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Contemplating 3-Hydroxypropionic Acid Biosynthesis in Klebsiella pneumoniae.

ABSTRACT: 3-Hydroxypropionic acid (3-HP) is a commercially valuable platform chemical from which an array of C3 compounds can be generated. Klebsiella pneumoniae has been considered a promising species for biological production of 3-HP. Despite a wealth of reports related to 3-HP biosynthesis in K. pneumoniae, its commercialization is still in infancy. The major hurdle hindering 3-HP overproduction lies in the poor understanding of glycerol dissimilation in K. pneumoniae. To surmount this problem, this review aims to portray a picture of 3-HP biosynthesis, involving 3-HP-synthesizing strains, biochemical attributes, metabolic pathways and key enzymes. Inspired by the state-of-the-art advances in metabolic engineering and synthetic biology, here we advocate protocols for overproducing 3-HP in K. pneumoniae. These protocols range from cofactor regeneration, alleviation of metabolite toxicity, genome editing, remodeling of transport system, to carbon flux partition via logic gate. The feasibility for these protocols was also discussed.

SUBMITTER: Li Y

PROVIDER: S-EPMC4363255 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Contemplating 3-Hydroxypropionic Acid Biosynthesis in Klebsiella pneumoniae.

Li Ying Y Tian Pingfang P

Indian journal of microbiology 20150115 2

3-Hydroxypropionic acid (3-HP) is a commercially valuable platform chemical from which an array of C3 compounds can be generated. Klebsiella pneumoniae has been considered a promising species for biological production of 3-HP. Despite a wealth of reports related to 3-HP biosynthesis in K. pneumoniae, its commercialization is still in infancy. The major hurdle hindering 3-HP overproduction lies in the poor understanding of glycerol dissimilation in K. pneumoniae. To surmount this problem, this re ...[more]

PMID: 25805899

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Insights into 3-hydroxypropionic acid biosynthesis revealed by overexpressing native glycerol dehydrogenase in Klebsiella pneumoniae.

Project description:In Klebsiella pneumoniae, glycerol dissimilation involves parallel oxidation and reduction pathways. Oxidation pathway provides adenosine triphosphate (ATP) and cofactors to sustain cell growth, while reduction pathway presents 3-hydroxypropionic acid (3-HP) and 1,3-propanediol(1,3-PDO), which are commercially attractive platform chemicals. Previous metabolic engineering of K. pneumoniae focused on the intensification of reduction pathway; however, it failed to overproduce 3-HP or 1,3-PDO. Contrary to this strategy, here we show that overexpression of glycerol dehydrogenase (dhaD), the first functional enzyme in oxidation pathway, can efficiently stimulate cell growth and facilitate 3-HP accumulation. Under microaerobic conditions, although metabolic burden arising from plasmid replication, the recombinant K. pneumoniae overexpressing dhaD grew actively and showed 60% enhancement of 3-HP compared to the control. In particular, overexpression of dhaD increased the activity of glycerol dehydratase, indicating the concerted action of two enzymes and the interdependence between glycerol oxidation and reduction pathways. Moreover, the strain overexpressing dhaD produced more lactic acid yet less acetic acid than the control, implying the interplay between dhaD expression and the formation of byproducts. Together, not only showing that intensifying glycerol oxidation pathway is beneficial to 3-HP production, this study also reveals the structural rigidity of dha operon that mediates glycerol dissimilation in K. pneumoniae.

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Gene Arrangements in Expression Vector Affect 3-Hydroxypropionic Acid Production in Klebsiella pneumoniae.

Project description:Biosynthesis of 3-hydroxypropionic acid (3-HP) typically involves two sequential reactions catalyzed by glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH). Although plasmid-dependent over-expression of the two enzymes is common, systematic investigation of gene arrangement in vector has not been reported. Here we show that gene arrangements have a noticeable influence on 3-HP production. Using Klebsiella pneumoniae as a host, three AldH-coding genes: ald4 from Saccharomyces cerevisiae, aldh from Escherichia coli, and puuC from host K. pneumoniae, were respectively ligated to dhaB. The recombinant Kp/pET-pk-ald4-dhaB (Kp refers to as K. pneumoniae, pk is a native promoter) produced the highest yield of 3-HP in comparison to both Kp/pET-pk-dhaB-ald4 and Kp/pET-pk-dhaB-pk-ald4, suggesting that the preferential expression of AldH can increase 3-HP production. Additionally, when different AldH-coding genes were respectively ligated downstream of dhaB, the recombinant Kp/pET-pk-dhaB-puuC produced more 3-HP than that by Kp/pET-pk-dhaB-aldh or Kp/pET-pk-dhaB-ald4, implying the intrinsic compatibility of native gene puuC with its host. These findings indicate the applicability of native AldH-coding gene and provide insights into strategies for metabolic engineering of multiple genes.

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Engineering Glucose-to-Glycerol Pathway in Klebsiella pneumoniae and Boosting 3-Hydroxypropionic Acid Production Through CRISPR Interference

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Heterologous Expression of Aldehyde Dehydrogenase from Saccharomyces cerevisiae in Klebsiella pneumoniae for 3-Hydroxypropionic Acid Production from Glycerol.

Project description:3-Hydroxypropionic acid (3-HP) is a commercially valuable platform compound. Klebsiella pneumoniae has been concerned as an appropriate host for 3-HP production because of its robust capacity to metabolize glycerol. Glycerol conversion to 3-HP in K. pneumoniae comprises two successive reactions: glycerol dehydratase catalyzes glycerol to 3-hydroxypropionaldehyde (3-HPA); aldehyde dehydrogenase catalyzes 3-HPA to 3-HP. Previous studies focusing on inducible expression of aldehyde dehydrogenase have shown defects of high cost of inducer and low catalytic activity due to inclusion body. Here we show a different strategy that a native promoter in the host K. pneumoniae was used to drive the heterologous expression of aldehyde dehydrogenase gene ald4 from Saccharomyces cerevisiae. The 3-HP yield of the recombinant reached a peak of 4.23 g/L at log phase, but it decreased during later period of fermentation. Except the validation of high activity of ald4, particularly, the 3-HP formation was uncovered to be closely coupled with cell division, and the lacking of NAD and ATP at latter fermentation phase became the bottleneck for cell growth and 3-HP accumulation. Furthermore, 3-HP is postulated to be converted to 3-HPA via feedback inhibition or other metabolite via unknown mechanism. Since glycerol dissimilation is a common mechanism in a variety of bacteria, the expression strategy using native promoter and implications may provide significant insight into the metabolic engineering for 3-HP production.

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Anodic electro-fermentation of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae L17 in a bioelectrochemical system.

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RmpA regulation of capsular polysaccharide biosynthesis in Klebsiella pneumoniae CG43.

Project description:Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5' noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur.

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