PART-IMPROVEMENT
We improved the isobutanol production upon the project of HUBU-Wuhan team in igem2018, with new isobutanol production module which contains different optimized promoters and RBS. Our metabolic pathway of isobutanol production is constructed in non-model organism Zymomonas mobilis. We intend to construct isobutanol production modules which are not only suitable in Zymomonas mobilis, but also compatible in other microorganisms.
PART ONE kdcA
HUBU-Wuhan team in igem2018:
The codon-optimized kdcA gene was cloned into the shuttle vector pEZ15Asp generating the plasmid pEZ-KT, in which kdcA is driven by the tetracycline-inducible promoter Ptet. pEZ-KT was then introduced into Z. mobilis ZM4 to generate the recombinant strain ZM4-KT.Experimental results are shown (Fig.1).
Fig.1-1 Glucose consumption, isobutanol production, and ethanol production of strain ZM 4-KT with different concentrations of tetracycline as an inducer.
The result showed that the kdcA gene can be expressed in Z. mobilis, and the isobutanol production was significantly improved with the increase of tetracycline concentrations. When the strain is induced with 2 μg/mL of tetracycline, isobutanol was accumulated to a maximum of 152.33 mg/L.
Our improvement:
To construct a stable isobutanol production strain, the codon-optimized kdcA gene driven by Ptet was integrated into the genome at either the chromosomal locus of ZMO0038 or the native plasmid locus pZM36-005, resulting in the recombinant strains of ZMQ1 and ZMQ2, respectively (Fig.1-2).
Fig.1-2 The construction of ZMQ1 and ZMQ2.
As shown in Fig.1-3, tetracycline concentrations of 0, 0.2, and 1μg/mL were used to induce the expression of kdcA for isobutanol production in ZMQ1 and ZMQ2. ZMQ1 reached a maximum isobutanol titer of 104.33 mg/L, which was 1.4 times higher than ZMQ2 with a titer of 74.67 mg/L with 1 μg/mL tetracycline induction 24-h post-inoculation. Obviously, the expression of the kdcA gene in the ZM4 genome at the ZMO0038 locus was better than in the native plasmid pZM36.
Fig.1-3 Isobutanol production and ethanol production of strain ZMQ1 and ZMQ2 with different concentrations of tetracycline as an inducer.
To avoid the use of tetracycline to achieve the isobutanol production, the Ptet promoter was replaced with constitutive strong promoter of Peno or Pgap, resulting in the recombinant strains of ZMQ3, ZMQ4 and ZMQ5, respectively (Fig.1-4).
Fig.1-4 The construction of ZMQ3, ZMQ4 and ZMQ5.
The titers of isobutanol production in ZMQ3, ZMQ4, and ZMQ5 were 144.33, 147.33, and 120.00 mg/L, respectively (Fig.1-5), which were all signifcantly higher than that in ZMQ1 and ZMQ2 (Fig.1-3). The results indicated that the use of a constitutive promoter driving the kdcA gene in the genome obviouly enhanced the isobutanol production, and the promoter Pgap was more effective than the promoter Peno.
Fig. 1-5 Isobutanol production and ethanol production of strain ZMQ3, ZMQ4, and ZMQ5.
PART TWO Bsals2-ilvC-ilvD
HUBU-Wuhan team in igem2018:
BBa_K2800020 (Fig.2-1) consists of an operon driven by an inducible promoter Ptet. It was employed to enhance the expression of the synthetic operon containing the Bsals2, ilvC, and ilvD genes involved in the isobutanol biosynthesis pathway. The composite part was cloned into the shuttle vector pEZ15Asp generating the plasmid A4, and then transformed into the recombinant ZMQ3 to generate the recombinant strain ZMQ3-A4.
Fig.2-1 Feature of the composite part of BBa_K2800020.
The fermentation performance of the recombinant strain ZMQ3-A4 is shown in Fig.2-2. Tetracycline concentrations of 0.2, 0.5, and 0.8 μg/mL were used to induce the expression of BsAls2-ilvC-ilvD for isobutanol production. The maximum isobutanol titer in ZMQ3-A4 reached 2.96 g/L after 24 h inoculation with 0.5 μg/mL tetracycline induction, which was signifcantly higher than the parental strain ZMQ3. The results indicated that the overexpression of the synthetic operon, als-ilvC-ilvD could enhance the isobutanol production.
Fig.2-2 Cell growth and isobutanol production of ZMQ3-A4 with different tetracycline. concentrations as an inducer.
Our improvement:
To increase the isobutanol production, two composite parts BBa_K4666033 and BBa_K4666034 were constructed improving upon BBa_K2800020 with another constitutive strong promoter Peno or Pgap inserted to enhance the expression of the last two genes ilvC and ilvD (Fig.2-3). The new parts were cloned into the shuttle vector pEZ15Asp saparetely generating the plasmid A5 and A8, and then transformed into the recombinant ZMQ3 to generate the recombinant strains ZMQ3-A5 and ZMQ3-A8, respectively.
Fig.2-3 Features of the new composite parts of BBa_K4666033 and the BBa_K4666034.
As shown in Fig.2-4, both the isobutanol production in ZMQ3-A5 and ZMQ3-A6 were signifcantly higher than that in the ZMQ3-A4, with the maximum isobutanol titer in ZMQ3-A5 reached 4.98 g/L and ZMQ-A8 reached 4.20 g/L. The results indicated that the improved composite part with another constitutive promoter inserted to drive the ilvC-ilvD genes was a practical strategy to enhance the isobutanol production in Z. mobilis, and especially, the promoter Peno (Peno-ilvC-ilvD ) was more effective than the promoter Pgap (Pgap-ilvC-ilvD ).
Fig.2-4 Cell growth and isobutanol production of ZMQ3-A5 and ZMQ3-A8 with different tetracycline concentrations as an inducer.