Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol: Reduction of acetate and other by-products
Introduction
Due to the rapid expansion of the biodiesel industry, a large amount of cheap crude glycerol has become available (Saxena et al., 2009). As glycerol is a good carbon source for microbial growth, microbial production of value-added chemicals from glycerol has gained much attention (Dharmadi et al., 2006). Among those chemicals, 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO) are specially important. 3-HP, one of the United States Department of Energy’s top 12 value-added chemicals producible from biomass, can be used for the production of acrylic acid, 1,3-PDO, 3-hydroxypropionaldehyde (3-HPA), and malonic acid (Paster et al., 2003). These compounds are utilized in the manufacture or fabrication of adhesives, polymers, fibers, packing materials, resins, food preservatives, and cleaning agents (Della Pina et al., 2011, Zhang et al., 2004). Similarly, 1,3-PDO has important applications in the synthesis or production of polytrimethylene terephthalate (PTT) (Biebl et al., 1998, Maervoet et al., 2010), food packaging and cosmetics. Due to their wide applications in many different fields, the market potentials of 3-HP and 1,3-PDO have been valued at more than 10 billion and 600 million USD, respectively (Molel et al., 2015).
Biological production of 3-HP and 1,3-PDO from glycerol proceeds by a relatively simple, two-step reaction for each (Raj et al., 2008). First, glycerol is converted to 3-HPA by glycerol dehydratase (GDHt) using coenzyme B12 as the cofactor. Second, the intermediate, 3-HPA, is further converted to 3-HP by NAD+-dependent aldehyde dehydrogenase (ALDH) (Toraya et al., 2008, Jo et al., 2008, Raj et al., 2010) or to 1,3-PDO by NAD(P)H-dependent 1,3-PDO oxidoreductase (1,3-PDOR) (Cao et al., 2006, Jarboe, 2011, Wang et al., 2003) (Fig. 1). So far, the highest production titers of 3-HP and 1,3-PDO have been 83.8 g/L (with Klebsiella pneumoniae) and 135 g/L (with Escherichia coli), respectively (Li et al., 2016, Li and Khanal, 2016). Nevertheless, microbial production of 3-HP or 1,3-PDO remains challenging. For instance, the limited regeneration and cellular availability of the cofactors, NAD(P)+ for 3-HP (Raj et al., 2010, Ko et al., 2012) and NAD(P)H for 1,3-PDO (Ashok et al., 2011), curtail the activity of ALDH and 1,3-PDOR, respectively, and their reduced activity leads to accumulation of toxic intermediate 3-HPA. For efficient NAD+ regeneration, the activity of electron transport chain (ETC) can be accelerated by increasing aeration, but this incurs a number of additional problems. First, GDHt can be rapidly inactivated by oxygen (Xu et al., 2009, Huang et al., 2016). Also, coenzyme B12, the cofactor for GDHt, is not sufficiently synthesized under high-aeration conditions in most of the natural 3-HPA producers such as K. pneumoniae and Lactobacillus sp. (Santos et al., 2008, Roth et al., 1996). Furthermore, high-aeration or -agitation conditions render industrial-scale production costly. For NAD(P)H regeneration in the 1,3-PDO production context, extra carbon sources should be oxidized in glycolysis, though this reduces the 1,3-PDO production yield.
