A genome-scale metabolic model of the effect of dissolved oxygen on 1,3-propanediol fermentation by Klebsiella pneumoniae.

Although 1,3-propanediol (1,3-PD) is usually considered an anaerobic fermentation product from glycerol by Klebsiella pneumoniae, microaerobic conditions proved to be more conducive to 1,3-PD production. In this study, a genome-scale metabolic model (GSMM) specific to K. pneumoniae KG2, a high 1.3-PD producer, was constructed. The iZY1242 model contains 2090 reactions, 1242 genes and 1433 metabolites. The model was not only able to accurately characterise cell growth, but also accurately simulate the fed-batch 1,3-PD fermentation process. Flux balance analyses by iZY1242 was performed to dissect the mechanism of stimulated 1,3-PD production under microaerobic conditions, and the maximum yield of 1,3-PD on glycerol was 0.83 mol/mol under optimal microaerobic conditions. Combined with experimental data, the iZY1242 model is a useful tool for establishing the best conditions for microaeration fermentation to produce 1,3-PD from glycerol in K. pneumoniae.

Mutation of rpoS is Beneficial for Suppressing Organic Acid Secretion During 1,3-Propandiol Biosynthesis in Klebsiella pneumoniae.

In this study, to reduce the formation of organic acid during 1,3-propanediol biosynthesis in Klebsiella pneumoniae, a method combining UV mutagenesis and high-throughput screening with pH color plates was employed to obtain K. pneumoniae mutants. When compared with the parent strain, the total organic acid formation by the mutant decreased, whereas 1,3-propanediol biosynthesis increased after 24 h anaerobic shake flask culture. Subsequently, genetic changes in the mutant were analyzed by whole-genome sequencing and verified by signal gene deletion. Mutation of the rpoS gene was confirmed to contribute to the regulation of organic acid synthesis in K. pneumoniae. Besides, rpoS deletion eliminated the formation of 2,3-butanediol, the main byproduct produced during 1,3-propanediol fermentation, indicating the role of rpoS in metabolic regulation in K. pneumoniae. Thus, a K. pneumoniae mutant was developed, which could produce lower organic acid during 1,3-propanediol fermentation due to an rpoS mutation in this study.

A new and effective genes-based method for phylogenetic analysis of Klebsiella pneumoniae.

The exponential increase in the number of genomes deposited in public databases can help us gain a more holistic understanding of the phylogeny and epidemiology of Klebsiella pneumoniae. However, inferring the evolutionary relationships of K. pneumoniae based on big genomic data is challenging for existing methods. In this study, core genes of K. pneumoniae were determined and analysed in terms of differences in GC content, mutation rate, size, and potential functions. We then developed a stable genes-based method for big data analysis and compared it with existing methods. Our new method achieved a higher resolution phylogenetic analysis of K. pneumoniae. Using this genes-based method, we explored global phylogenetic relationships based on a public database of nearly 953 genomes. The results provide useful information to facilitate the phylogenetic and epidemiological analysis of K. pneumoniae, and the findings are relevant for security applications.

Analysis of the Growth and Metabolites of a Pyruvate Dehydrogenase Complex- Deficient Klebsiella pneumoniae Mutant in a Glycerol-Based Medium.

To determine the role of pyruvate dehydrogenase complex (PDHC) in Klebsiella pneumoniae, the growth and metabolism of PDHC-deficient mutant in glycerol-based medium were analyzed and compared with those of other strains. Under aerobic conditions, the PDHC activity was fourfold higher than that of pyruvate formate lyase (PFL), and blocking of PDHC caused severe growth defect and pyruvate accumulation, indicating that the carbon flux through pyruvate to acetyl coenzyme A mainly depended on PDHC. Under anaerobic conditions, although the PDHC activity was only 50% of that of PFL, blocking of PDHC resulted in more growth defect than blocking of PFL. Subsequently, combined with the requirement of CO2 and intracellular redox status, it was presumed that the critical role of PDHC was to provide NADH for the anaerobic growth of K. pneumoniae. This presumption was confirmed in the PDHC-deficient mutant by further blocking one of the formate dehydrogenases, FdnGHI. Besides, based on our data, it can also be suggested that an improvement in the carbon flux in the PFL-deficient mutant could be an effective strategy to construct highyielding 1,3-propanediol-producing K. pneumoniae strain.

