2. Modification of Pseudomonas putida KT2440

P.putida KT2440 is one of bacteria which can utilize ethylene glycol (EG) at a high speed and meanwhile produces mcl-PHA. In 1988, Lageveen and his co-workers first found mcl-PHA in P.putida KT2440. And then, the metabolism of producing PHA in P.putida KT2440 was reasearched, which found the gene AcoA was the key gene in the procedure. José Manuel Borrero-de Acuña and his co-workers improved the yield by 33% by overexpressing AcoA.

From Björn Mückschel’s works, Ethylene Glycol Metabolism by Pseudomonas putida was found. The key enzymes were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only.

From those studies, we decided to overexpress AcoA and AceA in P.putida KT2440 to help utilize EG as energy source for its growth.

3. Modification of Bacillus subtilis

After some attempt in E.coli and yeast, we look for a new type of host cells- B.subtilis for more secretion. In our experiment, the genes encoding two enzymes are for the first time expressed in S.cerevisiae. Increased yields of PETase and MHETase enzymes are achieved when B. subtilis strains 168 and DB104 (deficient in two and three extracellular proteases, respectively^[1]) were transformed with the recombinant plasmid with the help of the enhanced promoter-p43.

4. A Controllable Lipid Producer

Cyanobacteria are excellent organisms for biofuel production. We thus have selected Cyanobacterium Synechocystis sp. PCC 6803 as the source of carbon in our mixed bacteria system. Our target is simply to make the cyanobacteria lyse at the appropriate time by transforming a plasmid contained three bacteriophage-derived lysis genes which were placed downstream of a nickel-inducible signal transduction system into the Synechocystis 6803.

5. Gene Knockout of Escherichia coli

In order to make S. cerevisiae and E.coli better survival, we need to change the energy source from glucose to xylose. When E. coli metabolizes xylose it excretes acetate, which is inhibitory to its own growth. S. cerevisiae cannot metabolize xylose but can use acetate as the sole carbon source without producing ethanol which is toxic to E.coli. For more acetate secretion, we successfully knocked off the atpF and atpH gene from the whole genome with the help of λ-red Recombination and I-SceI Cleavage.

1. Degradation of Terephthalate

Rhodococcus sp. strain RHA1 is thought to be capable of degrading a wide range of aromatic compounds including terephthalate acid (TPA). in 2006, a reliable pathway consisting of Distinct ring cleavage dioxygenase systems and protocatechuate (PCA) pathway was come up with, and the proposed degradation pathway for TPA is shown as below^[2].

Fig.3    Proposed degradation pathway for TPA^[2]

2. Degradation of Ethylene Glycol

By employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis, it is found that Pseudomonas putida KT2440 does not grow within 2 days of incubation, compared to Pseudomonas putida JM37 which can grow rapidly under the same conditions. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Postulated pathway for the metabolism of ethylene glycol in Pseudomonas putida strains KT2440 and JM37 is shown left^[3].

Fig.4   Postulated pathway for the metabolism of ethylene glycol in Pseudomonas putida strains KT2440 and JM37. The enzymes and/or metabolites identified in response to ethylene glycol in KT2440 are depicted in black. Additional pathways identified in strain JM37 are shown in gray. Detailed descriptions of the pathways are given in the text.^[3]

3. Production of Polyhydroxyalkanoate

Pseudomonas putida is a natural producer of medium chain length polyhydroxyalkanoates (mcl-PHA), a polymeric precursor of bioplastics. A genome-based in silico model for P. putida KT2440 metabolism was employed to identify potential genetic targets to be engineered for the improvement of mcl-PHA production using glucose as sole carbon source. Here, overproduction of pyruvate dehydrogenase subunit AcoA in the P. putida KT2440 wild type led to an increase of PHA production. In controlled bioreactor batch fermentations PHA production was increased by 33% in the acoA overexpressing wild type in comparison to P. putida KT2440. Transcriptome analyses of engineered PHA producing P. putida in comparison to its parental strains revealed the induction of genes encoding glucose 6-phosphate dehydrogenase and pyruvate dehydrogenase. In addition, NADPH seems to be quantitatively consumed for efficient PHA synthesis, since a direct relationship between low levels of NADPH and high concentrations of the biopolymer were observed. In contrast, intracellular levels of NADH were found increased in PHA producing organisms. Central metabolism of P. putida KT2440 is shown right^[4].

