Engineering Success

Overview

We constructed xylose reductase-expressing strains with different enzyme sources to catalyze xylose for xylitol production in E. coli BL21. The strategy of promoter engineering was utilized to improve the efficiency of conversion. We overexpressed the erythritol synthesis pathway enzyme in Yarrowia lipolytica. Based on a high-throughput screening strategy, we constructed fluorescent reporter system for characterizing erythritol production and improving erythritol production in Y. lipolytica.

Cycle 1: Construction of xylose reductase expression plasmid

Design

Through literature reading, we selected xylose reductase from Kluyveromyces sp. IIPE453 (KsXR, BBa_K4941010, GenBank: KJ563917.1), xylose reductase from Pichia stipites (PsXR, BBa_K4941009, GenBank: X59465.1), and xylose reductase from Debaryomyces nepalensis NCYC 3413 (DnXR, BBa_K4941008, GenBank: KT239024.1).

Build

In order to verify the catalytic performance of three different sources of xylose reductase in E. coli BL21, we need to construct three xylose reductase expression plasmids, including pET28a-KsXR-kana (BBa_K4941034), pET28a-PsXR-kana (BBa_K4941033), pET28a-DnXR-kana (BBa_K4941035). Here we take the process of constructing pET28a-PsXR as an example: i) Firstly, the DNA fragment of PsXR (BBa_K4941009) was amplified by PCR with primers PsXR_F and PsXR_R; ii) Next, the DNA fragment of the vector plasmid pET28a (BBa_K4941040) was amplified by PCR to obtain linearized pET28a; iii) Then, the linearized pET28a and PCR-amplified PsXR fragment were assembled by Gibson to obtain pET28a-PsXR; iv) The Gibson-assembled reaction mixture was transformed into Escherichia coli DH5α; and v) Positive transformants were sequenced by Sangon Biotech (Shanghai, China). In order to be able to screen transformants using the resistance screening markers, we replaced the kan resistance marker in the pET28a-PsXR plasmid using Gibson assembly (replaced with the Cm resistance marker).

Test

The correctly sequenced xylose reductase expression plasmids pET28a-KsXR-kana (BBa_K4941034), pET28a-PsXR-kana (BBa_K4941033), pET28a-DnXR-kana (BBa_K4941035)), and T7 RNAP expression plasmids (BBa_K4941063) were transferred into E. coli BL21. Here is an example of the transformation of the T7RNAP plasmid (BBa_K4941063) and pet28a-PsXR-kana plasmid:

1. Firstly, in order to screen the transformants using resistance screening markers, we replaced the kan resistance marker in the pET28a-PsXR-kana plasmid using Gibson assembly (replacing it with the Cm resistance marker) to obtain the strain pET28a-PsXR (BBa_K4941100) (using CM as the screening marker);
2. Next, the T7 RNAP expression plasmid PlacUV5 (BBa_K4941063) was transformed into BL21 and plated on LB plates containing kan resistance using the chemical transformation method;
3. The grown positive transformants were selected and inoculated into 5 mL LB tubes containing kan resistance for incubation, and the pET28a-PsXR plasmid was then transformed into these transformants using the chemical transformation method. The transformed cells were plated on LB plates containing both kan and Cm resistance;
4. Positive transformants were selected for colony PCR confirmation and prepared for subsequent fermentation tests.

The transformants were selected and transferred into the liquid medium and cultured in a shaker at 37 ℃ and 220 rpm for 12 hours. The seed solution was then transferred into 30 ml TB medium and cultured at 37 ℃ and 220 rpm for 3 hours. After that, the expression of xylose reductase was induced by adding 30 ul of IPTG inducer and the culture was continued at 28 ℃ and 160 rpm for 16 hours. Subsequently, xylose was added to a concentration of 5 g/L, and the reaction was carried out at 30 ℃ and 220 rpm for 36 hours. Samples were taken every 12 hours for HPLC detection.

