As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG.

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L-1 styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses. Physiological changes are one of the main factors responsible for the high OS tolerance in mutant strains. Moreover, the P-type ATPase GOX_RS04415 and the LysR family transcriptional regulator GOX_RS04700 were also verified as critical genes for styrene oxide tolerance. The tolerance mechanisms of OSs can be used in biocatalytic chassis cell factories to synthesize compounds and degrade environmental pollutants. This study provides new insights into the mechanisms underlying the toxicological response to OS stress and offers potential targets for enhancing the solvent tolerance of G. oxydans.

L-Sorbosone dehydrogenase (SNDH) is a key enzyme involved in the biosynthesis of 2-keto-L-gulonic acid , which is a direct precursor for the industrial scale production of vitamin C. Elucidating the structure and the catalytic mechanism is essential for improving SNDH performance. By solving the crystal structures of SNDH from Gluconobacter oxydans WSH-004, a reversible disulfide bond between Cys295 and the catalytic Cys296 residues is discovered. It allowed SNDH to switch between oxidation and reduction states, resulting in opening or closing the substrate pocket. Moreover, the Cys296 is found to affect the NADP+ binding pose with SNDH. Combining the in vitro biochemical and site-directed mutagenesis studies, the redox-based dynamic regulation and the catalytic mechanisms of SNDH are proposed. Moreover, the mutants with enhanced activity are obtained by extending substrate channels. This study not only elucidates the physiological control mechanism of the dehydrogenase, but also provides a theoretical basis for engineering similar enzymes.

Direct production of 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) from D-glucose through 2,5-diketo-D-gluconic acid (2,5-DKG) is a promising alternative route. To explore the pathway of producing 2-KLG from D-glucose, Gluconobacter oxydans ATCC9937 was selected as a chassis strain. It was found that the chassis strain naturally has the ability to synthesize 2-KLG from D-glucose, and a new 2,5-DKG reductase (DKGR) was found on its genome. Several major issues limiting production were identified, including the insufficient catalytic capacity of DKGR, poor transmembrane movement of 2,5-DKG and imbalanced D-glucose consumption flux inside and outside of the host strain cells. By identifying novel DKGR and 2,5-DKG transporter, the whole 2-KLG biosynthesis pathway was systematically enhanced by balancing intracellular and extracellular D-glucose metabolic flux. The engineered strain produced 30.5 g/L 2-KLG with a conversion ratio of 39.0%. The results pave the way for a more economical large-scale fermentation process for vitamin C.

In this study, the synthesis of xylonic acid from xylose by Gluconobacter oxydans NL71 has been investigated. According to the relationship between oxygen transfer rate and oxygen uptake rate, three different kinetic models of product formation were established and the nonlinear fitting was carried out. The results showed that G. oxydans has critical dissolved oxygen under different strain concentrations, and the relationship between respiration intensity and dissolved oxygen conformed to the Monod equation [Formula: see text]. The maximum reaction rate per unit cell mass and the theoretical maximum specific productivity of G. oxydans obtained by the kinetic model are 0.042 mol/L/h and 6.97 g/gx/h, respectively. These results will assist in determining the best balance between the airflow rate and cell concentration in the reaction and improve the production efficiency of xylonic acid.

Pre-hydrolysate liquor, as an inevitable by-product, contains a large amount of xylose, and is therefore an inexpensive feedstock that can be upgraded to value-added chemical xylonic acid. However, inhibitors, simultaneously formed in lignocellulose pretreatment process, are regarded as the major obstacle for effectively bio-converting xylose in pre-hydrolysate into xylonic acid. In this study, Gluconobacter oxydans, with highly selective and efficient, was employed for xylonic acid production; the impacts of five typical toxic inhibitory compounds on xylonic acid productivity and the activity of the membrane-bound dehydrogenase were evaluated. The results revealed that the inhibitors showed different degrees of influence toward xylonic acid production, and the order of inhibitory effect for acidic inhibitors was formic acid > acetic acid > levulinic acid; the inhibitory effect of aldehyde inhibitors was furfural > 5-hydroxymethyl-furfural. This study provides an important basis of metabolic modification and detoxification process for enhancing inhibitor tolerance and xylonic acid productivity.

