conditions yielded to a 6.2-fold lower lipase activity after 24 h (data not shown).
Additional copy of CalB (pEYK1-3AB-CalB) was transformed to JMY7539, resulting JMY7991 strain in order to see if the multi-copy expression is increasing the production of lipase. Cell growth kinetics of strain JMY7991 (multi-copy) was found similar to that of RIY368 (mono-copy) (Figure 3.9 (A)). It seems that the additional expression of heterologous gene does not alter host strain metabolism. As shown in Figure 3.9 and Table 3.5, lipase activity of JMY7991 reached its highest level (45,125 ± 2,144 U mL−1) after 24 h of culture, again after entire consumption of glycerol and assimilation of erythritol. At the maximal value, JMY7991 lipase activity was 1.6-fold higher than the one of JMY7989, which is consistent with the ratios of volumetric and specific production rates.
Figure 3.9. Dynamics of culture of strains JMY7989 (mono-copy) and JMY7991 (multi-copy) in DASGIP bioreactors.
Cells were grown for 48 h at 28 °C in YNBG2E medium. (A) Growth curve and lipase activity of strains JMY7989 and JMY7991. Values are means and standard deviations of duplicate experiments. (B) SDS-PAGE gel of 5 μL of supernatant (containing 40 U of lipase CalB, sample taken at 24 h). Protein sizes are indicated on the left-hand side.
The project of CalB production was a collaborative work with the group of professor Patrick Fickers at the University of Liège – Gembloux Agro – BioTech in Belgium. I have participated in the construction of the plasmids and Y. lipolytica strains, and the screening of CalB-expressing strains described in the part 3.3.1. The analysis of gene expression and CalB production during fermentation described in the part 3.3.2. and 3.3.3. were performed by the collaborating group.

Discussion

Traditionally, promoters and their regulatory elements are studied through deletion or point-mutation and assessment of gene expression, as exemplified by the research in which the regulatory motifs of XPR2, TEF1, and POX2 promoters in Y. lipolytica were determined [Madzak et al. 2000; Blazeck et al. 2011; Blazeck et al. 2013; Hussain et al. 2015]. As the number of available genomes increases and the costs of sequencing decrease, researchers can more frequently employ strategies such as phylogenetic footprinting, which is a powerful tool for identifying CRMs with regulatory functions of interest. In this study, we employed phylogenetic footprinting within the Yarrowia clade to explore the cis-regulatory modules of the EYD1 and EYK1 genes involved in the catabolism of erythritol.
From the mutation of each CRM in EYD1 promoter, we discovered that both CRM-eyd1 and CRM-eyd2 are important for effective expression and induction, regardless of genetic background. Between the conserved motifs A and B of the EYD1 promoter, motif A seemed to be more involved in erythritol-based induction. Trassaert and colleagues obtained similar results after introducing a mutation into the conserved motifs A (pEYK300aB) and B (pEYK300Ab) of the inducible EYK1 promoter [Trassaert et al. 2017]. When grown in minimal YNB medium containing 1% erythritol, the strain carrying the pEYK300A*B-YFP cassette with the mutated motif A displayed a decreased level of YFP expression compared to that of the unmutated pEYK300 (683 and 3,536 SFU after 60 h, respectively). In contrast, when motif B was mutated, induction levels were higher under the same conditions (8,389 and 3,536 SFU after 60 h, respectively).
Expression levels have been found to be dependent on UAS copy numbers, which have ranged from 4 tandem copies of UAS1B-xpr2 to as many as 32 copies of UAS1B-xpr2 [Madzak et al. 2000; Blazeck et al. 2011; Blazeck et al. 2012]. However, this relationship was not observed for the EYK1 and EYD1 hybrid promoters examined in this study. It was found that an increased number of UAS1-eyk1 copies increased promoter strength when the EYK1 wild-type strain (JMY1212) was grown on glucose or erythritol (Figure 3.4 and Table 3.2) and that four tandem repeats seemed optimal. Similar results were obtained in the eyk1Δ mutant strain (JMY7126), but the optimal expression was reached with 3 tandem repeats. This result reflects that the stronger expression by erythritol induction may lead to a saturation of expression.
For the hybrid promoter in which the core promoter was exchanged (i.e., EYK1-4AB-coreTEF vs. EYK1-4AB), expression levels were higher, while induction levels were lower. Indeed, when the coreTEF hybrid promoter was used, expression increased 10- and 2-fold, respectively, in the EYK1 WT (JMY1212) and eyk1Δ mutant (JMY7126) grown on glucose. When erythritol was used as an inducer, hybrid promoter strength increased less than when glucose medium was used (2-fold in the EYK1 WT (JMY1212), 5-fold in the eyk1Δ mutant (JMY7126)). It seems that while the core TEF is able to act in a similar way as the core elements of the erythritol-inducible promoter, the strength of its inducible response is less than that of the native EYK1 promoter. The hybrid promoter could be further improved by exchanging the core promoters or by employing a combination of TATA boxes from other inducible promoters [Redden and Alper, 2015; Shabbir Hussain et al. 2016].
Promoter strength was improved in the eyk1Δ mutant (JMY7126) utilizing erythritol as a free inducer, not as a carbon source. With the presence of erythritol in the medium, a 45.8-fold increase in EYK1-2AB promoter strength was observed in the eyk1Δ mutant (JMY7126) while a 5.9-fold increase under same promoter in the EYK1 WT (JMY1212). Under the same condition, the EYD1 promoter showed a 682.5-fold and a 13.4-fold increase of expression in the eyk1Δ mutant (JMY7126) and the EYK1 WT (JMY1212), respectively. The EYD1 promoter is a very tight promoter with very low expression levels without an inducer, and a significant increase of expression level (680-fold) with an inducer in the eyk1Δ strain.
As a proof of concept, lipase CalB was expressed in mono-copy in strain JMY7126 under the control of three types of erythritol inducible promoters. With hybrid inducible promoters, pEYK1-3AB and pHU8EYK, the expression of CalB was increased by 2.5- and 2.7-fold compared to the constitutive expression with pTEF. However, the similar expression of CalB with pEYK1-3AB and pHU8EYK showed an inconsistent result compared to the expression of the fluorescent reporter protein, which showed a significantly higher fluorescence level with pHU8EYK than pEYK1-3AB [Trassaert et al. 2017; unpublished data]. A similar disparity between the promoter strength and heterologous protein production was also observed from a previous study on the expression of xylanase C [Dulermo et al. 2017]. It is possible that excessive protein production due to the stronger promoter could lead to the processing burden in protein folding and secretion machinery, resulting in a saturated production level of heterologous protein [Dulermo et al. 2017; Ahmad et al. 2014]. This underlines the necessity of the separation the growth phase and the production phase to relive the processing burden in heterologous protein production, which can be controlled by the inducible promoter.
In brief, the identification of CRMs, the design of a broad range of hybrid promoters, and application of hybrid promoters for producing the protein of interest were demonstrated in this chapter. These new promoters that respond to erythritol could be very useful in not only fundamental research to understand the architecture of promoter but also the recombinant protein production, as is the case of the Gal1 promoter in S. cerevisiae. Process development shall expand the potentialities of the proposed expression system even further, and the combination may greatly improve the production of recombinant proteins in Y. lipolytica.