The results showed that BL21-pET28a-PsXR catalyzed the production of 4.6 g/L xylitol from the substrate 5 g/L xylose after 36 hours. It was demonstrated that the xylose reductase derived from Pichia stipitis has the ability to catalyze xylose to produce xylitol in the E. coli BL21 protein expression system, and xylitol production through fermentation in E. coli is achieved

Learn

The key issue in this work is to obtain the xylose reductase with the strongest catalytic capacity in expression system by assaying the activity of xylose reductase from different sources.

Cycle 2: Promoter engineering to optimize xylose reductase expression

Design

With our newly constructed T7RNAP expression library based on the lacUV5 promoter mutant, we optimized the expression using the pET expression system against xylose reductase (PsXR, GenBank: X59465.1) of Pichia stipitis origin. This was done to find the most suitable expression intensity in order to further improve the conversion to xylose and increase xylitol production.

Build

In order to verify the effect of three catalytic properties of the pET expression system on PsXR by modulating the intensity of T7RNAP expression, and thus affecting the catalytic properties of PsXR, we simultaneously transformed T7RNAP expression plasmids and pet28a-PsXR containing different lacUV5 promoters into E. coli BL21 hosts. Here is an example of the transformation process for the T7RNAP plasmid driven by PlacUV5MB7 (BBa_K4941056) and the pET28a-PsXR plasmid:

1. Firstly, plasmid PlacUV5MB7-T7RNAP-T7t (BBa_K4941083) was transformed into BL21 using the chemical transformation method and plated onto LB plates containing kanamycin resistance;
2. Next, the positively grown transformants were selected and transferred into Kanamycin-resistant 5 mL LB tubes for 3-4 hours to allow the receptor cells to grow. The pET28a-PsXR plasmid was then transformed into the above transformants using the chemical transformation method and plated onto LB plates containing both Kanamycin and Chloramphenicol resistances;
3. Positive transformants were selected for colony PCR test confirmation and prepared for subsequent fermentation testing.

Test

Three transformants containing different lacUV5 promoters were selected and transfected into 96-well culture plates containing LB liquid medium. The plates were then cultured in a shaker at 37 °C and 220 rpm for 12 hours. The seed solution was further transferred into 48-well culture plates containing 2 ml of fermentation medium and cultured at 37 °C and 220 rpm for 3 hours. Afterwards, 30 μl of IPTG was added as an inducer, and the culture was continued at 28 °C and 160 rpm for 16 hours to allow for the expression of xylose reductase.For xylose reductase expression, the plates were incubated at 16°C and 160 rpm for 16 hours. Subsequently, xylose was added to a final concentration of 8 g/L, and the reaction was carried out at 30°C and 220 rpm for 36 hours. HPLC detection was performed to analyze the reaction.

The results showed that MB7-PsXR successfully catalyzed the production of 6.8 g/L of xylitol from the substrate of 8 g/L xylose after 36 hours. This outcome demonstrates the feasibility of the strategy employed, which involved optimizing the pET expression system through the construction of a promoter library for T7RNAP expression.

Learn

The key objective of this study was to optimize the expression intensity of the pET expression system, specifically the activity of xylose reductase. This was achieved by utilizing a T7RNAP expression library based on a mutated lacUV5 promoter. The aim was to identify the combination that exhibited the optimal expression intensity during fermentation.

Cycle 3: Construction of expression plasmids for erythrosine 4-phosphate phosphatase and erythrosine reductase

Design

A natural pathway for erythritol synthesis exists in Y. lipolytica, with the genes Yida and ER playing important roles. Yida encodes the enzyme 4-phosphate erythritol phosphatase, which converts erythrose 4-phosphate to erythrose. ER, on the other hand, encodes erythritol reductase, which converts erythrose to erythritol. However, the yield of erythritol was too low to be detected using HPLC conditions. It was hypothesized that the expression levels of Yida and ER might be insufficient, leading to undetectable erythritol production. To address this issue, we attempted to overexpress LhYida (BBa_K4941041), which is an erythritol 4-phosphate phosphatase derived from Lactobacillus helveticus, as well as ylER (BBa_K4941019) in Y. lipolytica. The goal was to enhance the expression of erythrose 4-phosphate (E4P) and facilitate the de novo synthesis of erythritol, thereby increasing its yield.

