2KGA Production Research Articles

The Functional Characterization of the 6-Phosphogluconate Dehydratase Operon in 2-Ketogluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01

Genus Pseudomonas bacteria mainly consume glucose through the Entner–Doudoroff (ED) route due to a lack of a functional Embden–Meyerhof–Parnas (EMP) pathway. In the present study, a 6-phosphogluconate dehydratase (edd) operon in the ED route was well investigated to find its structural characteristics and roles in the regulation of glucose consumption and 2-ketogluconic acid (2KGA) metabolism in the industrial 2KGA-producer P. plecoglossicida JUIM01. The edd operon contained four structural genes of edd, glk, gltR, and gtrS, encoding 6-PG dehydratase Edd, glucokinase Glk, response regulatory factor GltR, and histidine kinase GtrS, respectively. A promoter region was observed in the 5′-upstream of the edd gene, with a transcriptional start site located 129 bp upstream of the edd gene and in a pseudo-palindromic sequence of 5′-TTGTN7ACAA-3′ specifically binding to the transcription factor HexR. The knockout of the edd gene showed a remarkably negative effect on cell growth and re-growth using 2KGA as a substrate, beneficial to 2KGA production, with an increase of 8%. The deletion of glk had no significant effect on the cell growth or glucose metabolism, while showing an adverse impact on the 2KGA production, with a decrease of 5%. The outputs of the present study would provide a theoretical basis for 2KGA-producer improvement with metabolic engineering strategies and the development and optimization of P. plecoglossicida as the chassis cells.

Exploring the response mechanism of Pseudomonas plecoglossicida to high-temperature stress by transcriptomic analyses for 2-keto gluconic acid production

High temperatures, particularly in summer, lead to decreased yields in the industrial application of Pseudomonas plecoglossicida for 2-keto gluconic acid (2KGA) fermentation. To address this, the alterations in the transcriptomics of P. plecoglossicida in response to high-temperature stress were examined at temperatures of 32 °C, 36 °C, and 40 °C. The analysis of differential expression revealed substantial discrepancies in the number of differentially expressed genes (DEGs) at 36 °C (357) and 40 °C (1,487), primarily affecting vital biological functions. Elevated temperatures resulted in a shift in the metabolic processing of glucose, transitioning from extracellular oxidation to intracellular phosphorylation. Notable changes were observed in metabolic pathways, including the pentose phosphate pathway and tricarboxylic acid cycle. A significant observation was the decline in the activity of genes associated with extracellular glucose oxidation, accompanied by an increase in the activity of genes involved in intracellular phosphorylation pathway. This indicates a prompt and dynamic response to high-temperature stress. The investigation revealed notable alterations in genes linked to glucose metabolism, emphasizing the strain's adaptive capabilities to endure high temperatures. The reveal of adaptations are crucial for optimizing 2KGA production in challenging industrial environments.

Recent Advances in 2-Keto-l-gulonic Acid Production Using Mixed-Culture Fermentation and Future Prospects.

Vitamin C, also known as ascorbic acid, is an essential vitamin that cannot be synthesized by the human body and must be acquired through our diet. At present, the precursor of vitamin C, 2-keto-l-gulonic acid (2-KGA), is typically produced via a two-step fermentation process utilizing three bacterial strains. The second step of this traditional two-step fermentation method involves mixed-culture fermentation employing 2-KGA-producing bacteria (Ketogulonicigenium vulgare) along with associated bacteria. Because K. vulgare has defects in various metabolic pathways, associated bacteria are needed to provide key substances to promote K. vulgare growth and 2-KGA production. Unlike previous reviews where the main focus was the interaction between associated bacteria and K. vulgare, this Review presents the latest scientific research from the perspective of the metabolic pathways associated with 2-KGA production by K. vulgare and the mechanism underlying the interaction between K. vulgare and the associated bacteria. In addition, the dehydrogenases that are responsible for 2-KGA production, the 2-KGA synthesis pathway, strategies for simplifying 2-KGA production via a one-step fermentation route, and, finally, future prospects and research goals in vitamin C production are also presented.

