Part:BBa_K4432122

idi and dsx expression operon under the control of the T5 promoter

This part is an expression cassette of the E. coli 's idi (BBa_K3166068) and dxs (BBa_K3166061) genes under the control of the T5 promoter regulated by LacI (BBa_K4432000) and custom-made RBSes (BBa_K4432012 and BBa_K4432013, respectively).

Usage and Biology

E. coli is not able to naturally produce carotenoids, but is able to synthesize the farnesyl diphosphate (FPP) which is a key intermediate in the isoprenoids biosynthesis. In E. coli, FPP production is achieved via the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway which is a ten steps pathway starting from pyruvate and glyceraldehyde 3-phosphate. An alternative, the mevalonate pathway starting with acetyl, is present in eukaryotic organisms (including examples of fungi and algae) and archaea, using as Acetyl-CoA precursor.

Microbial biosynthesis of carotenoids is a well-studied and optimized metabolic engineering case thanks to the introduction of a biosynthetic pathway to classical E. coli chassis. The first metabolic engineering approaches for the carotenoid biosynthesis rapidly included optimizing the flux through the FPP to increase production yields. Over-expressing endogenous rate limiting enzymes of the MEP pathway [1,2] or expressing heterologous mevalonate pathway [3] have both been used. The latter gave better results so far, but MEP pathway is nonetheless promising because its theoretical yield is higher [4].

To maximize FPP production through the MEP pathway, understanding its regulation is critical to bypass cellular control, since it is an endogenous pathway [5]. The new tools provided by the systems biology field established that the steps catalyzed by the 1-deoxyxylulose-5-phosphate synthase (dxs) (BBa_K3166061) and isopentenyl diphosphate isomerase (idi) (BBa_K3166068) enzymes are limiting steps of the MEP pathway [6]. Indeed, overexpression of dxs and idi enzymes increased carotenoids production yields in E. coli [7].

To overexpress the E. coli idi and dxs genes and thus increase the amount of FPP precursor available, we assembled through the Golden Gate technique, this part that is expression vector in the both pSB1A3 and pSB3T5 backbones in which the idi and dxs gene were placed under the control of a hybrid T5 promoter regulated by LacI. For this, the idi and dxs gene sequences were PCR-amplified from the genome of E. coli equipped by custom-made RBSes that we specifically designed using the online tools provided by Salis’s De Novo DNA company.

References

[1] Kim SW, Keasling JD. Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production. Biotechnology and Bioengineering (2001) 72: 408–415.

[2] Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control through promoter engineering. Proceedings of the National Academy of Sciences of the United States of America (2005) 102: 12678–12683.

[3] Yoon S-H, Lee S-H, Das A, Ryu H-K, Jang H-J, Kim J-Y, Oh D-K, Keasling JD, Kim S-W. Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. Journal of Biotechnology (2009) 140: 218–226.

[4] Ajikumar PK, Xiao W-H, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G. Isoprenoid pathway optimization for Taxol precursor overproduction in Escherichia coli. Science (New York, N.Y.) (2010) 330: 70–74.

[5] Banerjee A, Sharkey TD. Methylerythritol 4-phosphate (MEP) pathway metabolic regulation. Natural Product Reports (2014) 31: 1043–1055.

[6] Volke DC, Rohwer J, Fischer R, Jennewein S. Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis. Microbial Cell Factories (2019) 18: 192.

[7] Albrecht M, Misawa N, Sandmann G. Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin. Biotechnology Letters (1999) 21: 791–795.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 500
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 500
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 674
  Illegal BamHI site found at 681
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 500
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 500
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1435