Part:BBa_K3241040

Pgap-PhbC-PhbA-PhbB

This part is a new part, which consists of BBa_K3241000 BBa_K3241022 BBa_K3241112 BBa_K2800032 BBa_K2800013. It is used to construct the PHB biosynthetic pathway in Zymomonas mobilis .

Design Notes

Zymomonas mobilis is the model strain of ethanol production. It's mainly metabolize glucose, fructose and sucrose through ED(entner-doudoroff) pathway to produce ethanol. Here，we introduced phbA, phbB, and phbC genes from Ralstonia eutropha to modify its metabolic pathway.When Z.mobilis disassemble sugar molecules to produce pyruvate, One part of the Metabolic flux produces ethanol under the catalysis of pyruvate decarboxylase and ethanol dehydrogenase, and in another part pyruvate oxidation decarboxylation to acetyl CoA.Acetyl coenzyme A translates into PHB under the catalysis of β-ketothiolase, acetoacetyl-CoA Reductase and PHB polymerase.

Fig.1 Heterogenous PHB biosynthesis pathway

Fig.2 PHB synthesizes key enzymes

Part one: basic fermentation

We used the promter Ptet to explore the growth of recombinant strains under the induction of different concentrations of tetracycline.(the original can refer to BBa_K3241029)

Fig.3 Original design.

Fig.4 Growth Curve.

Fig.5 PHB Production.

The expressions of PhbA PhbB and PhbC have little influence on the growth of wild-type Z. mobilis.And we have successfully detected PHB in Z. mobilis by HPLC.But the problem is that the productin of PHB is very low.

Part two: Optimization of PHB metabolic pathway

Fig.6 Metabolic pathway optimization diagram.

The strong promoter Pgap was selected as the biological element to optimize the metabolic pathway of PHB by increasing the content of precursor acetyl-coA and NADPH. We selected several endogenous genes related to these two influencing factors.And enhanced their expression separately.

Fig.7 Parts.

For details of the originals, please refer to BBa_K3241041 BBa_K3241042 BBa_K3241043 BBa_K3241044

Fig.8 Growth curves of different recombinant strains.

Fig.8 The production of different recombinant strains.

Conclusion: By enhancing the expression of ZMO0367(BBa_K3241023) ZMO1329（BBa_K3241024）, we successfully increased the production of PHB in Z .mobilis and the maximum production is 7%.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 465
  Illegal NheI site found at 1341
  Illegal NheI site found at 3257
  Illegal NotI site found at 2
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 495
  Illegal NgoMIV site found at 2356
  Illegal NgoMIV site found at 2441
  Illegal NgoMIV site found at 2846
  Illegal NgoMIV site found at 2975
  Illegal AgeI site found at 600
  Illegal AgeI site found at 3379
  Illegal AgeI site found at 4051
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 421
  Illegal BsaI site found at 1142
  Illegal BsaI site found at 2272