Improved Production of Pyrroloquinoline Quinone by Simultaneous Augmentation of Its Synthesis Gene Expression and Glucose Metabolism in Klebsiella pneumoniae

Mi, Zhiwei; Cheng, Jingchao; Zhao, Peng; Tian, Pingfang; Tan, Tianwei

doi:10.1007/s00284-020-01918-3

Cited by 6 publications

(2 citation statements)

References 38 publications

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“…Although the understanding of the PQQ biosynthetic pathway made it possible to achieve a high production of PQQ, only a limited increase was achieved by stimulating the expression of PQQ synthesis genes (Klinman and Bonnot 2014 ; Shen et al 2012 ; Zhu and Klinman 2020 ). Mi et al constructed a cassette with repetitive tac promoters to overexpress PQQ synthesis genes in Klebsiella pneumoniae , but PQQ production reached only 0.78 mg/L in a 5-L bioreactor (Mi et al 2020 ). Until now, there is no report of PQQ high-yield H. denitrificans strain by genetic manipulation due to the insufficient genetic engineering tools and the uncleared metabolic network and regulation mechanism of PQQ biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

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

Liang,

Tang,

Chen

et al. 2024

Appl Microbiol Biotechnol

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Pyrroloquinoline quinone (PQQ) is a natural antioxidant with diverse applications in food and pharmaceutical industries. A lot of effort has been devoted toward the discovery of PQQ high-producing microbial species and characterization of biosynthesis, but it is still challenging to achieve a high PQQ yield. In this study, a combined strategy of random mutagenesis and adaptive laboratory evolution (ALE) with fermentation optimization was applied to improve PQQ production in Hyphomicrobium denitrificans H4-45. A mutant strain AE-9 was obtained after nearly 400 generations of UV-LiCl mutagenesis, followed by an ALE process, which was conducted with a consecutive increase of oxidative stress generated by kanamycin, sodium sulfide, and potassium tellurite. In the flask culture condition, the PQQ production in mutant strain AE-9 had an 80.4% increase, and the cell density increased by 14.9% when compared with that of the initial strain H4-45. Moreover, batch and fed-batch fermentation processes were optimized to further improve PQQ production by pH control strategy, methanol and H2O2 feed flow, and segmented fermentation process. Finally, the highest PQQ production and productivity of the mutant strain AE-9 reached 307 mg/L and 4.26 mg/L/h in a 3.7-L bioreactor, respectively. Whole genome sequencing analysis showed that genetic mutations in the ftfL gene and thiC gene might contribute to improving PQQ production by enhancing methanol consumption and cell growth in the AE-9 strain. Our study provided a systematic strategy to obtain a PQQ high-producing mutant strain and achieve high production of PQQ in fermentation. These practical methods could be applicable to improve the production of other antioxidant compounds with uncleared regulation mechanisms. Key points • Improvement of PQQ production by UV-LiCl mutagenesis combined with adaptive laboratory evolution (ALE) and fermentation optimization. • A consecutive increase of oxidative stress could be used as the antagonistic factor for ALE to enhance PQQ production. • Mutations in the ftfL gene and thiC gene indicated that PQQ production might be increased by enhancing methanol consumption and cell growth.

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Section: Discussionmentioning

confidence: 99%

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

Liang,

Tang,

Chen

et al. 2024

Appl Microbiol Biotechnol

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“…Mi et al further overexpressed PQQ synthesis-related genes from three repeats of the tac promoter in a K. pneumoniae strain. After adding sufficient glucose to activate the direct glucose oxidation pathway, 0.78 mg/L PQQ was generated in a 5-L bioreactor [24]. Ye et al overexpressed the pqqABCDE gene cluster and tldD gene under the control of an endogenous constitutive promoter in a G. oxydans strain without pyruvate decarboxylase-encoding genes, and a PQQ yield of 51.32 ± 0.8997 mg/L was achieved after the optimization of carbon sources and culture conditions [25].…”

Section: Introductionmentioning

confidence: 99%

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

Ren

Yang²,

Ding³

et al. 2023

Biotechnol Biofuels

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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.

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Microbial synthesis of pyrroloquinoline quinone

Gao,

Wang,

Yang

et al. 2023

World J Microbiol Biotechnol

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Improved Production of Pyrroloquinoline Quinone by Simultaneous Augmentation of Its Synthesis Gene Expression and Glucose Metabolism in Klebsiella pneumoniae

Cited by 6 publications

References 38 publications

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

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

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

Microbial synthesis of pyrroloquinoline quinone

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