Build

To enable the de novo synthesis of erythritol in Y. lipolytica, we selected the Yida and ER genes from Y. lipolytica and constructed the LhYida-ylER expression plasmid pYLXP’-LhYida-ylER (BBa_K4941105). We also constructed single gene expression plasmids, namely pYLXP’-LhYida (BBa_K4941041), pYLXP’-ylER (BBa_K4941037), and pYLXP’-ura (BBa_K4941030). Let's take pYLXP’-LhYida as an example:

1. The target fragments LhYida (BBa_K4941020), pTEF (BBa_K4941012), and XPR2 (BBa_K4941014) were obtained using PCR and designed with 4bp sticky ends for plasmid assembly;
2. Plasmid pYLXP' (BBa_K4941039) was digested with nuclease BsaI to obtain linearized pYLXP' (BBa_K4941039);
3. The resulting reaction mixture from the golden gate assembly was transformed into E. coli DH5α.iv) The colonies were verified by PCR and sent for sequencing by Sangon Biotech (Shanghai, China) to confirm the correct sequence.

After obtaining the single gene expression plasmids, we proceeded to construct the pYLXP’-LhYida-ylER expression plasmid:

1. Firstly, plasmids were digested with AvrII and NotI to obtain PTEF1-LhYida-XPR1 (BBa_K4941044) and PTEF-URA-XPR2 (BBa_K4941026) fragments for plasmid assembly;
2. Plasmid pYLXP-ylER (BBa_K4941037) was digested with NheI and NotI to obtain linearized pYLXP-ylER0.
3. The linearized pYLXP-ylER and the two standardized fragments were ligated using T4 ligation;
4. The resulting T4 assembly reaction mixture was transformed into Escherichia coli DH5α.v) Colonies were verified by PCR and sent for sequencing by Sangon Biotech (Shanghai, China) to confirm successful construction and sequence accuracy.

Test

Next, we proceeded to integrate the LhYida-ylER expression fragment (BBa_K4941106) into the genome of Y. lipolytica po1g in order to achieve de novo synthesis of erythritol. For successful integration, we employed the lithium acetate transformation method, following a standard protocol previously reported [1, 2]. Here is a brief overview of the protocol:

1. During the exponential growth phase (16-24 hours), 1 mL of the Y. lipolytica culture was obtained from 2 mL of YPD medium (containing yeast extract 10 g/L, peptone 20 g/L, and dextrose 20 g/L) in a 14 mL shaker tube;

2. The bacterial cells were then washed twice with 100 mM phosphate buffer (pH 7.0);

3. The cells were resuspended in 105 µL of transformation solution, consisting of 90 µL of 50% PEG4000 solution, 5 µL of lithium acetate (2M), 5 µL of boiled single-stranded DNA (denatured salmon spermatozoa), and 5 µL of DNA products (including 200-500 ng of plasmid, linear plasmid, or DNA fragment);

4. The mixture was thoroughly mixed and heated, followed by incubation for 1 hour at 37°C in a water bath;

5. After incubation, the mixture was coated onto selected solid plates. It is important to note that the transformation mixtures needed to be oscillated every 15 minutes for 15 seconds during the incubation at 37°C;

6. The selected marker for integration in this study was Uracil. As a result, we successfully integrated the LhYida-ylER fragments into the genome of Y. lipolytica po1g, obtaining the engineered strain po1g-LhYida-ylER (po1g-1).

Subsequently, we selected positive transformants for fermentation experiments. The strain was transferred to YNB-URA medium and cultured for 48 hours to obtain the seed liquid. Samples were taken after transferring 500 µL of seed liquid to 30 mL of fermentation medium, which was then cultured at 30°C and 220 rpm on a shaker for 120 hours. The content of erythritol was detected using HPLC. The results revealed that the engineered strain po1g-1 overexpressing LhYida and ylER achieved a yield of 2.1 g/L of erythritol, thus successfully realizing de novo synthesis of erythritol in Y. lipolytica.