As a solution to the aforementioned problems, simultaneous production of 3-HP and 1,3-PDO in a single cell has been suggested (Ashok et al., 2011, Huang et al., 2012). If 3-HP and 1,3-PDO are co-produced, the cofactor generated from one product can be used for the synthesis of the other product, so that the whole system becomes redox-balanced. In other words, NAD(P)H generated from 3-HP production can be used for the production of 1,3-PDO or vice versa (Kumar et al., 2012) (Fig. 1). Co-production is also expected to reduce accumulation of 3-HPA, because two enzymes, ALDH and 1,3-PDOR, consume the toxic intermediate simultaneously (Kumar et al., 2013). Additionally, co-production can be performed under low-aeration conditions that enhance the synthesis of coenzyme B12 and reduce the energy expenditure during large-scale production. Ashok et al., 2011, Huang et al., 2012, Huang et al., 2013 experimentally examined the concept of co-production with K. pneumoniae which has several advantages over other strains. K. pneumoniae grows faster on glycerol, harbors the native 1,3-PDO production pathways (GDHt encoded by dhaB and 1,3-PDOR encoded by dhaT) (Wang et al., 2003, Pérez et al., 2008, Seo et al., 2010), and produces coenzyme B12 under anaerobic/microaerobic conditions. Huang et al. (2013) could produce 48.9 g/L 3-HP and 25.3 g/L 1,3-PDO with a co-production yield of 0.66 mol/mol in 28 h under microaerobic conditions. Although this co-production titer was satisfactory, it was much lower than the individual production titer of 3-HP or 1,3-PDO. An important reason, speculatively, is excessive accumulation of by-products such as lactate, ethanol and acetate, among others (Huang et al., 2012, Huang et al., 2013). 25 g/L lactate, 2 g/L ethanol and 9 g/L acetate were accumulated in 24 h. Similarly, Ashok et al. (2011) reported accumulation of 22 g/L lactate, 3 g/L ethanol and 2.5 g/L acetate within 24 h during co-production of 3-HP and 1,3-PDO. Production of lactate and ethanol at high concentrations is toxic to cells and, furthermore, consumes NADH which otherwise could be used for 1,3-PDO production. Especially, acetic acid is highly toxic even at moderate concentrations (De Mey et al., 2007). Elimination or reduction of these by-products could potentially improve the co-production of 3-HP and 1,3-PDO.
This study was undertaken with the goal of improving 3-HP and 1,3-PDO co-production by K. pneumoniae J2B isolated in our laboratory and does not produce pathogenic lipopolysaccharides (Arasu et al., 2011). By-products such as lactate, ethanol, succinate and acetate levels were reduced by disrupting the corresponding enzymes leading to their production. However, when the pta-ackA gene of the acetate pathway was disrupted, cell growth, glycerol assimilation as well as 3-HP and 1,3-PDO production were all significantly reduced. To reduce acetate formation while leaving pta-ackA intact, we attempted the following: (i) reduction of glycerol assimilation through the glycolytic pathway (i.e., overflow metabolism) by disruption of glycerol kinase (GlpK) or glycerol dehydrogenase (DhaD); (ii) increase of glycerol flow towards 1,3-PDO and 3-HP by over-expression of DhaT and/or DhaB, and (iii) variation of the aeration rate during cultivation. Various mutant strains were constructed and evaluated for co-production of 3-HP and 1,3-PDO and formation of by-products on the flask and bioreactor scales. Our work should provide important insight into the metabolism of K. pneumoniae as it relates to 3-HP and 1,3-PDO co-production from glycerol.
Section snippets
Materials
The genomic DNA isolation kit and pGEM-T vector were purchased from Promega (Madison, WI, USA). Pfu-X polymerase for PCR amplification of DNA fragments was obtained from Solgent (Seoul, Korea). Synthesis of oligonucleotides for PCR and DNA sequencing was performed by Macrogen Co. Ltd. (Seoul, Korea). Yeast Extract (Cat. 212750), Bacto tryptone (Cat. 211705) and Bacto agar (Cat.214010) were acquired from Difco (Becton Dickinson; Franklin Lakes, NJ, USA). 3-HP was purchased from Tokyo Kasei Kogyo
Improvement of co-production by deletion of by-product-formation pathways
To improve co-production titer and yield, lactate dehydrogenase (ldhA), succinate dehydrogenase (frdA) and alcohol dehydrogenase (adhE) were deleted from the wild-type K. pneumoniae J2B (designated as Kp01). The resulting Kp01ΔldhAΔfrdAΔadhE (designated as Kp03) was further mutated in the acetate pathway by disruption of pyruvate oxidase (poxB), yielding Kp04, or both poxB and acetyl-CoA kinase (pta-ackA), thereby resulting in Kp05. These mutant strains (Kp03, Kp04 and Kp05) along with the
Conclusion
This study investigated the metabolic engineering of K. pneumoniae for improved 3-HP and 1,3-PDO co-production. Various attempts such as down-regulation of assimilatory glycerol metabolism and up-regulation of the co-production pathway showed co-production improvement and acetate-accumulation reduction on the flask scale, with the highest co-production yield, >0.82 mol/mol. However, the positive results were not fully repeated in bioreactor experiments, due to the excessive acetate accumulation
Acknowledgements
This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2011-0031361).
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