Team:Jiangnan China/Engineering3

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The core of our project is to construct a recombinant strain of Starmerella bombicola to produce the sophorolipid with our expectation. Step by step, we faced a lot of problems and failures. With careful consideration and effective improvement, finally we achieved engineering success.

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Engineering strains of Starmerella bombicola

Background:

There are two main types of sophorolipids (acid type and lactone type). Acid type includes better biosurfactant activity, while lactone type includes better bacteriostatic effect. However, the sopholipids produced by wild-type S. bombicola is a random mix of these two types.[10] The separation of these two types of sophorolipids is difficult, so there is no pure type sophorolipid on the market.

Research:

We did some research on how S. bombicola produces sophorolipids.[11] There has been some research on the sophorolipids metabolic pathway of S. bombicola (Fig.15). We can indicate that the lactone sophorolipid is generated by the acid sophorolipid under the action of lactonase (SBLE).

Fig.15 The metabolic pathway of sophorolipids in S. bombicola

Imagine

The yield of these two types of sophorolipids can improved via using a strong promoter to over-express UDP-glucosyltransferase B (UGTB). A pure acid type can be produced via knocking out lactonase (SBLE) gene, and a pure lactone one can be produced via using a strong promoter to over-express lactonase (SBLE).

Design

Choose Pxa1 as the editing site. Knock in three strong promoters Ptef1, Peno and Pgki to over-express UDP-glucosyltransferase B (UGTB).

Knock out the lactonase gene (SBLE) by adding a terminator to terminate its expression.

Choose SBLE as the editing site. Knock in three strong promoters Ptef1, Peno and Pgki to over-express lactonase (SBLE).

Add the self-excising hygromycin marker cassette to the repair template, which can be used to excise hygromycin resistance gene after induction of galactose.

The galactose-induced promoter Pgalk was used to control the expression of recombinase. Two recombinant site were designed on both side of the hygromycin resistance gene.

To ensure the effectiveness of plasmid, the Cas9 protein and sgRNA expression fragment was constructed dividually. Use the Ptef1 to transiently express Cas9 protein and sgRNA.

Bulid

Cas9 and sgRNA expression cassette:

The Ptef1 and the fragment “sg-Pxa1-13-Tsyn7” was amplified by PCR.

The fragment “Cas9-Tsyn7” was fused with Ptef1 by fusion PCR.

The fragment “sg-Pxa1-13-Tsyn7” was fused with Ptef1 by fusion PCR.

These two fragments “Ptef1-Cas9-Tsyn7” and “Ptef1-sg-Pxa1-13” were linked to the vector T-Simple-19 to construct the recombinant vector.

Then by taking the recombinant vector as the template, the Cas9 and sgRNA expression can be acquired by PCR.

Fig.16 The Cas9 and sgRNA expression cassette

SBLE knock-out cassette:

By taking the vector with the SBLE gene as the template, the homologous arms upSBLE and doSBLE were obtained by reverse PCR.

The DNA sequences of six site and recombinase were chemically synthesized by the company (Jinweizhi, suzhou, China).

The six site fragment was acquired by enzyme digest and gel recovery.

The recombinant vector was acquired by linking the homologous arms with the six gene.

The Pgalk, fragment “Rec-Tgki” and hygromycin resistance gene were amplified by PCR. These fragments were fused by fusion PCR.

Then the recombinant vector was acquired by linking the fragment with the basic vector.

Then by taking the recombinant vector as the template, the SBLE knock-out cassette can be acquired by PCR.

Fig.17 The self-excising hygromycin marker used in editing SBLE

UGTB, SBLE over-expression cassette (Take UGTB over-expression by Ptef1 as a sample):

By taking the vector with the UGTB gene as the template, the homologous arms upPXA1 and doPXA1 were obtained by reverse PCR.

The DNA sequences of six site and recombinase were chemically synthesized by the company (Jinweizhi, suzhou, China).

The six site fragment was acquired by enzyme digest and gel recovery.

The recombinant vector was acquired by linking the homologous arms with the six gene.

The Pgalk, fragment “Rec-Tgki” and hygromycin resistance gene were amplified by PCR. These fragments were fused by fusion PCR.

The Ptef1 and the fragment “UGTB-Tsyn7” were amplified by PCR and then were fused by fusion PCR.

Then link these two fragments to the basic vector, we can acquire the recombinant vector.

Then by taking the recombinant vector as the template, the UGTB over-expression cassette can be acquired by PCR.

Click the picture to go to the Parts Pages

Fig.18 Over-expression of UGTB by using Ptef1 as the promoter

The differences between them are the editing site and target gene. Over-expression of UGTB is editing the PXA1 site, while over-expression of SBLE is editing the SBLE site.

Test

Then we transformed the corresponding knock-out or over-expression fragments and the Cas9 and sgRNA expression cassette to the wild-type Starmerella bombicola.

After incubation and fermentation, test the yield and acid/lactone ratio of sophorolipids produced by different strains.