Learn

Although the erythritol synthesis pathway exists in the wild-type Y. lipolytica, the expression of the pathway enzymes is insufficient, resulting in a low yield of erythritol that cannot be detected by HPLC. Therefore, in this project, we successfully achieved the de novo synthesis of erythritol in Y. lipolytica by overexpressing the endogenous LhYida and ylER, which enhanced the expression of E4P for erythritol production.

Cycle 4: Construction of the fluorescent reporter system for characterizing erythritol production

Design

The fluorescent reporter system has been widely employed to assess protein expression levels [1] [2]. Inducible promoters generate varying intensities depending on the concentration of the inducer, thereby modifying the expression level of the protein [3]. Jean Marc Nicaud [4] combined an erythritol-inducible promoter with a luciferase reporter system that correlates fluorescence intensity with erythritol concentration. Consequently, erythritol concentration was determined using a more straightforward and intuitive method. Building upon this, we utilized an erythritol-inducible promoter to express the luciferase-encoding gene. Our aim was to construct an erythritol-luciferase reporter system in Y. lipolytica and assess erythritol yield through fluorescence intensity.

Build

To construct the fluorescent reporter system for characterizing erythritol production, we created the expression plasmid pylxp-pEYK1-Nluc (BBa_K4941036) which contains the Nluc (BBa_K4941022) gene driven by an erythritol-inducible promoter (pEYK1, BBa_K4941036). The construction process involved the following steps:

1. The target fragments Nluc (BBa_K4941022), pEYK1 (BBa_K4941023), and XPR2 (BBa_K4941014) were obtained using the PCR method, with 4bp sticky ends added to both ends for plasmid assembly；

2. Plasmid pYLXP' (BBa_K4941039) was then digested with nuclease BsaI, resulting in linearized pYLXP' (BBa_K4941039)；

3. The linearized pYLXP' and the standardized processed fragments were then ligated together using Golden Gate assembly；

4. The resulting reaction mixture from the Golden Gate assembly was transformed into Escherichia coli DH5α；

5. Corrected colonies were verified using PCR and subsequently sequenced by Sangon Biotech (Shanghai, China).

Test

Next, we proceeded to transform the pylxp-pEYK1-Nluc (BBa_K4941036) plasmid into the Y. lipolytica po1g strain. For the transformation, we utilized the lithium acetate method, as previously described in studies by Islam, Zhisheng et al. (2015) and Xu, Wu et al. (2022). In our project, we employed leucine as the selection marker. The transformation was successful, resulting in the creation of the engineered strain po1g pylxp-pEYK1-Nluc (po1g-2).

Subsequently, we proceeded to select positive transformants for fermentation experiments. The strain was transferred to YNB-leu medium and cultured for 48 hours to obtain the seed liquid. Then, 500 μl of the seed liquid was transferred to 30 ml of fermentation medium with varying concentrations of erythritol (0 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L). The samples were taken after 48 hours of culturing in a shaking bed at 30°C and 220 rpm. The luciferase substrate was added to initiate the reaction, and a kinetic assay was performed to compare the fluorescence intensity of the fermentation broths with different erythritol concentrations.

The results demonstrated a notable variation in the fluorescence intensity of the fermentation broth upon the addition of different concentrations of erythritol, as depicted in Figure 7. The fluorescence intensity provided a visual representation of the varying erythritol concentrations within the fermentation broth.

Learn

In this project, we successfully constructed a fluorescent reporter system to characterize erythritol production by correlating the concentration of erythritol in the fermentation broth with the fluorescent intensity. This approach allowed us to identify and obtain engineered strains with higher erythritol production through the detection of fluorescent intensity.