2-Keto-gluconate production and purification by thermotolerant acetic acid bacterium Nguyenibacter vanlangensis KKS-R1

2-Keto-gluconic acid (2-KGA) is a pivotal intermediate in the production process of ascorbic acid, commonly known as vitamin C. The bacterium Nguyenibacter vanlangensis KKS-R1 is an acetic acid bacterium (AAB) that has been selectively screened for its ability to produce 2-KGA among twenty-five bacterial isolates at high temperatures up to 40°C. This thermotolerant property makes it advantageous for various industries. Therefore, it has garnered interest for its potential application in the production of 2-KGA and purification of the 2-KGA product. This study evaluated the production of 2-KGA by N. vanlangensis KKS-R1 using 1% gluconate as the substrate. The 2-KGA was determined by thin-layer chromatography, Lanning and Cohen’s method, and high-performance liquid chromatography. After fermentation in a minimal liquid medium for 24 hours, the bacterium showcased its prowess by producing 2-KGA concentrations of 7.5 g/L. However, the purity of the 2-KGA production is paramount for industrial applications. In addressing this, a rigorous chromatographic purification regimen was employed. This involved two sequential stages of ion exchange chromatography, DEAE-Sephacel and DOWEX 1X4, followed by SuperdexTM S-200 column chromatography. The collective efficacy of these methodologies yielded a product with an impressive purity index of 71.02%. The 2-KGA product was then subjected to a decolorization process using activated carbon and freeze-dried. This results in the compound being a pure white powder. This investigation indicates that N. vanlangensis KKS-R1 shows good potential as a 2-KGA producer at high temperatures. Under proper purification techniques. These findings contribute to developing purified processes and producing 2-KGA for industrial efficiency.

Symbiosis Mechanism of Associated Bacteria on 2-keto-L-gulonic Acid Production via Mixed Fermentation: A Review

Vitamin C, a water-soluble vitamin with strong reducing power, cannot be synthesized by the human body and participates in a variety of important biochemical reactions. Vitamin C is widely used in the pharmaceutical, food, health care, beverage, cosmetics, and feed industries, with a huge market demand. The classical two-step fermentation method is the mainstream technology for vitamin C production. D-sorbitol is transformed into L-sorbose by Gluconobacter oxydans in the first step of fermentation; then, L-sorbose is transformed into 2-keto-L-gulonic acid (2-KGA) by a coculture system composed of Ketogulonicigenium vulgare and associated bacteria; and finally, 2-KGA is transformed into vitamin C through chemical transformation. The conversion of L-sorbose into 2-KGA in the second fermentation step is performed by K. vulgare. However, considering the slow growth and low 2-KGA production of K. vulgare when cultured alone, it is necessary to add an associated bacteria to stimulate K. vulgare growth and 2-KGA production. Although the mechanism by which the associated bacteria promote K. vulgare growth and 2-KGA production has extensively been studied, this remains a hot topic in related fields. Based on the latest achievements and research, this review summarizes the metabolic characteristics of K. vulgare and associated bacteria and elucidates the mechanism by which the associated bacteria promote the growth and 2-KGA production of K. vulgare.

Cascading and precise regulation of the selective bio-production of 2- or 5-ketogluconic acid from glucose with whole-cell catalysis technology

Ketogluconic acid is widely applied in several industries as the bio-precursor of vitamin C and tartaric acid, while the selective production of 2-kGA or 5-kGA from glucose is so far still a challenge for chemical and biotechnical processes.

Genomic Analysis of Pseudomonas asiatica JP233: An Efficient Phosphate-Solubilizing Bacterium.

The bacterium Pseudomonas sp. strain JP233 has been reported to efficiently solubilize sparingly soluble inorganic phosphate, promote plant growth and significantly reduce phosphorus (P) leaching loss from soil. The production of 2-keto gluconic acid (2KGA) by strain JP233 was identified as the main active metabolite responsible for phosphate solubilization. However, the genetic basis of phosphate solubilization and plant-growth promotion remained unclear. As a result, the genome of JP233 was sequenced and analyzed in this study. The JP233 genome consists of a circular chromosome with a size of 5,617,746 bp and a GC content of 62.86%. No plasmids were detected in the genome. There were 5097 protein-coding sequences (CDSs) predicted in the genome. Phylogenetic analyses based on genomes of related Pseudomonas spp. identified strain JP233 as Pseudomonas asiatica. Comparative pangenomic analysis among 9 P. asiatica strains identified 4080 core gene clusters and 111 singleton genes present only in JP233. Genes associated with 2KGA production detected in strain JP233, included those encoding glucose dehydrogenase, pyrroloquinoline quinone and gluoconate dehydrogenase. Genes associated with mechanisms of plant-growth promotion and nutrient acquisition detected in JP233 included those involved in IAA biosynthesis, ethylene catabolism and siderophore production. Numerous genes associated with other properties beneficial to plant growth were also detected in JP233, included those involved in production of acetoin, 2,3-butanediol, trehalose, and resistance to heavy metals. This study provides the genetic basis to elucidate the plant-growth promoting and bio-remediation properties of strain JP233 and its potential applications in agriculture and industry.

