As a manufacture project, what we concern most is the yield of the final product or the intermediate that positively related to the final product. Therefore, this year, Engineering Cycle consisting of designing, building, testing, and learning from the data collected was adopted in the optimization of our expression strategy.

    This year, our team wants to develop an engineered metabolic pathway that can produce phosphate using phosphite as the substrate. The strategy adopted to achieve this goal is co-expression of phosphite dehydrogenase and polyphosphate kinase from the lac promoter in plasmid pBBR1MCS2 [1]. Although phosphite dehydrogenase catalyzes the oxidation of phosphite to phosphate, solo overexpression of this enzyme in chassis cannot realize the manufacture of phosphate. Because once the amount of oxidized phosphite is sufficient to support the growth of chassis, it would not put more effort in this process. Consistent with previous reports, overexpression of phosphite dehydrogenase derived from Ralstonia sp. strain 4506 (RPD) [2] in chassis E. coli K12 confirmed this biological phenomenon (Figures 1 and 2).

    The key to the success of our strategy is polyphosphate kinase, which catalyzes the synthesis of polyphosphate (polyP) using ATP as the substrate [3, 4]. When this enzyme is overexpressed in E. coli K12, a significant amount of intracellular ATP will be redirected into polyP synthesis, during which the terminal phosphate residue from ATP will be deprived and sequestered in the form of polyP. However, regeneration of ATP depends on the assimilation of free phosphate. Under such circumstances, the chassis will be pushed to oxidize more phosphite, favoring its cellular ATP regeneration if phosphite is the only available phosphorus source.

    Given that polyphosphate kinase is the key player in our engineered metabolic pathway, we spent a lot of time on it at the beginning of our project, aiming at obtaining a highly active version of this enzyme (please refer to our new Part BBa_K4257000 for detailed information) [5]. After we got this done by site-directed mutagenesis of native polyphosphate kinase from E. coli, we naturally cloned aforementioned RPD behind this mutant polyphosphate kinase (PPK-M) in pBBR1MCS2 (Figures 3 and 4).

    The effect of our strategy was then tested by supernatant phosphite determination and polyP staining soon after we got the engineered E. coli K12 that harbors pBBR1MCS2/PPK-M+RPD. When we found that K12/PPK-M+RPD consumed more phosphite and produced intracellular polyP granules (Figure 5), we knew that our strategy works, because polyP is the polymer of phosphate and more polyP equals to more final product.

    However, things changed after we performed collaboration with team NNU-China. During interacting, we learned from them that the order of configuration for the same genes in expression vector may affect the final outcomes [6]. As far as our design was concerned, another order of configuration, bringing RPD prior to PPK-M, had not been tested yet. Given that phosphite oxidation is the first step in our designed pathway, the configuration RPD+PPK-M matches the order of such metabolic flux much better. Therefore, we started to rebuild our expression system by constructing pBBR1MCS2/RPD+PPK-M (Figure 6).

    Eventually, it turns out that K12/RPD+PPK-M performs better than K12/PPK-M+RPD. We confirmed the success of Engineering by subjecting two engineered strains to quantitative assessment and comparison at different levels, including the gene transcription, biomass yield, phosphite utilization, and intracellular polyP accumulation.

    Transcriptional analysis indicated that the gene placed in front showed elevated levels of expression (Figure 7).

    Quantification of biomass yield (expressed as maximum OD600) showed that, as far as growth of engineered strains was concerned, both designs had no discriminable differences (Figure 8).

    Under such circumstances, K12/RPD+PPK-M consumed approximately 25% more supernatant phosphite than K12/PPK-M+RPD (Figure 9), indicating an elevated intracellular polyP yield.

    In consistent with biomass and supernatant phosphite tests, the redesigned K12/RPD+PPK-M indeed produced more intracellular polyP, which can be distinguished by the naked eye after the cells were stained by toluidine blue (Figure 10).

[1] M.E. Kovach, P.H. Elzer, D.S. Hill, G.T. Robertson, M.A. Farris, R.M. Roop II, K.M. Peterson, Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes, Gene 166(1) (1995) 175-176.
[2] R. Hirota, S.-t. Yamane, T. Fujibuchi, K. Motomura, T. Ishida, T. Ikeda, A. Kuroda, Isolation and characterization of a soluble and thermostable phosphite dehydrogenase from Ralstonia sp. strain 4506, Journal of Bioscience Bioengineering 113(4) (2012) 445-450.
[3] N.N. Rao, M.R. Gómez-García, A. Kornberg, Inorganic polyphosphate: essential for growth and survival, Annual Review of Biochemistry 78 (2009) 605-647.
[4] M.R. Brown, A. Kornberg, The long and short of it–polyphosphate, PPK and bacterial survival, Trends in Biochemical Sciences 33(6) (2008) 284-290.
[5] A.K. Rudat, A. Pokhrel, T.J. Green, M. Gray, Mutations in Escherichia coli polyphosphate kinase that lead to dramatically increased in vivo polyphosphate levels, Journal of Bacteriology 200(6) (2018) e00697-17.
[6] A. Lechner, E. Brunk, J.D. Keasling, The need for integrated approaches in metabolic engineering, Cold Spring Harbor Perspectives in Biology 8(11) (2016) a023903.