Fig.5    Central metabolism of P. putida KT2440^[4]

4. Advantages of B. subtilis strains

Bacillus subtilis has an excellent secretion ability, displays fast growth, and is a nonpathogenic bacterium free of endotoxin [5]. It can, therefore, be used in food, enzyme, and pharmaceutical industries and can replace Escherichia coli for protein expression. Furthermore, the extracellular heterogeneous proteins secreted from B. subtilis are more convenient for recovery and purification in large-scale production during downstream processing [6].

5. Enhanced promoter-p43

In order to increase secretion, some enhanced promoters are necessary. However, native gene in a high-copy number plasmid was found to be unstable in B. subtilis[5]. To optimize the production and the stability of the expression vectors, both the promoter and the signal sequence of PETase were replaced by B. subtilis P43 promoter, a constitutively expressed promoter. This overcame the plasmid instability problem.

Fig.6  construction of plasmid of cooperation of p43+psacB promoter^[7]

6. the cooperation of two promoters-p43+p_sacB

Interestingly, the cooperation of two promoters in B. subtilis are easily found. For example, An endoglucanase from Bacillus akibai I-1 was successfully overexpressed in Bacillus subtilis 168 by the help of p43 promoter and the expression level of the recombinant enzyme was greatly enhanced by using the sucrose-inducible psacB promoter[7].The construction of plasmid is in the Figure 1 . Thus, we are willing to try whether the combination of p43 and psacB can make a difference in the secretion of enzyme.

1. Construction of Reporting System

We use a common expression vector plasmid, pUC19, in E.coli to load our device, which consists of heterologous gene part (in this circumstance, PETase gene) and inclusion body reporting part. First of all, we transform the plasmid with part BBa_K339007 from the kit shipped to us using the protocol in the instruction from iGEM official website. Then we use PCR to amplify this part with restriction endonuclease cutting sites Xba1 and Pst1 respectively on sense and anti-sense primers. Then we use corresponding restriction endonuclease to cut the part and plasmid pUC19 and then use T4 DNA ligase to link them together. The next step is to transform the PETase gene into the same plasmid. The initial gene synthetized does not has promoter and terminator so it cannot express. We have to cut the PETase gene and plasmid pET21A with BamH1 and Sal1 enzyme and link them together to transform the PETase gene into pET21A and then use PCR to amplify the T7 promoter-PETase gene-T7 terminator fragment added the restriction endonuclease cutting sites EcoR1 and Sac1. In this way, after we cut the recombinant plasmid pUC19 and T7 promoter-PETase gene-T7 terminator fragment with corresponding restriction endonucleases and link them together, we can obtain the complete device we want.

                             Fig.8. The construction process of our reporting system

                 Fig.9. The construction process of our cell lysis based regulation system

5. Construction of Cell Lysis Based Regulation System

This system has a great similarity to the reporting system above. Therefore it is easy to construct because we only need to change the RFP gene to the ddpX gene. However, there is no restriction endonuclease cutting site between the CpxR and RFP gene sequence according to the part map from the iGEM official website, so we have to use PCR to amplify the CpxR promoter solely and add restriction endonuclease cutting sites Xba1 and BamH1 respectively in both end. The ddpX gene is obtained from the E.coli genome using colony PCR and the BamH1 and EcoR1 restriction endonuclease cutting sites are added respectively to both end. Then the three fragments, CpxR promoter, ddpX gene, and cut plasmid pET21a are linked together. Then the whole part is amplified by PCR with Xba1 and Pst1 restriction endonuclease cutting sites added respectively to both end. This way, we can easily cut down the former CpxR-RFP fragment and add the new CpxR-ddpX fragment to the plasmid pUC19.

9. Construction of TPA Positive Feedback Based Regulation System

We use common plasmids of Saccharomyces cerevisiae, pRS413, pRS415 and pYES2, to respectively load the TPA transporting protein gene, TPA regulation protein gene and TPA induced RFP gene. First of all, we use PCR to amplify all of these fragments and add different restriction endonuclease cutting sites. Then we cut the plasmids with corresponding restriction endonucleases. Then these cut fragments are linked according to the designed order and transformed into Saccharomyces cerevisiae. We screen for the correctly transformed cell by using the Sc-Ura-Leu-His plate.

          Fig.10. The construction process of our TPA Positive Feedback Based regulation system