Development of efficient 5-ketogluconate production system by Gluconobacter japonicus.

5-Ketogluconate (5KGA) is a precursor for synthesizing tartrate, a valuable compound used in several industries. In a previous study, Gluconobacter japonicus NBRC 3271 mutant strain D2, which lacks two membranous gluconate 2-dehydrogenases, was shown to produce 5KGA but not 2-ketogluconate from a mixture of glucose and gluconate. In this study, we aimed to develop an efficient 5KGA production system using G. japonicus D2 as the parental strain. D2 produced 5KGA from glucose in a jar fermentor culture; however, 5KGA levels were reduced during the late phase of cultivation. To increase the potential of D2 for 5KGA production, the cytoplasmic metabolism related to the utilization of 5KGA and gluconate was modified; the gno and gntK genes encoding 5KGA reductase and gluconokinase, respectively, were deleted from D2, generating D4. Improved 5KGA production was observed in D4 compared to that in D2, but a significant amount of gluconate remained at the end of cultivation, leading to an unsatisfied yield of 0.83mol (mol glucose)-1. The conversion of gluconate to 5KGA is catalyzed by pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH), which easily forms an apoenzyme by releasing PQQ and calcium ions. Thus, the effects of CaCl2 addition to the culture medium on 5KGA production by D4 were investigated. We demonstrated that 1mM CaCl2 addition positively affected the maintenance of the PQQ-GLDH activity toward gluconate and consequently enhanced 5KGA production, and the yield reached 0.97mol (mol glucose)-1. KEY POINTS: • An efficient 5KGA production system was developed with Gluconobacter japonicus. • Deleting the gno and gntK genes blocked the catabolism of 5KGA and gluconate. • The addition of 1mM CaCl2 efficiently improved the conversion of glucose to 5KGA.

Characterization of a transcriptional regulator PtxS from Pseudomonas plecoglossicida for regulating 2-ketogluconic acid metabolism

Homologs of PtxS are ubiquitous transcriptional regulators controlling the expression of the glucose dehydrogenase and kgu operon to globally regulate the 2-ketogluconic acid (2KGA) metabolism in Pseudomonas. In the present study, a PtxS from a 2KGA industrial producer Pseudomonas plecoglossicida JUIM01 (PpPtxS) was heterologously expressed in E. coli BL21(DE3), then structurally and functionally characterized. The obtained results showed that PpPtxS was a 36.65-kDa LacI-family transcriptional regulator. 2KGA was the sole effector of PpPtxS. Glucose negatively affected the molecular binding of PpPtxS and 2KGA, and gluconic acid inhibited the PpPtxS-2KGA binding reaction. PpPtxS in water solution mainly existed as a dimer and bound to two molecules of 2KGA. The effector 2KGA mainly bound to the region close to the C-terminal of PpPtxS by interacting with the 299th to the 301st amino acids (Ala, Gln, Pro, Thr, Glu and Arg). PpPtxS specifically recognized and bound to a 14-bp palindrome sequence (5′-TGAAACCGGTTTCA-3′) due to its conserved HTH motif at the N-terminal. The characterization of PpPtxS in this study would provide a theoretical guidance for the industrial production of 2KGA.

2-Keto-D-gluconic acid and prodigiosin producing by a Serratia marcescens

Microbial fermentation has become the main method to produce target compound. In this study, a 2-Keto-D-gluconic acid (2-KGA) producing mutant strain was obtained by mutation with rational screening methods. Meanwhile, prodigiosin was produced when the nitrogen source in the medium was changed to peptone and its fermentation conditions were evaluated to achieve high-efficient accumulation. The mutant strain SDSPY-136 was firstly identified as Serratia marcescens by morphological observation and 16S rDNA sequencing. The 2-KGA synthetic capacity of S. marcescens SDSPY-136 was evaluated by shake fermentation with 110 g/L glucose as substrates. For fermentation, 2-KGA yield, conversation rate and purity of SDSPY-136 reached 104.60 g/L, 95.10%, 99.11% in 72 h. The red pigment was extracted from the fermentation broth using acidic methanol and identified as prodigiosin by FT-IR. The optimal conditions were as follows: glycerol 20 g/L, peptone 20 g/L, MgSO415 g/L, pH 6.0, a 2% (v/v) inoculum, 30 °C and 200 rpm of shaking culture. Eventually, prodigiosin reached a yield of 9.89 g/Lafter shake culturing for 50 h under this condition. The mutant S. marcescens SDSPY-136 was shown to be promising for 2-KGA and prodigiosin production and a suitable object for prodigiosin metabolism research of S. marcescens.