BACKGROUND: (R)-mandelic acid (R-MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R-MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains.
RESULTS: Herein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R-MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans. With a new screened strong endogenous promoter P12780, the production of R-MA was improved to 10.26 g/L compared to 7.36 g/L of using Plac. As R-MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R-MA tolerance. The adapted strain that can tolerate 6 g/L R-MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R-MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R-MA stress condition, we identified five critical genes that were associated with R-MA tolerance, among which AcrA overexpression could further improve R-MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate.
CONCLUSIONS: The microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.

Gluconobacter oxydans, is used in biotechnology because of its ability to oxidize a wide variety of carbohydrates, alcohols, and polyols in a stereo- and regio-selective manner by membrane-bound dehydrogenases located in periplasmic space. These reactions obey the well-known Bertrand-Hudson\'s rule. In our previous study (BBA-General Subjects, 2021, 1865:129740), we discovered that Gluconobacter species, including G. oxydans and G. cerinus strain can regio-selectively oxidize the C-3 and C-5 hydroxyl groups of D-galactitol to rare sugars D-tagatose and L-xylo-3-hexulose, which represents an exception to Bertrand Hudson\'s rule. The enzyme catalyzing this reaction is located in periplasmic space or membrane-bound and is PQQ (pyrroloquinoline quinine) and Ca2+-dependent; we were encouraged to determine which type of enzyme(s) catalyze this unique reaction.
Enzyme was identified by complementation of multi-deletion strain of Gluconobacter oxydans 621H with all putative membrane-bound dehydrogenase genes.
In this study, we identified this gene encoding the membrane-bound PQQ-dependent dehydrogenase that catalyzes the unique galactitol oxidation reaction in its 3\'-OH and 5\'-OH. Complement experiments in multi-deletion G. oxydans BP.9 strains established that the enzyme mSLDH (encoded by GOX0855-0854, sldB-sldA) is responsible for galactitol\'s unique oxidation reaction. Additionally, we demonstrated that the small subunit SldB of mSLDH was membrane-bound and served as an anchor protein by fusing it to a red fluorescent protein (mRubby), and heterologously expressed in E. coli and the yeast Yarrowia lipolytica. The SldB subunit was required to maintain the holo-enzymatic activity that catalyzes the conversion of D-galactitol to L-xylo-3-hexulose and D-tagatose. The large subunit SldA encoded by GOX0854 was also characterized, and it was discovered that its 24 amino acids signal peptide is required for the dehydrogenation activity of the mSLDH protein.
In this study, the main membrane-bound polyol dehydrogenase mSLDH in G. oxydans 621H was proved to catalyze the unique galactitol oxidation, which represents an exception to the Bertrand Hudson\'s rule, and broadens its substrate ranges of mSLDH. Further deciphering the explicit enzymatic mechanism will prove this theory.

In this work, the effect of activated carbon particles on the production of xylonic acid from xylose by Gluconobacter oxydans in a stirred tank bioreactor was investigated. The enhancement of the oxygen transfer coefficient by activated carbon particles was experimentally evaluated under different solids volume fractions, agitation and aeration rates conditions. The experimental conditions optimized by response surface methodology (agitation speed 800 rpm, aeration rate 7 L min-1, and activated carbon 0.002%) showed a maximum oxygen transfer coefficient of 520.7 h-1, 40.4% higher than the control runs without activated carbon particles. Under the maximum oxygen transfer coefficient condition, the xylonic acid titer reached 108.2 g/L with a volumetric productivity of 13.53 g L-1 h-1 and a specific productivity of 6.52 g/gx/h. In conclusion, the addition of activated carbon particles effectively enhanced the oxygen mass transfer rate. These results demonstrate that activated carbon particles enhanced cultivation for xylonic acid production an inexpensive and attractive alternative.

In industrial production, the precursor of l-ascorbic acid (L-AA, also referred to as vitamin C), 2-keto-l-gulonic acid (2-KLG), is mainly produced using a classic two-step fermentation process performed by Gluconobacter oxydans, Bacillus megaterium, and Ketogulonicigenium vulgare. In the second step of the two-step fermentation process, the microbial consortium of K. vulgare and B. megaterium is used to achieve 2-KLG production. K. vulgare can transform l-sorbose to 2-KLG, but the yield of 2-KLG is much lower in the monoculture than in the coculture fermentation system. The relationship between the two strains is too diverse to analyze and has been a hot topic in the field of vitamin C fermentation. With the development of omics technology, the relationships between the two strains are well explained; nevertheless, the cell-cell communication is unclear. In this review, based on current omics results, the interactions between the two strains are summarized, and the potential cell-cell communications between the two strains are discussed, which will shed a light on the further understanding of synthetic consortia.