Comparison of Three Xylose Pathways in Pseudomonas putida KT2440 for the Synthesis of Valuable Products

Abstract

Pseudomonas putida KT2440 is a well-established chassis in industrial biotechnology. To increase the substrate spectrum, we implemented three alternative xylose utilization pathways, namely the Isomerase, Weimberg, and Dahms pathways. The synthetic operons contain genes from Escherichia coli and Pseudomonas taiwanensis. For isolating the Dahms pathway in P. putida KT2440 two genes (PP_2836 and PP_4283), encoding an endogenous enzyme of the Weimberg pathway and a regulator for glycolaldehyde degradation, were deleted. Before and after adaptive laboratory evolution, these strains were characterized in terms of growth and synthesis of mono-rhamnolipids and pyocyanin. The engineered strain using the Weimberg pathway reached the highest maximal growth rate of 0.30 h^-1. After adaptive laboratory evolution the lag phase was reduced significantly. The highest titers of 720 mg L^-1 mono-rhamnolipids and 30 mg L^-1 pyocyanin were reached by the evolved strain using the Weimberg or an engineered strain using the Isomerase pathway, respectively. The different stoichiometries of the three xylose utilization pathways may allow engineering of tailored chassis for valuable bioproduct synthesis.

Keywords: Pseudomonas putida; flux balance analysis; heterologous production; metabolic engineering; phenazine; pyocyanin; rhamnolipid; xylose.

Figure 1

Introduction of xylose metabolism pathways into the central carbon metabolism of P. putida KT2440 with the presumed endogenous enzymes and heterologous enzymes from E. coli DH5α and P. taiwanensis VLB120. The blue lines indicate the central carbon metabolism based on (Sudarsan et al., 2014). The PP numbers represent the locus tag in P. putida KT2440, the PVLB numbers represent the locus tag in P. taiwanensis VLB120, and the b numbers represent the locus tag in E. coli K12, of which E. coli DH5α is derived from. ADP, adenosine diphosphate; ATP, adenosine triphosphate; NAD⁺, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; PQQ, pyrroloquinoline quinone; PQQH₂, reduced pyrroloquinoline quinone.

Figure 2

Growth of P. putida KT2440, its derivate xylose consuming strains, and the xylose-adapted strains in minimal medium containing 10 g L⁻¹ xylose. Consumption of xylose (, dark gray), formation of xylonate (, light gray), and cell dry weight (•, black) of (A) P. putida KT2440, (B) P. putida KT2440 pIso, (C) P. putida KT2440 pWeim, (D) P. putida KT2440ΔΔ pDahms, (E) P. putida KT2440 pWeim2, and (F) P. putida KT2440ΔΔ pDahms2. Error bars indicate deviation from the mean (n = 3).

Figure 3

ALE and growth characteristics of isolates of P. putida KT2440 pWeim on xylose. (A) ALE of P. putida KT2440 pWeim on xylose as sole carbon source. A single representative culture is shown, (B) Comparison of growth for P. putida KT2440 pWeim (•, green) and five isolates from the second ALE culture on xylose. Error bars indicate deviation from the mean (n = 3).

Figure 4

Depiction of the labeling of metabolites resulting from labeled xylose. Shown is the stable isotope introduction into the central carbon metabolism of P. putida KT2440. Orange lines indicate the Dahms pathway, green lines indicate the Weimberg pathway, and dashed lines indicate several enzymatic steps. Orange dots indicate the labeling resulting from the Dahms pathway and green dots indicate the labeling resulting from the Weimberg pathway.

Figure 5

Fractional labeling of amino acids during growth in minimal medium containing 50% 1-¹³C-xylose. Error bars indicate deviation from the mean of three technical replicates from one growth experiment (n = 3). Ala, alanine; Glx, glutamate and deaminated glutamine; Lys, lysine; Pro, proline; Val, valine.

Figure 6

Mono-rhamnolipid and pyocyanin production from xylose by engineered P. putida strains. The minimal medium contained 10 g L⁻¹ xylose. Shown are the product titers of mono-rhamnolipids (A), the product titers of pyocyanin (B), the yield of mono-rhamnolipids on xylose (C), and the yield of pyocyanin on xylose (D). Error bars indicate deviation from the mean (n = 3).