A 2-ketogluconate kinase KguK in Pseudomonas plecoglossicida JUIM01: Enzymatic characterization and its role in 2-keto-d-gluconic acid metabolism

Our previous study has revealed that Pseudomonas plecoglossicida JUIM01 produced 2-keto-d-gluconic acid (2KGA) and re-utilized 2KGA as an alternative carbon source to support cell growth after complete consumption of glucose. Phosphorylation of intracellular 2KGA to 2-keto-6-phosphogluconic acid by 2-ketogluconate kinase (KguK) was regarded as the first step of 2KGA catabolism in Pseudomonas cytoplasm. In the present study, a kguK gene encoding 2-ketogluconate kinase from P. plecoglossicida JUIM01 was cloned and heterologously expressed in Pichia pastoris. The recombinant KguK showed the highest activity at 30–33 °C and pH 7.7, and high stability at 33 °C. Under the optimal conditions of 30 °C and pH 7.7 with addition of 5 mM Mg2+, the purified and concentrated (~30 folds) KguK had a specific activity of 3649.6 U/g and a Michaelis constant for 2KGA of 8.7 × 10−4 M. Knockout of kguK could retard but not completely inhibit 2KGA catabolism, indicating other existing 2KGA utilization pathway(s). The kguK-knockout P. plecoglossicida significantly reduced 2KGA re-utilization without negative effects on cell growth, glucose consumption or 2KGA production. The outputs demonstrated kguK knockout could be an effective strategy to develop the alternative 2KGA high-producing Pseudomonas strains to avoid the decrease of 2KGA yield caused by its re-utilization.

Production of 2-keto-gluconic acid from glucose by immobilized Pseudomonas plecoglossicida resting cells.

2-Keto-d-gluconic acid (2KGA) is an important organic acid derived from d-glucose and is used to produce the food antioxidant erythorbic acid. To improve the 2KGA production performance and cell reusability, various carriers such as calcium alginate, k-carrageenan, chitosan, and poly(vinyl alcohol)-alginate were evaluated to immobilize Pseudomonas plecoglossicida JUIM01 resting cells. Calcium alginate was shown to be a suitable carrier since the immobilized cells had the highest number of reuse times and produced the highest 2KGA concentration of 171.77g/L, with a productivity of 3.58g/L·h and conversion ratio of 98.38%. The cell concentration, cultivation temperature, aeration rate and initial glucose concentration were further optimized in a 5-L airlift bioreactor to obtain the best 2KGA production performance by calcium alginate-immobilized P. plecoglossicida cells. Under the optimal conditions including a cell concentration of 4.0g/L, glucose concentration of 126.0g/L, temperature of 34°C and aeration rate of 2.8 L/min, 134.45g/L 2KGA was produced by alginate-immobilized P. plecoglossicida cells within 30h, with a total productivity of 4.48g/L·h and yield of 1.07g/g (conversion ratio of over 99.0%). The immobilized cells maintained a stable conversion capacity after nine reuses and 25days of storage at 4°C, which indicated that calcium alginate immobilization of P. plecoglossicida cells had industrial practicability for 2KGA production.

Directing cell catalysis of glucose to 2-keto-d-gluconic acid using Gluconobacter oxydans NL71

2-Keto-d-gluconic acid (2-KGA) is a high-value-added product from lignocellulosic straw-based biorefining process obtained via cell catalysis using Gluconobacter oxydans (G. oxydans); nonetheless, the economical production has some limitations because of the formation of undesirable mixture products of keto-gluconic acid from glucose. In this study, it was demonstrated that pulse addition of hydrogen peroxide (H2O2) to synthetic media was beneficial to improve the conversion rate of glucose to 2-KGA by 1.47-fold in G. oxydans. Moreover, some lignocellulosic degradation compounds served as substitute and played the role of H2O2 on corn stover hydrolysate for the production of 2-KGA. By conducting qRT-PCR analysis, eight dehydrogenase genes were identified in G. oxydans, which were responsible for regulating 2-KGA production. Moreover, most of the identified genes were found to produce membrane-bound glucose dehydrogenase and gluconate 2-dehydrogenase, and were up-regulated by adding H2O2 or degradation compounds. These up-regulated genes could be responsible for directing cell catalysis of glucose to 2-KGA in G. oxydans.

