Strain Improvement For Biological Production Of Pyrroloquinoline Quinone

Unlike nicotinamide nucleotides (NAD, NADP) and riboflavin nucleotides (FMN, FAD), pyrroloquinoline quinone (PQQ) is a novel cofactor. Due to its unique biological functions, PQQ has attracted much attention in recent years. Since the PQQ biosynthesis mechanism has not been completely deciphered, genetically modifying the strain encounters various techniqual barriers. Towards high PQQ production, here, the strain Klebsiella pneumoniae DSM2026was modified by the following methods and the main achievements are listed below.1. We optimized the medium of three high-yielding strains U1, U2and L1，which were previously obtained in our lab by UV mutagenesis, lithium chloride mutagenesis and UV-LiC1mutagenesis.2. The conditions for genome shuffling of K. pneumoniae were optimized in this study. Based on available reports on PQQ biosynthesis, along with exploration on growth attributes and cellular architecture of K. pneumoniae, the genome shuffling conditions were systemically optimized. Below are the optimum conditions:Protoplast was prepared by enzymatic hydrolysis at37℃for1.5-2h, shaken once every15min; the lysozyme concentration was0.1mg/mL; the protoplast was inactivated by heat treatment for25min at55℃, and finally subjected to ultraviolet irradiation for5min. The optimized conditions for protoplast fusion include:0.1mL of hypertonic solution,0.1mL of newly prepared calcium phosphate solution,0.8mL of PEG6000, and incubation at37℃for5-6min.3. U1, U2and L1, the three high production strains, were subjected to genome shuffling under the aforementioned conditions. After three rounds of genome shuffling followed by high throughput screening, we acquired a high-yielding strain F3-8from3000clones. Subsequently, this strain was fermented in LB and M9media to determine its cultivation condition and PQQ yield. HPLC analysis showed that the highest PQQ concentration was418mg/L, which is nearly5.4and10-fold of that in starting strain and wild type strain.4. The sigma factor coding gene rpoD was cloned by PCR from the genomic DNA of K. pneumoniae, and its mutant library was constructed via error-prone PCR. Next the mutant library were ligated to expression vector pET-PK and subsequently transformed into K. pneumoniae DSM2026, leading to a number of positive clones. By high throughput screening from about1500positive clones, a PQQ-yielding strain was obtained and designated as K.p (pET-PK-trpoD). In batch fermentation, this strain produced PQQ of76mg/mL, which is10%enhancement compred to the control that carrying unmutated rpoD gene. DNA sequencing showed that the mutation rates of rpoD at nucleotide and protein sequence levels were2.5and0.5%, respectively. Secondary structure prediction showed the changes in the number of alpha helix, extended strand, beta turn, and random coil. We thus speculate that the mutation of sigma factor may affect the binding to promoters, thereby affecting the transcription of PQQ genes, reallocating metabolic flux, and intensifying PQQ biosynthesis.5. PQQ biosynthesis usually involves4-7genes. In K. pneumoniae, total six genes pqqABCDEF participate in PQQ synthesis. In this study, the six genes were rearranged to determine the influences of gene number, arrangement, and nucleic acid sequence on the production of PQQ. Furthermore, the experimental results were profoundly systematically analyzed.