Ren Yang, Yang Xinwei, Ding Lingtao, Liu Dongfang, Tao Yong, Huang Jianzhong, Ke Chongrong
National and Local United Engineering Research Center of Industrial Microbiology and Fermentation Technology, Engineering Research Center of Industrial Microbiology, Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, 350117, Fujian, China.
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, 100101, China.
Biotechnol Biofuels Bioprod. 2023 Jan 19;16(1):11. doi: 10.1186/s13068-023-02261-y.
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.
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.
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.
吡咯喹啉醌（PQQ）是细菌脱氢酶的一种辅因子，与线粒体生成、繁殖、生长和衰老等生物学过程相关。由于化学合成成本极高且微生物合成产率低，筛选有效的菌株并开发提高PQQ产量的动态发酵策略是满足大规模工业需求的重要举措。
通过整合常压室温等离子体（ARTP）诱变、适应性实验室进化和高通量筛选策略，获得了一株高产PQQ的突变菌株——脱氮生丝微菌FJNU-A26。随后，采用单因素法和响应面法对发酵培养基进行系统优化，使PQQ浓度从1.02 g/L提高到1.37 g/L。利用qRT-PCR进行转录分析表明，当应用ARTP-ALE衍生的突变体时，参与PQQ生物合成的基因表达显著上调。此外，引入了一种新的两阶段pH控制策略来解决pH值对细胞生长和PQQ产量影响不一致的问题。这些综合策略使PQQ浓度比初始菌株FJNU-6提高了148%，在5 L发酵罐中进行144 h补料分批发酵后，PQQ浓度达到1.52 g/L，产量为40.3 mg/g DCW。
上述特性表明FJNU-A26是工业生产PQQ的有效候选菌株，且这些综合策略可轻松扩展到其他微生物用于大规模生产PQQ。