Two-Stage Semi-Continuous 2-Keto-Gluconic Acid (2KGA) Production by Pseudomonas plecoglossicida JUIM01 From Rice Starch Hydrolyzate

A two-stage semi-continuous strategy for producing 2-keto-gluconic acid (2KGA) by Pseudomonas plecoglossicida JUIM01 from rice starch hydrolyzate (RSH) has been developed. The initial glucose concentration (140 g/L) was selected for first-stage fermentation due to its highest 2KGA productivity of 7.58 g/(L⋅h), cell weight of 3.91 g/L, and residual glucose concentration of 25.00 g/L. Followed by removing 70.0% (v/v) of the first-stage broth and feeding 400.0 g/L of glucose to the second-stage fermentor, a total of 50680.0 g glucose was consumed, and 50005.20 g 2KGA was obtained with a yield of 0.9867 g/g by P. plecoglossicida JUIM01 after a 3-cycle two-stage semi-continuous fermentation. Our results indicated that the developed two-stage semi-continuous fermentation could be industrially applied due to its high 2KGA concentration, 2KGA yield and operation efficiency.

Efficient biosynthesis of 2-keto-D-gluconic acid by fed-batch culture of metabolically engineered Gluconobacter japonicus

2-keto-d-gluconic acid (2-KGA) is a key precursor for synthesising vitamin C and isovitamin C. However, phage contamination is as constant problem in industrial production of 2-KGA using Pseudomonas fluorescens. Gluconobacter holds promise for producing 2-KGA due to impressive resistance to hypertonicity and acids, and high utilisation of glucose. In this study, the 2-KGA synthesis pathway was regulated to enhance production of 2-KGA and reduce accumulation of the by-products 5-keto-d-gluconic acid (5-KGA) and d-gluconic acid (D-GA) in the 2-KGA producer Gluconobacter japonicus CGMCC 1.49. Knocking out the ga5dh-1 gene from a competitive pathway and overexpressing the ga2dh-A gene from the 2-KGA synthesis pathway via homologous recombination increased the titre of 2-KGA by 63.81% in shake flasks. Additionally, accumulation of 5-KGA was decreased by 63.52% with the resulting G. japonicas-Δga5dh-1-ga2dh-A strain. Using an intermittent fed-batch mode in a 3 L fermenter, 2-KGA reached 235.3 g L−1 with a 91.1% glucose conversion rate. Scaling up in a 15 L fermenter led to stable 2-KGA titre with productivity of 2.99 g L−1 h−1, 11.99% higher than in the 3 L fermenter, and D-GA and 5-KGA by-products were completely converted to 2-KGA.

Biosynthesis of α-lipoic acid in Gluconobacter oxydans increases the production of vitamin C by one-step fermentation

In a one-step fermentation system of vitamin C production with Gluconobacter oxydans and Ketogulonicigenium vulgare, a functional module of α-lipoic acid biosynthesis was constructed in G. oxydans. The engineered G. oxydans was co-cultured with K. vulgare to enhance the growth and 2-keto-L-gulonic acid (2-KGA) production of K. vulgare. This one-step fermentation system alleviated the growth inhibition during the mono-culture of K. vulgare and strengthened the interaction between the two bacteria. Moreover, the yield of vitamin C precursor (2-KGA) increased to 73.34 g/L (the control group was 59.09 g/L), and the conversion of D-sorbitol to 2-KGA increased to 86.0%. This study provides a new idea for further optimizing the one-step fermentation system of vitamin C production.

A Membrane-Bound Gluconate Dehydrogenase from 2-Keto-d-Gluconic Acid Industrial Producing Strain Pseudomonas plecoglossicida JUIM01: Purification, Characterization, and Gene Identification

