Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose

Abstract

Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose.

Keywords: Pseudomonas putida; metabolic engineering; sucrose metabolism.

Figure 1

Growth and sugar consumption/production of different P. putida strains grown in M9 minimal medium in shake flasks. OD (blue circles), sucrose (orange triangles), glucose (light blue diamonds), and fructose (red squares) were measured in each experiment. (a) P. putida (pSEVA224‐cscA) with 3 g/L sucrose. (b) P. putida (pSEVA224‐cscAB) with 3 g/L sucrose (c) P. putida eYFP with 1.5 g/L glucose and fructose each (d) P. putida eYFP with 3 g/L sucrose

Figure 2

Relative growth rates of different P. putida eYFP strains with genomic integration of a polycistronic cscAB construct via pSST2. Cells were grown in M9 medium with 3 g/L sucrose in a microplate reader and OD ₆₀₀ was measured every 20 min. Growth rates were determined in the log‐linear parts of the growth curves and normalized to the respective mean growth rate of each experiment. Standard deviations were calculated from these normalized growth rates of three independent experiments

Figure 3

Growth and sucrose consumption by (a) P. putida PP_0075::cscA and (b) P. putida PP_3398::cscAB. Cells were grown in M9 minimal medium in shake flasks with 3 g/L sucrose. OD (blue circles), sucrose (orange triangles), glucose (light blue diamonds), and fructose (red squares) were measured in each experiment. Shown are representative curves of three independent experiments

Figure 4

CscB production and localization in P. putida and E. coli. Confocal fluorescence microscopy of (a) P. putida (pVLT_gfp), (b) P. putida (pVLT_cscB‐GFP), (c) E. coli (pVLT_gfp), and (d) E. coli (pVLT_cscB‐GFP). Pictures were taken with an Olympus FluoView 1000. Arrows point to the cells where the membrane localization rings are most clear

Figure 5

Growth of C. necator wt (a), C. necator::miniTn5‐cscA (b), and C. necator::miniTn5‐cscAB (c) with either fructose (red dashed line), sucrose (light blue dotted line), or glucose (solid dark blue line), or glucose/fructose (orange dotted and dashed line) as the single carbon source. Note that the presence of CscA is sufficient to allow growth on sucrose, whereas the expression of cscB additionally allows growth on glucose

Figure 6

CscA activity of P. putida PP_0075::cscA. Sucrose cleavage rates were determined from the cell extracts, the culture supernatants, and the whole cells of P. putida PP_0075::cscA. The mean activity of the supernatants is shown in relation to the optical density at the time of harvest, mean activity of the whole cells and cell extracts in relation to the optical density of the cells used for the assay. The mean values and the corresponding standard deviations of three independent experiments are depicted