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.

Oxidative stress resistance prompts pyrroloquinoline quinone biosynthesis in Hyphomicrobium denitrificans H4-45

“…strain was mutated by UV, NTG, EMS, and LiCl-UV for 11 consecutive generations, and the PQQ concentration increased from 11.21 to 19.33 mg/L (Zhao et al 2021 ). By integrating ARTP mutagenesis and methanol tolerance of ALE, an H. denitrificans mutant strain was obtained to increase PQQ production from 29.6 to 69.5 mg/L (Ren et al 2023 ). In this study, an H. denitrificans strain isolated by our lab was mutated by integrating UV-LiCl mutagenesis and ALE with oxidative stress, and the PQQ production increased from 17.14 to 30.96 mg/L.…”

“…Methanol dehydrogenase (MDH) in methylotrophic bacteria catalyzes the first step of methanol utilization, and PQQ serves as a crucial coenzyme for MDH (Keltjens et al 2014 ). Among them, H. denitrificans showed great potential for PQQ production and has been used as the only industrial strain to produce PQQ by several biopharmaceutical companies (Ren et al 2023 ). However, the concentration and productivity of PQQ were relatively low in the wild strain.…”

“…ALE is an increasingly popular technique for applying pressure on a microbial population to promote the acquisition of mutations that will improve cell fitness (Sandberg et al 2019 ). A combined strategy composing mutagenesis approaches and ALE has improved the production of PQQ (Ren et al 2023 ) and many other compounds, including astaxanthin (Jiang et al 2020 ), 1,3-propanediol (Yun et al 2022 ), 4-hydroxyphenylacetic acid (Shen et al 2022b ), and S-adenosyl-L-methionine (Weng et al 2022 ). The mutations and selections are important due to the potential of breaking the limits of increasing PQQ productivity and could obtain an inherent property of genetic material.…”