The membrane-bound gluconate dehydrogenase (mGADH) is a critical enzyme for 2-keto-D-gluconic acid (2KGA) production in Pseudomonas plecoglossicida JUIM01. The purified native flavin adenine dinucleotide-dependent mGADH (FAD-mGADH) was consisted of a gamma subunit, a flavoprotein subunit, and a cytochrome c subunit with molecular mass of ~ 27, 65, and 47kDa, respectively. The specific activity of FAD-mGADH was determined as 90.71U/mg at optimum pH and temperature of 6.0 and 35°C. The Km and Vmax values of calcium D-gluconate were 0.631mM and 0.734mM/min. The metal ions Mg2+ and Mn2+ showed slight positive effects on FAD-mGADH activity. On the other hand, a 3868-bp-length gad gene cluster was amplified and expressed in Escherichia coli BL21(DE3). The recombinant protein showed the same molecular weight and enzyme activity as the native FAD-mGADH, which confirmed it as a FAD-mGADH encoding gene. The flavoprotein subunit and the cytochrome c subunit containing a putative FAD-binding motif and three possible heme-binding motifs concluded from alignment results of mGADHs. This study characterized the native and recombinant FAD-mGADH and would provide the basis for further genetic modification of Pseudomonas plecoglossicida JUIM01 with the intention of 2KGA productivity improvement.

Integrated proteomic and metabolomic analysis of a reconstructed three-species microbial consortium for one-step fermentation of 2-keto-L-gulonic acid, the precursor of vitamin C.

Microbial consortia, with the merits of strong stability, robustness, and multi-function, played critical roles in human health, bioenergy, and food manufacture, etc. On the basis of 'build a consortium to understand it', a novel microbial consortium consisted of Gluconobacter oxydans, Ketogulonicigenium vulgare and Bacillus endophyticus was reconstructed to produce 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. With this synthetic consortium, 73.7g/L 2-KGA was obtained within 30h, which is comparable to the conventional industrial method. A combined time-series proteomic and metabolomic analysis of the fermentation process was conducted to further investigate the cell-cell interaction. The results suggested that the existence of B. endophyticus and G. oxydans together promoted the growth of K. vulgare by supplying additional nutrients, and promoted the 2-KGA production by supplying more substrate. Meanwhile, the growth of B. endophyticus and G. oxydans was compromised from the competition of the nutrients by K. vulgare, enabling the efficient production of 2-KGA. This study provides valuable guidance for further study of synthetic microbial consortia.

Reconstruction and analysis of carbon metabolic pathway of Ketogulonicigenium vulgare SPU B805 by genome and transcriptome

Ketogulonicigenium vulgare has been widely used in vitamin C two-step fermentation. Four K. vulgare strains (WSH-001, Y25, Hbe602 and SKV) have been completely genome-sequenced, however, less attention was paid to elucidate the reason for the differences in 2-KGA yield on genetic level. Here, a novel K. vulgare SPU B805 with higher 2-keto-L-gulonic acid (2-KGA) yield, was genome-sequenced to confirm harboring one circular chromosome with plasmid free. Comparative genome analyses showed that the absence of plasmid 2 was an important factor for its high 2-KGA productivity. The amino acid biosynthetic pathways in strain SPU B805 are much more complete than those in other K. vulgare strains. Meanwhile, strain SPU B805 harbored a complete PPP and TCA route, as well as a disabled EMP and ED pathway, same as to strain SKV, whereas strain WSH-001, Y25 and Hbe602 harbored complete PPP, ED, TCA pathway and a nonfunctional EMP pathway. The transcriptome of strain SPU B805 validated the carbon metabolism in cytoplasm mainly through the PPP pathway due to its higher transcriptional levels. This is the first time to elucidate the underlying mechanism for the difference in 2-KGA yield, and it is of great significance for strain improvement in the industrial fermentation.

Enhancing 2-Ketogluconate Production of Pseudomonas plecoglossicida JUIM01 by Maintaining the Carbon Catabolite Repression of 2-Ketogluconate Metabolism.

2-Ketogluconate (2KGA) is an organic acid that is important for pharmaceutical, cosmetic, and environmental applications. Pseudomonas plecoglossicida JUIM01 strain is an important industrial 2KGA producer in China. In this paper, we found that P. plecoglossicida JUIM01 could convert glucose to 2KGA extracellularly, and the formed 2KGA was subsequently consumed after glucose was exhausted during the fermentation process. Experiments of glucose and 2KGA supplementation during fermentation process revealed that, only when glucose was exhausted, the strain started to consume the product 2KGA. Then, the mechanism of this phenomenon was investigated at transcription and protein levels, and the results indicated that P. plecoglossicida JUIM01 possesses carbon catabolite repression of 2KGA metabolism by glucose. Next, increasing the supply of glucose could attenuate 2KGA consumption and enhance the 2KGA yield from glucose. Finally, fed-batch fermentation of P. plecoglossicida JUIM01 resulted in 205.67 g/L of 2KGA with a productivity of 6.86 g/L/h and yield of 0.953 g/g glucose. These results can provide references for the industrial fermentation production of 2KGA and other fermentation products.