Second-generation biofuels are organic molecules that can be synthesized by using wastes of other processes as starting material. These organic molecules offer a green alternative source of fuel in respect to the oil-derived ones, for their renewability and low emissions. Isobutanol is a good example of this kind of application.

In this article, published in the Journal of Industrial Microbiology & Biotechnology (2020), K. Novak et al explore the best conditions of growth for E. coli cultures (engineered strains), from whose fermentation isobutanol can be recovered, and suggest the use of cheese whey as alternative complex media. 

To achieve this goal, the researchers:

  • Studied the best background strain: in other words, they need to find the best strain of E.coli compatible with the transformation and fermentation conditions. They tested strains with different isobutanol tolerance and with different metabolic backgrounds (pre-existing enzymes).
  • Used constitutive promoters: the gene or genes inserted in E. coli needed to be under constitutive promoters. This is important because, even if inducible promoters are useful to fine-tune the expression of genes at different times of the fermentation, they also require particular compounds to act as inducers that are often expensive.
  • Evaluated the utilization of cheese whey as the main component of the media
  • Wanted to improve the overall yield and titer of isobutanol


The transformation:

The selected pathway is pyruvate dependent and involves five enzymes, 2 of which (2 and 3) are naturally present in E.coli.

  1. Acetolactate synthase (AlsS or BudB): two versions are available, one coming from Bacillus subtilis, and another one from Enterobacter cloacae.
  2. Ketol-acid reductoisomerase (IlvC): a modified version of a naturally occurring enzyme, that used a different cofactor, allowing the fermentation to be performed in absence of oxygen.
  3. Dihydroxy-acid dehydratase (IlvD)
  4. α -ketoisovalerate decarboxylase (KdcA)
  5. Alcohol dehydrogenase (AdhA): another dehydrogenase is available in E. coli but a new version from Lactococcus lactis has been used, again, for different cofactor usage.

Multiple genes have been put in a single plasmid vector (created by Golden Gate assembly) and propagated by using E. coli BL21(DE3), a widely used strain for the production of large amounts of plasmids. Each gene has been put downstream a different constitutive promoter, each of which with different strength. The result was a library of eight different vectors different combinations of the five genes.  Then each vector has been tested in background strains.

The strains:

Three main strains were tested:

  • E. coli W: the strain was adapted by cultivation on increasing isobutanol concentrations.
  • E. coli W ΔldhA ΔadhE Δpta ΔfrdA (Δ4): strain lacking specific genes that could interfere with isobutanol accumulation (look at the metabolic network in the article).
  • E. coli K12-BW25113: a strain previously reported to be able to produce isobutanol on complex media. Production in defined media was not achieved in this study, so the focus was mainly on the other two strains.

For each test, isobutanol production was tested by using a defined media containing glucose as C-source.


(DASbox® Mini Bioreactor system)

Results:

Which was the best strain-construct combination?

The higher titer and total yield were obtained by expressing a plasmid containing all the enzymes under a strong promoter (114), with BudB as the first enzyme of the pathway, in the Δ4 strain. It was found that the KO of enzymes involved in secondary metabolisms helps in increasing the production of isobutanol, even if the number of secondary products (mainly acids) was still high.

Which fermentation conditions showed the best results?

Δ4 strain was used for large-scale experiments, pulsate fed-batch system was used as an alternative to continuous culture, which researchers were not able to establish. The presence of oxygen helped in obtaining higher isobutanol production and glucose usage. In the best performing batch, isobutanol was produced at 15.6 g/L concentration.

To recover isobutanol from the growth media, gas was bubbled in the reactor and, to recover the product, the gas stream was flushed into cold citric acid and water solution.

Cheese whey as alternative media

The performance of Δ4 strain on cheese whey was evaluated by comparing it with processes performed on defined media containing glucose or lactose. Isobutanol production in the alternative media was generally higher, possibly due to the presence of lactate (that can also be used as C-source by E. coli) and other more complex organic molecules. Isobutanol yield reached 39% of the theoretical maximum, which is a 1.5- to 2.8-fold increase compared to lignocellulosic hydrolysates, which was the other alternative for complex media.



The complete article can be found in the Journal of Industrial Microbiology & Biotechnology, it is open access, and I strongly suggest taking a look at the entire article, particularly for the additional information about methods, fermentation conditions, and economical aspects of cheese whey utilization. Moreover, additional materials, pictures, and tables are available for anyone who is interested.

NOVAK, Katharina, et al. Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material. Journal of Industrial Microbiology & Biotechnology: Official Journal of the Society for Industrial Microbiology and Biotechnology, 2020, 47.12: 1117-1132.