Regulation of Pyruvate Formate Lyase-Deficient Klebsiella pneumoniae for Efficient 1,3-Propanediol Bioproduction.

Anaerobic growth defect of pyruvate formate lyase (PFL)-deficient Klebsiella pneumoniae limits its industrial application, and the reason for this growth defect was analyzed in this study. The obtained evidences, combined with normal intracellular redox status and no further inhibition by adhE deletion, strongly suggested that growth defect in PFL-deficient K. pneumoniae was probably caused by lack of carbon flux from pyruvate to acetyl-CoA (AcCoA). Correspondingly, the anaerobic growth of PFL-deficient K. pneumoniae was promoted by deletion of pdhR, a negative transcriptional regulator gene for AcCoA generation. Through the regulation of pdhR deletion, the PFL-deficient K. pneumoniae exhibited highly efficient 1,3-propanediol production. Besides, in a 2-L fed-batch fermentation process, the cell growth of PFL-deficient K. pneumoniae strain almost recovered, when compared with that of the normal strain, and the 1,3-propanediol yield increased by 14%, while the byproducts acetate and 2,3-butanediol contents decreased by 29% and 24%, respectively.

Inactivating NADH:quinone oxidoreductases affects the growth and metabolism of Klebsiella pneumoniae.

NADH:quinone oxidoreductases (NQOs) act as the electron entry sites in bacterial respiration and oxidize intracellular NADH that is essential for the synthesis of numerous molecules. Klebsiella pneumoniae contains three NQOs (NDH-1, NDH-2, and NQR). The effects of inactivating these NQOs, separately and together, on cell metabolism were investigated under different culture conditions. Defective growth was evident in NDH-1-NDH-2 double and NDH-1-NDH-2-NQR triple deficient mutants, which was probably due to damage to the respiratory chain. The results also showed that K. pneumoniae can flexibly use NQOs to maintain normal growth in single NQO-deficient mutants. And more interestingly, under aerobic conditions, inactivating NDH-1 resulted in a high intracellular NADH:NAD+ ratio, which was proven to be beneficial for 2,3-butanediol production. Compared with the parent strain, 2,3-butanediol production by the NDH-1-deficient mutant was increased by 46% and 62% in glycerol- and glucose-based media, respectively. Thus, our findings provide a practical strategy for metabolic engineering of respiratory chains to promote the biosynthesis of 2,3-butanediol in K. pneumoniae.

Deleting pck improves growth and suppresses by-product formation during 1,3-propanediol fermentation by Klebsiella pneumoniae.

AIMS: To investigate the role of phosphoenolpyruvate (PEP) carboxylation in cell metabolism in Klebsiella pneumoniae. METHODS AND RESULTS: The effects of deleting pck, which encodes PEP carboxykinase (PCK), and/or ppc, which encodes PEP carboxylase (PPC), on growth, enzyme activity and metabolite formation of Kl. pneumoniae were investigated. A self-regulatory mechanism of PEP carboxylation was found in ppc- or pck-deficient mutants, which resulted in almost no change in succinate formation. However, almost no growth was observed in a ppc- and pck-deficient mutant. Interestingly, only deleting pck affected the energy metabolism and promoted aerobic cell growth. Under micro-aerobic conditions, although there was only a small (8·1%) increase of 1,3-propanediol production by such pck-deficient mutant during a 2-l fed-batch process, the by-products 2,3-butanediol and acetate significantly decreased by 73·0% and 54·8%, respectively, compared with those in the parent strain. CONCLUSIONS: PEP carboxylation could be a critical anaplerotic reaction for converting C3 to C4 metabolites in the central metabolism of Kl. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to identify the critical role of PEP carboxylation, as well as those of PPC and PCK, which are responsible for this reaction, in Kl. pneumoniae. In addition, the pck-deficient mutant was proven to be a valuable 1,3-propanediol producer.

Deletion of poxB, pta, and ackA improves 1,3-propanediol production by Klebsiella pneumoniae.