Table.1 The yield of sophorolipids produced by different strains

Chart.1 The yield of sophorolipids

Fig.19 The sophorolipids production

(Left: Lactone type; Right: Acid type)

Learn:

After over-expressing UGTB, the yield could be increased a lot than that of the control. Pure acid sophorolipid could be produced via knocking out lactonase (SBLE) gene, and pure lactone one can be produced via using a strong promoter to over-express lactonase (SBLE).

Improve:

Different types of sophorolipids have different functions. The combination of these two types may increase their effect. So we wanted to regulate the lactone/acid ratio of sophorolipids.

Research:

We did some research on the effect of different types of sophorolipid. The acid type has better surfactant activity , while the lactone type has better bacteriostatic effect. We also found out that all of the sophorolipids on the market are a mixture of these two types without determining proportion.[12] How to control the ratio of acid/lactone type has also been researched.

Imagine:

We have obtained 14 promoters with different intensity. Using promoters of different intensity can control the expression level of key genes. And then controlling the ratio of lactone/acid types is possible.

Design:

If we randomly combined these promoters in pairs, there will be 225 (15×15) combinations. To minimize this work, we constructed a qualitative linear path model, based on Michaelis–Menten kinetics.

Firstly, we normalized the promoter transcription level and translation level data, and then weied them to obtain the promoter equivalent parameters. Because of the complexity of membrane transportation process and the qualitative nature of the model, we mainly used Michaelis-Menten equation[13] as the theoretical basis of membrane transportation dynamics in design of linear path model. That is to say, the relationship between transporters and substrate concentration and substrate affinity is mainly considered. The promoter equivalent parameters μi andμj were added to reaction 𝒗𝟒 to generate Acid SL and reacton 𝒗5 to generate Lactone SL, respectively. Then, by virtue of the law of conservation of mass, we constructed a set of ordinary differential equations, and use ODE45 in MATLAB R2020a to solve the equations numerically. When the simulated results entered the metastable state, we can calculate the ratio of acid and lactone type of sophorolipid in a particular promoter combination. Then, according to the predicted trend of the model and the requirement of acid and lactone type ratio in the actual production process, a certain number of promoter combinations were selected for quantitative experimental verification.

Fig.20 Design of model

With the forecast of the model, we can reduce the promoters combination to limited pairs.Then we can use these combination of promoters to control the expression level of UGTB and SBLE.

Build:

Similar to the UGTB, SBLE over-expression cassette, the difference was the promoter behind the target gene. Replace the promoters of UGTB and SBLE with different promoters.

Fig.21 The expression fragments of UGTB by using different promoters

Fig.22 The expression fragments of SBLE by using different promoters

Test:

Then we transformed the corresponding expression fragments and the Cas9 and sgRNA expression cassette to the wild-type Starmerella bombicola.

After incubation and fermentation, test the yield and the lactone/acid ratio of sophorolipids of different combinations. Then we will find the strain producing the sophrolipids with our expectation.

Learn:

By the combination of different promoters we can achieve the control of the proportion of sophorolipids. However, every combination of promoters corresponding to a ratio need a gene-editing process, which is complex and time-consuming.

Improve:

Dynamic regulation: Use of regulable promoters to control the proportion of different types of sophorolipids.

Research:

We do some research on some applications of regulable promoters.

Imagine:

The dynamic regulation of the lactone/acid ratio can be realized via replacing the normal promoters behind the target gene with the temperature-sensitive promoters.

Design:

Temperature-sensitive promoter[14] PHT is used to control the expression of UGTB and PLT is used to control the expression of SBLE. Making temperature adjustment range from 20℃ to 30℃. PHT will display its highest activity under a relatively high temperature(30℃), while PLT, will display highest activity at relatively low temperature(20℃).

Culture the recombinant S. bombicola at 30°C. At this time, PHT has the maximum transcriptional activity and UGTB gene is expressed in large quantities, while PLT has an activity of 0 at 30°C, SBLE does not express. So single acid sophorolipids accumulated in large amounts. Then the temperature is lowered to 20°C. At this time, the PLT is the highest and the PHT activity is 0. The acid-type sophorolipids accumulated in the previous stage are gradually converted into lactone-type sophorolipids. By controlling the time of incubation can control the ratio of lactone/acid sophorolipids.

Establish a model of lactone-type sophorolipid production-SBLE enzyme activity, PLT temperature-SBLE activity to guide the determination of the incubation temperature T1 required to produce a specific ratio of sophorolipids. By controlling the temperature T1 can control the ratio of lactone/acid sophorolipids.

Build:

Replace the promoters of UGTB and SBLE with the temperature-sensitive promoters PHT and PLT.

Test:

Test the yield and lactone/acid ratio of the sophorolipids produced by engineered S. bombicola.

Learn:

The temperature-sensitive promoters could achieve the dynamic regulation of the lactone/acid ratio.

References:

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[9]. Biotechnology - Gene Editing; Researchers at Noble Research Institute LLC Target Gene Editing (Improving the Genome Editing Efficiency of Crispr/cas9 In Arabidopsis Andmedicago Truncatula)[J]. Biotech Week,2020.

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