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• Synthase Screening
• Pathway Construction
• Constructing E. coli

Design

Design

1. Squalene Synthase (SQS) Clone

Choosing a suitable enzyme plays an important role in synthesizing the target product. Evaluating and eliminating the unnecessary structures, moreover, can dramatically improve the enzyme’s expression, solubility, and activity.

In order to compare the squalene yield of different SQSs, we planned to clone the SQSs from Yarrowia lipolytica, Saccharomyces cerevisiae, Bacillus subtilis, and human beings.

2. Truncating YSS

Membrane proteins of eukaryotes need to be located on specific plasma membrane of organelles in cells to play a part, but E. coli doesn’t have plasma membrane that eukaryote membrane proteins need to locate on. So, the expression of plasma membrane in E. coli will affect its folding correctly, reducing its viability. In the amino acid sequence of eukaryote membrane proteins, there is a transmembrane domain on n-terminus or c-terminus that can be used to locate the hydrophobic amino acid sequence of membrane, which is a factor that affect its correct folding in E. coli. By erasing the transmembrane domain of eukaryote membrane proteins is one of the common strategies used to realize correct folding in E. coli as well as increase the catalyzing efficiency.

So far, SQS from human being, Thermosynechococcus elongatus and Streptomyces peucetius ATCC 27952 is introduced into E. coli to synthesize squalene biologically. However, Yarrowia lipolytica is a new type of chassis cell widely used in the biological synthesis of terpenoid in the recent years.

By the software prediction of transmembrane domain of amino acid sequence of YSS, as shown below, it can be found that YSS has a transmembrane domain in the c-terminus, amino acid sequence if YSS402. In order to discover whether the transmembrane domain of YSS will affect the biological viability of E. coli, amino acid sequence of YSS402 and YSS40226 will be respectively erased.

TMHMM2.0 outside 1 402

TMHMM2.0 TMhelix 403 425

TMHMM2.0 inside 426 445

3. Establishing the Screening Method

It was reported in the documents that dehydrosqualene desaturase CrtN from Staphylococcus aureus can transform squalene into carotenoid, lead CrtN and SQS genes into E.coli at the same time, and extract with acetone the carotenoid which is produced in thallus. As a result, to estimate the squalene production, we planned to evaluate the 470nm light absorbance of the extracted samples with ultraviolet spectrophotometer. This method is fast and convenient, capable of screening productive strains.

1.The Construction of MVA Pathway

Shown below is the genes in Plasmid P35151 bought from addgene.org(Plasmid information at http://www.addgene.org/35151/), including 6 genes in the MVA Pathway: AtoB, HMGS, tHMGR, MK, PMK and PMD.

Construct Plasmid pMVA1 and pMVA2 according to the MVA Pathway in Farnesene-producing strains as reported by the document.

pMVA1:

Plasmid pBBR1MCS is used as the vector, while the lac operon as a medium-strength promoter. The replicon is replaced by the p15A replicon. This would contain the first three genes of atoB, HMGS and tHMGR in the MVA Pathway.

pMVA2:

Plasmid pBBR1MCS-2 is used as the vector, and the lac operon is once again the promoter. The replicon is the original pBBR1MCS. This would contain the four other genes in the MVA Pathway: MK, PMK, PMD and idi.

Unlike Plasmid p35151, this method constructs the 6 genes in the MVA Pathway separately on two plasmids, all of which are taken from Plasmid p35151, providing us with the results below:

pMVA1：pBBR1MCS-1(p15A)-AtoB-HMGS-tHMGR

pMVA2：pBBR1MCS-2-MK-PMK-PMD-Idi

2. Overexpression of MEP pathway

According to previous studies, dxs and idi, two important regulatory enzymes in MEP pathway, plays an important role in strengthening IPP/DMAPP flux in MEP pathway. The researches also find out that stimulating IspG can remarkably decrease cell growth as well as limit the production of β-Carotene. The overexpression of IspG will cause HMBPP, a kind of intermediate, to accumulate, disturbing the mechanism in E.coli to synthesize nucleotide and protein. However, stimulating downstream kinases IspH can solve the problem of HMPBB accumulation, erasing the negative effect caused by IspG overexpression. The balance stimulation can erase the accumulation of HMBPP and MECPP, enabling the carbon flux to move backwards from DXP to create a more effective MEP pathway. As a result, we try to overexpress the key kinases Idi, Dxs, IspG and IspH in MEP pathway.

3. Construction of carotenoid Escherichia coli containing MVA pathway and MEP pathway

Introduce the constructed plasmid with MVA and the MEP pathway into E. coli BL21 (DE3). The plasmid combinations are as follows:

Combination I: p35151/pET-IIAY-CN, strain H1

Combination II: p35151/pMEP1/pET-HCN, strain H2

Combination III: pMVA1/pMVA2/ pET-IIAY-CN, strain H3

Combination IV: pMVA1/pMM/pET-HCN strain H4

Substitute the chassis cells and introduce the constructed plasmid with MVA and MEP pathway into E. coli XL1-Blue. The plasmid combinations are as follows:

Combination I: p35151/pUC-IIAY-CN, strain H5

Combination II: p35151/pMEP1/pUC-HCN, strain H6

Combination III: pMVA1/pMVA2/ pUC-IIAY-CN, strain H7

Combination IV: pMVA1/pMM/pUC-HCN, strain H8

Subject the strains H1-H8 to a TB medium with shake flask fermentation, and extract the carotenoids produced by the fermentation process with acetone. The level of carotenoid production is compared by measuring the absorbance at 470 nm, and the optimal combination of the chassis cells and plasmid is finally determined.

III. Constructing the squalene-producing E. coli

The best combination of plasmids was introduced into the appropriate chassis cells. CrtN gene was removed from the plasmid combination, while other genes remained unchanged. The successfully established strains were cultured in shaking flask with TB medium. Squalene was extracted from the bacteria and detected by HPLC. The squalene obtained through biosynthesis was verified using mass spectrometry and nuclear magnetic resonance spectroscopy.

The fermentation of high-yield squalene-producing E. coli

The fermentation conditions of the strains with high squalene yield were optimized, and the fermentation was finally carried out using the 5L high-density fermentation tank.

References:

(1) Production of squalene by squalene synthases and their truncated mutants in Escherichia coli

(2) In Vitro Reconstitution of Mevalonate Pathway and Targeted Engineering of Farnesene Overproduction in Escherichia coli

(3) Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli

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