Cycle 5: Screening of Erythrose-4P phosphatase

Design

The successful construction of the fluorescent reporter system played a crucial role in our ability to effectively screen erythritol-producing strains. In order to enhance the expression of Erythrose-4P phosphatase (Yida) for increased erythritol production, we selected this enzyme from various sources. These selected enzymes were then integrated into Y. lipolytica for erythritol production. We utilized the expression of pYLXP-pEYK1-Nluc (BBa_K4941036) to evaluate the erythritol production of each engineered strain based on fluorescence intensity.

Build

We selected EcYida (BBa_K4297032) from E. coli [1] and StYida (BBa_K4297028) from Streptococcus thermophilus (BBa_K4297032). Through PCR, we obtained the target fragments EcYida (BBa_K4941018) and StYida (BBa_K4297028). Reference to the construction of pylxp-LhYida-ylER (BBa_K4941105), we obtained two expression plasmids, pylxp-EcYida-ylER (BBa_K4941104) and pylxp-StYida-ylER (BBa_K4941107).

Test

Next, we proceeded to transform the expression fragments EcYida-ylER (BBa_K4941103) and StYida-ylER (BBa_K4941108) into the genome of Y. lipolytica po1g, enabling integration of their expression [1, 2]. In our project, the selection marker used was Uracil. Consequently, we successfully integrated the EcYida-ylER and StYida-ylER expression fragments into the genome of Y. lipolytica po1g, resulting in the engineered strains po1g-EcYida-ylER plate (po1g-3) and po1g-StYida-ylER plate (po1g-4).

Subsequently, we utilized the lithium acetate transformation method to introduce pylxp-pEYK1-Nluc into the mixed bacteria of po1g-1, po1g-3, and po1g-4 separately. The strains were then transferred to YNB-URA medium and cultured for 48 hours to obtain the fermentation broth. The fermentation broth was cultivated at 30°C and 220rpm. In order to initiate the reaction, the luciferase substrate was added, and a kinetic assay was performed to compare the fluorescence intensity of the fermentation broths of the three engineered strains. The strain with the highest yield was selected for further analysis. The chosen engineered strains were subsequently transferred into YNB-leu fermentation broth and allowed to ferment for 120 hours. The fermentation broth was then collected for HPLC detetion. According to the results, strain po1g-3, expressing EcYida-ylER, exhibited the highest erythritol yield, as depicted in Figure 9. The HPLC assay confirmed that the erythritol yield reached 5.9 g/L.

Learn

In this project, we employed the EcYida fragment from E. coli and the ylER fragment from Y. lipolytica. The expression construct EcYida-ylER was integrated into the genome of Y. lipolytica po1g, resulting in the successful expression of the desired genes. Ultimately, the engineered strain po1g-3 displayed a remarkable erythritol yield, reaching 5.9 g/L.

Reference

1. Islam, Z.U., et al., Microbial conversion of pyrolytic products to biofuels: a novel and sustainable approach toward second-generation biofuels. J Ind Microbiol Biotechnol, 2015. 42(12): p. 1557-79.

2. Xu, Y., et al., Sustainable bioproduction of natural sugar substitutes: Strategies and challenges. Trends in Food Science & Technology, 2022. 129: p. 512-527.

3. Biewenga, L., B. Rosier, and M. Merkx, Engineering with NanoLuc: a playground for the development of bioluminescent protein switches and sensors. Biochem Soc Trans, 2020. 48(6): p. 2643-2655.

4. Wang, X., et al., Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun, 2021. 12(1): p. 1411.

5. Qiu, C., H. Zhai, and J. Hou, Biosensors design in yeast and applications in metabolic engineering. FEMS Yeast Res, 2019. 19(8).

6. Trassaert, M., et al., New inducible promoter for gene expression and synthetic biology in Yarrowia lipolytica. Microb Cell Fact, 2017. 16(1): p. 141.

7. Carly, F., et al., Enhancing erythritol productivity in Yarrowia lipolytica using metabolic engineering. Metab Eng, 2017. 42: p. 19-24.