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Improve propionate tolerance

Introduction

Propionate, a key substrate for OCFA production, can be used by Y. lipolytica as a sole carbon source in lipid accumulation [Fontanille et al. 2012; Kolouchová et al. 2015; Gao et al. 2017]. However, it is also known that propionate has an inhibitory effect on the cell growth at concentrations above 5 g/L in several previous studies. For this reason, it is necessary to improve microbial resistance to propionate when used as a substrate for lipid accumulation. Very few studies have looked at the tolerance and utilization of propionate by oleaginous yeast for lipid production. Consequently, it is required to identify propionate tolerant genes and have a better understanding of the molecular and regulatory responses of yeast to propionate for utilizing propionate as a substrate for biomass and OCFA production.

Table of contents :

CHAPTER 1. INTRODUCTION
1.1. CONTEXT OF THE STUDY
1.2. BACKGROUND
1.2.1. Yarrowia lipolytica
1.2.2. Metabolic engineering tools for Y. lipolytica
1.2.3. The biotechnological applications of Y. lipolytica
1.2.4. Lipid production in Y. lipolytica
1.2.5. Odd-chain fatty acids (OCFAs)
1.3. OBJECTIVES
CHAPTER 2. MATERIALS AND METHODS
2.1. STRAINS, MEDIA, AND GROWTH CONDITIONS
2.2. CLONING
2.2.1. General molecular biology
2.2.2. Gene synthesis
2.2.3. Plasmid construction
2.3. CONSTRUCTION OF Y. lipolytica STRAIN
2.3.1. Transformation
2.3.2. Verification of construction in Y. lipolytica
2.3.3. Re-use of marker
2.4. ANALYSIS
2.4.1. Growth
2.4.2. Fluorescence
2.4.3. Metabolites
2.4.4. Lipids
2.4.5. Morphology
2.4.6. Gene and protein sequence
CHAPTER 3. DEVELOPMENT OF SYNTHETIC BIOLOGICAL TOOLS -SYNTHETIC INDUCIBLE PROMOTER
3.1. INTRODUCTION
3.2. ENGINEERING OF ERYTHRITOL-INDUCIBLE PROMOTERS
3.3. APPLICATION OF THE SYNTHETIC INDUCIBLE PROMOTER FOR RECOMBINANT PROTEIN PRODUCTION; THE EXPRESSION OF CALB
3.4. DISCUSSION
CHAPTER 4. PRODUCTION OF ODD-CHAIN FATTY ACIDS (OCFAS)
4.1. IMPROVE PROPIONATE TOLERANCE
4.1.1. Introduction
4.1.2. Results
4.1.3. Discussion
4.2. INCREASE THE PRODUCTION OF OCFAS
4.2.1. Introduction
4.2.2. Results and discussion
CHAPTER 5. ENGINEERING PRECURSOR POOLS FOR INCREASING LIPID PRODUCTION
5.1. INTRODUCTION
5.2. INCREASING MALONYL-COA POOL
5.3. INCREASING ACETYL-COA POOL
5.4. INCREASING PROPIONYL-COA POOL FOR THE OCFA PRODUCTION
5.5. DISCUSSION
CHAPTER 6. DE NOVO PRODUCTION OF OCFAS FROM GLUCOSE 
6.1. INTRODUCTION
6.2. RESULTS
6.3. DISCUSSION
CHAPTER 7. CONCLUSION AND PERSPECTIVES …

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