Adaptive evolutionary strategy coupled with an optimized biosynthesis process for the efficient production of pyrroloquinoline quinone from methanol

Abstract

Background: Pyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements.

Results: A high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room‑temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter.

Conclusion: The characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

Keywords: Adaptive laboratory evolution (ALE); Hyphomicrobium denitrificans; Pyrroloquinoline quinone; Response surface methodology (RSM); Two-stage pH control strategy.

Fig. 1

High-yield PQQ-producing strains were achieved using a high-throughput screening system after ARTP mutagenesis with ALE

Fig. 2.

3D response surface plots of interactions on the production of PQQ at p < 0.01. A 3D surface graph of AC interaction; B 3D surface graph of CD interaction; C 3D surface graph of BC interaction

Fig. 3

Fed-batch fermentation using a two-stage oxygen supply strategy in a 5-L fermentation tank. A Fed-batch fermentation by the wild strain FJNU-6; B fed-batch fermentation by the mutant strain FJNU-A26

Fig. 4

Gene transcription in the ARTP-ALE mutant strain FJNU-A26 and wild strain FJNU-6 in fed-batch fermentation. A Expression of the pqqABCDE operon in mutant strain FJNU-A26; B expression of five pqqA copies in mutant strain FJNU-A26; C expression of α subunits of five methanol dehydrogenases in mutant strain FJNU-A26; D changes in relative transcription levels between the mutant strain (M) and the wild strain (W)

Fig. 5

Effects of pH on the specific cell growth rate and the specific PQQ formation rate. A Effects of pH on the specific cell growth rate, μ_x; B effects of pH on the specific PQQ formation rate, μ_p

Fig. 6

Fed-batch fermentation of mutant FJNU-A26 using an integrated fermentation strategy in a 5-L fermentation tank