Daun Jeong

Redistribution of Carbon Flux toward 2,3-Butanediol Production in Klebsiella pneumoniae by Metabolic Engineering

Klebsiella pneumoniae KCTC2242 has high potential in the production of a high-value chemical, 2,3-butanediol (2,3-BDO). However, accumulation of metabolites such as lactate during cell growth prevent large-scale production of 2,3-BDO. Consequently, we engineered K. pneumoniae to redistribute its carbon flux toward 2,3-BDO production. The ldhA gene deletion and gene overexpression (budA and budB) were conducted to block a pathway that competitively consumes reduced nicotinamide adenine dinucleotide and to redirect carbon flux toward 2,3-BDO biosynthesis, respectively. These steps allowed efficient glucose conversion to 2,3-BDO under slightly acidic conditions (pH 5.5). The engineered strain SGSB105 showed a 40% increase in 2,3-BDO production from glucose compared with that of the host strain, SGSB100. Genes closely related to 2,3-BDO biosynthesis were observed at the gene transcription level by cultivating the SGSB100, SGSB103, SGSB104, and SGSB105 strains under identical growth conditions. Transcription levels for budA, budB, and budC increased approximately 10% during the log phase of cell growth relative to that of SGSB100. Transcription levels of 2,3-BDO genes in SGSB105 remained high during the log and stationary phases. Thus, the carbon flux was redirected toward 2,3-BDO production. Data on batch culture and gene transcription provide insight into improving the metabolic network for 2,3-BDO biosynthesis for industrial applications.

Observation of 2,3-butanediol biosynthesis in Lys regulator mutated Klebsiella pneumoniae at gene transcription level

Microorganisms that produce 2,3-butanediol (2,3-BDO) are mostly mixed acid fermentation microorganisms, and they synthesize 2,3-BDO in order to suppress medium acidification. The 2,3-BDO operon (budBAC) is activated by the LysR regulator protein derived from the budR. In this study, the effect of the budR on 2,3-BDO-biosynthesis was observed at gene transcription level. The Klebsiella pneumoniae strains (wabG-deleted strain (SGSB100), budR over-expressed strain (SGSB101), and the budR-deleted strain (SGSB102)) were constructed. The resulting strains were cultivated in unified conditions. Samples were obtained at the lag-, log-, and stationary-phase of cell growth, and gene transcription levels of the budR, 2,3-BDO-biosynthesis-related (budB, budA, and budC), and acid-biosynthesis-related (ldhA and ack) genes were observed. During the lag-phase of cell growth in SGSB101, the budR transcription level increased approximately 8-fold, and 2,3-BDO production increased approximately 2-fold, when compared to SGSB100. Also in SGSB101 the transcription level of the acid-biosynthesis-related genes (ldhA and ack) increased approximately up to 11-fold during the lag-phase of cell growth compared to SGSB100. On contrast, in SGSB102 budR transcription was not detected, and the transcription level of the acid-biosynthesis-related genes (ldhA and ack) decreased approximately 70-fold during the lag-phase of cell growth compared to SGSB100. This is by far the first observation of 2,3-BDO regulation mechanism at gene transcription level in K. pneumoniae, and therefore may be useful for understanding and improving 2,3-BDO biosynthesis metabolic network.

A non-pathogenic and optically high concentrated (R,R)-2,3-butanediol biosynthesizing Klebsiella strain

The objective of this work was to construct a non-pathogenic Klebsiella pneumonia strain that can produce optically high concentrated (R,R)-2,3-BDO. A K. pneumonia mutant lacking the pathogenic factor was used as the host strain. In order to construct a K. pneumonia strain that would biosynthesize high concentrated (R,R)-2,3-BDO, gene deletion and over-expression methods were combined; firstly, the 2,3-BDO dehydrogenase (budC) gene was deleted to re-direct utilization of the carbon source to (R,R)-2,3-BDO biosynthesis; secondly, the two glycerol dehydrogenase (GDH) enzymes in K. pneumonia (DhaD and GldA) were over-expressed to maximize (R,R)-2,3-BDO biosynthesis; and thirdly, the lactate dehydrogenase (ldhA) gene was deleted to minimize the accumulation of lactate. SGSB112, a non-pathogenic strain of K. pneumonia that can produce optically high concentrated (R,R)-2,3-BDO, was constructed as above. Approximately 36% of the carbon source was converted to (R,R)-2,3-BDO by SGSB112, achieving a production of 61gL(-1) (R,R)-2,3-BDO in a fed-batch fermentation. On the other hand, meso-2,3-BDO was produced 1.4gL(-1) and (S,S)-2,3-BDO was not detected. This study provides an insight into 2,3-BDO biosynthesis in K. pneumonia and demonstrates the achievement of high-yield production of optically high concentrated (R,R)-2,3-BDO through constructing a strain by genetic modification and metabolic engineering.

The influence of budA deletion on glucose metabolism related in 2,3-butanediol production by Klebsiella pneumoniae

Klebsiella pneumoniae (K. pneumoniae), which is a promising microorganism for industrial bulk production of 2,3-butanediol (2,3-BDO), naturally converts glucose to 2,3-BDO. The 2,3-BDO biosynthesis from glucose is composed of three steps; α-acetolactate biosynthesis by α-acetolactate synthase (budB); acetoin biosynthesis by α-acetolactate decarboxylase (budA); and 2,3-BDO biosynthesis by acetoin reductase (budC). In an effort to understand the influence of blocked 2,3-BDO pathway on K. pneumoniae glucose metabolism by budA deletion, we constructed K. pneumoniaeΔwabGΔbudA (SGSB106). Carbon flux distribution analysis, transcriptome analysis and extracellular amino acid concentration analysis were carried out to understand the effects of the budA deletion, and K. pneumoniaeΔwabG (SGSB100) was used as a control strain. Approximately 50.3% decrease in CO2 emission; and approximately 3.8-fold increase in amino acid production was observed in SGSB106. In addition to, among the amino acids, valine production significantly increased, suggesting that the branched-chain amino acid biosynthesis (BACC) in SGSB106 was activated by deletion of budA. Furthermore, whole genome transcriptome analysis of SGSB106 and SGSB100, correlates with the results from carbon distribution and amino acids concentration analyses.

Deletion of the budBAC operon in Klebsiella pneumoniae to understand the physiological role of 2,3-butanediol biosynthesis

ABSTRACT Klebsiella pneumoniae is known to produce 2,3-butanediol (2,3-BDO), a valuable chemical. In K. pneumoniae, the 2,3-BDO operon (budBAC) is involved in the production of 2,3-BDO. To observe the physiological role of the 2,3-BDO operon in a mixed acid fermentation, we constructed a budBAC-deleted strain (SGSB109). The production of extracellular metabolites, CO2 emission, carbon distribution, and NADH/NAD+ balance of SGSB109 were compared with the parent strain (SGSB100). When comparing the carbon distribution at 15 hr, four significant differences were observed: in 2,3-BDO biosynthesis, lactate and acetate production (lactate and acetate production increased 2.3-fold and 4.1-fold in SGSB109 compared to SGSB100), CO2 emission (higher in SGSB100), and carbon substrate uptake (higher in SGSB100). Previous studies on the inactivation of the 2,3-BDO operon were focused on the increase of 1,3-propanediol production. Few studies have been done observing the role of 2,3-BDO biosynthesis. This study provides a prime insight into the role of 2,3-BDO biosynthesis of K. pneumoniae.