To date, few studies have focused on reducing the toxic by-product acetate during 1,3-propanediol production by Klebsiella pneumoniae. In this study, the effects of deleting the poxB, pta, and ackA genes, which are involved in the two main acetate synthesis pathways, on cell growth and 1,3-propanediol production were investigated. Although acetate synthesis via pyruvate oxidase (PoxB, encoded by poxB) generally seems unnecessary and wasteful, PoxB was shown to play an important role in K. pneumoniae. Deletion of poxB severely inhibited cell growth, and the poxB mutant exhibited an anomalously high accumulation of acetate in aerobic cultures and failed to produce an endogenous supply of carbon dioxide (CO2) in anaerobic cultures. It is interesting that both the aerobic and anaerobic growth defects of the poxB mutant were corrected by further deleting pta and ackA, which blocked the other main acetate synthesis pathway. The poxB-pta-ackA mutant excreted less acetate and showed an excellent ability to produce 1,3-propandiol. The final 1,3-propanediol yield and concentration in a 2-L fed-batch fermentation reached 0.66 (mol/mol) and 76.8 g/L, respectively, which were 16 and 15 % greater, respectively, than those of the parent strain.

Vector promoters used in Klebsiella pneumoniae.

Much effort has been devoted to the metabolic engineering of Klebsiella pneumoniae; however, our knowledge of the actual expression level of promoters used in K. pneumoniae is limited. In this study, the expression levels of three promoters were compared systematically by using the lacZ reporter gene with different carbon sources in K. pneumoniae. The results showed that, although promoters PT5 and Ptac designed for Escherichia coli were functional, PT5 appeared more efficient and the induction/repression ratio of Ptac was decreased extremely in K. pneumoniae. The basal level of Ptac for lacZ expression reached 396.5 U/mg, which was 9.5-fold higher compared with PT5 in LB medium, indicating Ptac can be used as an efficient "constitutive" promoter as well as an efficient induced promoter in K. pneumoniae. In different carbon sources medium, a newly constructed endogenous constitutive Pbud proved to be a stable and weak promoter. On the basis of our data, a set of Pbud and Ptac promoters could meet the broad range (about 1,000 orders of magnitude) of gene expression needed for engineered K. pneumoniae in glycerol-based medium.

Improvement of 1,3-propanediol production in Klebsiella pneumoniae by moderate expression of puuC (encoding an aldehyde dehydrogenase).

OBJECTIVE: To improve 1,3-propanediol production in Klebsiella pneumoniae, the effects of puuC expression in lactate- and lactate/2,3-butanediol-deficient strains were assessed. RESULTS: Overexpression of puuC (encoding an aldehyde dehydrogenase) inhibited 1,3-propanediol production and increased 3-hydroxypropionic acid formation in both lactate- and lactate/2,3-butanediol-deficient strains. An improvement in 1,3-propanediol production was only achieved in a lactate-deficient strain via moderate expression of puuC; at the end of the fermentation, 1,3-propanediol productivity increased by 14% compared with the control. Further comparative analysis of the metabolic flux distributions in different strains indicated that 3-hydroxypropionic acid formation could play a considerable role in cell metabolism in K. pneumoniae. CONCLUSION: An improvement in 3-hydroxypropionic acid formation would be beneficial for cell metabolism, which can be accomplished by enhancing 1,3-propanediol productivity in a lactate-deficient strain via moderate expression of puuC.

Acceleration effect of amino acid supplementation on glycerol assimilation by Escherichia coli in minimal medium.

Growth of Escherichia coli BL21 in a glycerol minimal medium was accelerated following supplementation with trace amounts of amino acid (0.35 mM). Of 12 amino acids tested, Arg and Ser gave the highest response, increasing cell growth by 63 and 53 %, respectively, compared to control cells. The ability of amino acids to accelerate cell growth was "switch-like" and was achieved by promoting glycerol utilization, which may be applied to shorten the long lag-phase when glycerol is used as carbon source. Acceleration of cell growth following amino acid supplementation was also observed using lactose minimal medium.

[1, 3-propanediol production under salt stress].

Through studying the process of glycerol fermentation to 1, 3-propanediol(1, 3-PD) by Klebsiella pneumoniae, it was found that the cell growth and product (or by-product) production were under salt stress. Cell growth and product formation kept high rate at low salt concentration. High salt concentration led to low growth of cells, final concentration of 1, 3-PD and conversion from glycerol to 1, 3-PD, and, 1, 3-propanediol oxidoreductase activity decreased. When the salt concentration in 5 m3 bioreactor was controlled under appropriate manner, the concentration of 1, 3-PD production was markedly enhanced. The final 1, 3-PD concentration ,the conversion of glycerol to 1, 3-PD and productivity were 64 g/L, 61% and 2.1 g/(L x h).