Team:Evry/Description/Chassis

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Let's PLAy project - Bioproduction of PLA

Project Description

Our chassis

As mentioned in Context, PLA has already been produced in Escherichia coli. E. coli is the most common chassis in synthetic biology and iGEM, because of advantages such as fast replication and well described metabolism [1]. However, when aiming to produce a compound, it is important to consider the compound characteristics and evaluate the adequacy of different chassis.

In the case of PLA, initially there were two characteristics which made us consider using a different chassis: the fact of having as precursor lactic acid and the fact of being a polymer.

E. coli is not ranked usually as a good producer of lactate. If having a look on the literature, some species reported to be good lactate producers are:

  • Wild type Lactobacillus casei RL20 → 72 g/L at 48h. 
When expressing the genes pfk and glk → 144.2 g/L at 48h 
[2].
  • B. subtilis MUR1 can produce 99.3 and 183.2 g/L of L-LA in 12 and 52 h respectively with a 98.5% substrate conversion yield and a maximum L-LA production rate of 16.1 g/l/h [3]
    • .
  • Good yield results of lactic acid have been observed in Pseudomonas putida, from the activities of its iLDH (L:22.1; D:66.6 nmol/min*mg) [4]
.

Besides, there are some organisms known to produce naturally polymers and have facility to do reactions of polymerization. Four major classes of polymers are produced by bacteria: polysaccharides, polyesters, polyamides and inorganic polyanhydrides. First biosynthesized polymers were discovered in Bacillus megaterium, but later on they have been studied in many Pseudomonas spp., such as Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida [5].

Taking into account that the polymerization step is the main bottleneck on PLA bioproduction [6], and that Pseudomonas has good lactate production [4] and facility for synthesis of polyesters [5], they seem to be the best candidate for optimization of PLA production in prokaryotic bacteria.

Pseudomonas putida

Pseudomonas spp. are described to be bacillus (rod-shape), gram-negative, oxidase + bacteria, with aerobic metabolism. Specifically, P. putida is well known in the laboratory for its uses on bioremediation of soil. Its optimal growth is at 25-30ºC and compared to its similar spp. Pseudomonas aeruginosa, P. putida lacks the genes of virulence, converting it in a non-pathogenic species. This, together with its ability to degrade organic compounds, contributes to the list of benefits that make this species highly useful in research [7][8].

One of its most interesting characteristics is the ability to produce Polyhydroxyalkanoates (PHA), a natural polyester. PHAs are thermoplastics, insolubles in water, non-toxic and they have a high degree of polymerization. Similarly to our PLA, they are interesting for biotechnological industry [7]

.

Our chassis: Pseudomonas putida KT2440

Above all P. putida, the strain KT2440 is the one which metabolism is more accurately described, and it is used as microbial laboratory work horse. There are reported good in silico models for the reactions of its pathways and a variety of experimental results on metabolic engineering [9][10].

Moreover, it is reported to be a GRAS (Generally Recognized As Safe) organism, providing another advantage on its use for biomaterial [8]. The PLA we are going to produce will be used in biomedical applications, or even in toys for children. Thus, safer reports of the organism can help guarantee safety of our product.

Comparison of P. putida with E. coli and Bacillus spp.

To sum up, the following table shows the advantages that P. putida can offer compared to the already used E. coli and the other candidate chassis Bacillus spp.


Characteristic E. coli B. subtilis P. putida KT2440
Ease of genetic manipulation

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Safety

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Lactate production

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Polymer formation

*

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Extra: Co-culture, a feasible idea?

Regarding iGEM, there have been projects on production of compounds with co-cultures of species. Indeed, UChile-OpenBio 2015 tried to produce PLA by splitting the pathway into different E. coli. Co-cultures are useful for splitting the metabolic charge and optimizing precursor production [11]. However, we have to take into account the characteristics of the compound of interest. In our case, we studied the possibility of producing first lactate with Lactobacillus spp., and making it grow in co-culture with Pseudomonas putida, who would do the polymerization. Species were compatible, but exchange of Lactyl-CoA, as it is a 24 carbon compound, would have needed extra membrane transporters. This, together with the possible limitations in physical interactions made us rely on Occam's razor and stick on single-bacteria production.


References

  1. iGEM: Registry of Standard Biological Parts.Escherichia coli chassis. Retrieved from: http://parts.igem.org/Escherichia_coli_chassis (29/09/2016)
  2. Gong, Y., Li, T., Li, S., Jiang, Z., Yang, Y., Huang, J., Liu, Z., Sun, H. Achieving High Yield of Lactic Acid for Antimicrobial Characterization in Cephalosporin-Resistant Lactobacillus by the Co-Expression of the Phosphofructokinase and Glucokinase. J Microbiol Biotechnol. 26(6):1148-61 (2016).
  3. Gao, T., Wong, Y., Ng, C., Ho, K. L-lactic acid production by Bacillus subtilis MUR1. Bioresour Technol, 121:105-110 (2012).
  4. Wang, Y., Min L., Zhang, Y., et al. Reconstruction of lactate utilization system in Pseudomonas putida KT2440: a novel biocatalyst for L-2-hydroxy-carboxylate production. Sci Reports, 4: 6939 (2014).
  5. Rehm, B.H.A. Bacterial polymers: biosynthesis, modifications and applications. Nat Microbio 8, 578-592 (2010)
  6. Jung, Y.K., Kim, T.Y., Park S.J. & Lee S.Y. Metabolic Engineering of Escherichia coli for the Production of PLA and Copolymers in E. coli. Biotech and Bioeng 105:1, 161-171 (2010)
  7. Poblete-Castro, I., Becker, J., Dohnt, K., Martins dos Santos, V., Wittmann, C. Industrial biotechnology of Pseudomonas putida and related species. Appl Microbiol Biotechnol (2012).
  8. Nikel, P.I., Martinez-Garcia, E., de Lorenzo, V.Biotechnological domestication of pseudomonads using synthetic biology. Nat Rev Microbiol 12, 368-379 (2014)
  9. K. E. Nelson, C.W., Paulsen, I.T., Dodson, R.J. et al. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4(12), 799–808(2002).
  10. Nogales, J., Palsson, B. Ø. & Thiele, I. A genome-scale metabolic reconstruction of Pseudomonas putida KT2440: iJN746 as a cell factory. BMC Syst Biol 2, 79 (2008).
  11. Zhang, H., Pereira, B., Li, Z., Stephanopoulos, G. Engineering Escherichia coli coculture systems for the production of biochemical products. Proc Natl Acad Sci USA, 112(27): 8266–8271 (2015).