Jinping Lin

Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.

We have expressed the pqqABCDE gene cluster from Gluconobacter oxydans, which is involved in pyrroloquinoline quinone (PQQ) biosynthesis, in Escherichia coli, resulting in PQQ accumulation in the medium. Since the gene cluster does not include the tldD gene needed for PQQ production, this result suggests that the E. coli tldD gene, which shows high homology to the G. oxydanstldD gene, carries out that function. The synthesis of PQQ activated d-glucose dehydrogenase in E. coli and the growth of the recombinant was improved. In an attempt to increase the production of PQQ, which acts as a vitamin or growth factor, we transformed E. coli with various recombinant plasmids, resulting in the overproduction of the PQQ synthesis enzymes and, consequently, PQQ accumulation—up to 6 mM—in the medium. This yield is 21.5-fold higher than that obtained in previous studies.

Membrane-Bound Pyrroloquinoline Quinone-Dependent Dehydrogenase in Gluconobacter oxydans M5, Responsible for Production of 6-(2-Hydroxyethyl) Amino-6-Deoxy-l-Sorbose

ABSTRACT A membrane-bound protein purified from Gluconobacter oxydans M5 was confirmed to be a pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase. Gene disruption and complementation experiments demonstrated that this enzyme is responsible for the oxidation of 1-(2-hydroxyethyl) amino-1-deoxy-d-sorbitol (1NSL) to 6-(2-hydroxyethyl) amino-6-deoxy-l-sorbose (6NSE), which is the precursor of an antidiabetic drug, miglitol.

Characterization of Enzymes in the Oxidation of 1,2-Propanediol to d-(−)-Lactic Acid by Gluconobacter oxydans DSM 2003

Although Gluconobacter oxydans can convert 1,2-propanediol to d-(−)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of d-(−)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in d-(−)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to d-(−)-lactic acid by G. oxydans DSM 2003.

Characterization of a novel dextran produced by Gluconobacter oxydans DSM 2003.

A novel water-soluble dextran was synthesized from maltodextrin by cell-free extract of Gluconobacter oxydans DSM 2003. The dextran was purified by size exclusion chromatography, and the structure was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and gas chromatography–mass spectrometer. Based on the spectral data, we found that the dextran contained only d-glucose residues. The ratio of nonreducing end glucopyranosyl (Glcp) to 6-linked Glcp to 4,6-linked Glcp was estimated to be 8.62:78.79:12.59 by methylation analysis. This result indicated the existence of a small proportion of α(1,4) branches in α(1,6) glucosyl linear chains. Here, we reported the first time a novel dextran was synthesized by G. oxydans DSM 2003.

Functions of Membrane-bound Alcohol Dehydrogenase and Aldehyde Dehydrogenase in the Bio-oxidation of Alcohols in Gluconobacter oxydans DSM 2003

In this study a new insight was provided to understand the functions of membrane-bound alcohol dehydrogenase (mADH) and aldehyde dehydrogenase (mALDH) in the bio-oxidation of primary alcohols, diols and poly alcohols using the resting cells of Gluconobacter oxydans DSM 2003 and its mutant strains as catalyst. The results demonstrated that though both mADH and mALDH participated in most of the oxidation of alcohols to their corresponding acid, the exact roles of these enzymes in each reaction might be different. For example, mADH played a key role in the oxidation of diols to its corresponding organic acid in G. oxydans, but it was dispensable when the primary alcohols were used as substrates. In contrast to mADH, mALDH appears to play a relatively minor role in organic acid-producing reactions because of the possible presence of other isoenzymes. Aldehydes were, however, found to be accumulated in the mALDH-deficient strain during the oxidation of alcohols.

Identification of the enzymes responsible for 3-hydroxypropionic acid formation and their use in improving 3-hydroxypropionic acid production in Gluconobacter oxydans DSM 2003

Gluconobacter oxydans can be efficiently used to produce 3-hydroxypropionic acid (3-HP) from 1,3-propanediol (1,3-PDO). However, the enzymes involved remain unclear. In this study, transcription analysis of two mutants of strain DSM 2003, obtained by UV-mutagenesis, revealed that membrane-bound alcohol dehydrogenase (mADH) and membrane-bound aldehyde dehydrogenase (mALDH) might be the main enzymes involved. Through deletion and complementation of the genes adhA and aldh, mADH and mALDH were verified as the main enzymes responsible for 3-HP production. Then mALDH was verified as the rate-limiting enzyme in 3-HP production. Since that overexpression of mADH had no effect on 3-HP production, whereas overexpression of mALDH increased 23.6% 3-HP production. Finally, the 3-HP titer of 45.8 g/L and the highest productivity 1.86 g/L/h were achieved when the two mutants DSM 2003/adhAB and DSM 2003/aldh were mixed at a ratio of 1:2 (cell density) and used as whole cell catalysts for 3-HP production.