Bioconversion offers a safer and more viable alternative with the opportunity to produce a variety of chemicals under milder conditions. However, there are currently only a very few examples of bio-based processes outcompeting petro-based process such as yeast-based ethanol, yeast- or lactic acid bacteria-based lactic acid andE. coli- based PDO (Patel et al., 2006). For these bio-based chemicals to be economically feasible, a high product concentration (above 100 g/L), productivity (over 2 g/L⋅h) and substrate-to-product yield (above 50%) are required. For the production of 3-HP, several biological routes and recombinant strains have already been developed and examined. However, as summarized in Table 14.3, the results with most recombinant strains are still not satisfactory for commercial production of 3-HP. There are several barriers to microbiological production of 3-HP that differ depending on the kind of substrates and/or pathways employed. The following section covers these challenges and offers some possible solutions while focusing on processes that use glycerol as substrate.
14.5.1 Toxicity and Tolerance
In general, the toxic effects of organic acids are related to their ability to diffuse across the cell membrane. Inside the cell, these compounds dissociate and disrupt pH as well as the anion pool in the cytoplasm. The acidification of cytoplasm can ruin the integrity of purine bases and result in denaturing of essential enzymes, both of which seriously impair cell viability. The export of organic acids requires high energy, especially under low extracellular pH. van Maris et al. (2004) reported that under low pH, the metabolic energy requirement for product export may equal or exceed the metabolic energy yielded from product formation, causing the metabolic energy for growth and other essential cellular functions to become deficient. Once cells cannot maintain their intracellular pH within a physiologically acceptable range, serious
TABLE14.33-HPProductionbyDifferentKlebsiellapneumoniaeandEscherichiacolistrains CarbonTiteraYP/SQPAeration Recombinantstrainsource(g/L)(mol/mol)(g/L⋅h)conditionReference Batchcultivation E.coliBL21_dhaB_aldHGlycerol0.60.480.02AerobicRajetal.(2008) K.pneumoniaeME-308_aldAGlycerol2.8(9.8)0.100.12MicroaerobicZhuetal.(2009) K.pneumoniaeAK_pduPGlycerol1.4(8.4)0.070.06AerobicLuoetal.(2011b) E.coliBL21_mcr_acc_pntABGlucose0.20.020.01AerobicRathnasinghetal. (2012) Fed-batchcultivation E.coliBL21_dhaB_aldHGlycerol31.00.350.43AerobicRajetal.(2009) E.coli SH254_dhaB_KGSADHGlycerol38.70.360.54AerobicRathnasinghetal. (2009) E.coliBX3_0240Glucose49.00.460.71AerobicLynchetal.(2011) E.coli BL21_dhaB_dhaR_aldHGlucoseand glycerol14.3(3.9)0.150.26AerobicKwaketal.(2012) K.pneumoniaeDSM2026 ΔdhaT_puuCGlycerol16.0(16.3)0.230.67AerobicAshoketal.(2011) (continued
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TABLE14.3(Continued) CarbonTiteraYP/SQPAeration Recombinantstrainsource(g/L)(mol/mol)(g/L⋅h)conditionReference K.pneumoniaeAK_aldHkGlycerol6.8(22.7)–0.23AerobicLuoetal.(2011a) K.pneumoniaeCuGlycerol1.9(7.8)0.100.08AerobicLuoetal.(2012) K.pneumoniae WM3pUC18kan_aldHecGlycerol24.4(49.3)0.181.02AnaerobicHuangetal. K.pneumoniaeWM3 pUC18kan_aldHecGlycerol48.9(25.3)0.411.75MicroaerobicHuangetal. K.pneumoniaeJ2B ΔdhaT_KGSADHGlycerol16.3(5.8)0.400.30AerobicKoetal.(2012) K.pneumoniae ΔdhaTΔyqhD_dhaB_puuCGlycerol28.1(3.3)0.400.58AerobicAshoketal. (2013a) K.pneumoniae ΔglpKΔdhaT_puuCGlycerol22.0(5.9)0.300.46AnaerobicAshoketal. (2013b) K.pneumoniae J2B_KGSADHGlycerol11.3(15.9)0.270.94AnaerobicKumaretal. (2012a) K.pneumoniae J2BΔldhA_KGSADHGlycerol22.7(23.5)0.350.38AerobicKumaretal. (2012b) PDO,1,3-propanediol;3-HP,3-hydroxypropionicacid. aThevaluesshownintheparenthesesarethePDOconcentrationsobtainedalongwith3-HPincoproductionstudies.
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damage to cellular metabolism and finally cell death occur (Holyoak et al., 1996; Brul and Coote, 1999; Halm et al., 2004). This situation is further aggravated when the metabolic pathway leading to product formation has no net ATP production. However, the mechanisms of most of the transport processes and their energy requirements are still not clear. Transport phenomena such as substrate uptake and product export are important factors that must be investigated extensively to improve most acid production processes. The problems associated with acid toxicity can be partly solved by adapting the microorganisms to low pH and conducting fermentation at pH values lower than the pKa of the acid. This would also circumvent the use of large amounts of the acid titrant, thereby lowering the overall production cost. However, the final 3-HP concentration may be lower (Sauer et al., 2008).
Similar to other acids, 3-HP causes pH-based growth inhibition. In addition, anion-specific interference with cellular metabolism has been observed. Warnecke et al. (2010) reported that 3-HP toxicity was related to inhibition of the chorismate and threonine super-pathways. Specifically, they tested the 3-HP tolerance of 10 different clones homologously expressing the enzyme(s) involved in chorismate and threonine super-pathways and observed a significant increase in 3-HP tolerance when measured by the minimum inhibitory concentration (MIC). Furthermore, they found that 3-HP tolerance was improved when some metabolites in the chorismate or threonine super- pathways were added to the culture medium. Taken together, the results of their study suggest that alleviation of the inhibition of either of these super-pathways can mitigate the toxicity of 3-HP.
3-HPA is a toxic intermediary compound produced during glycerol-based 3-HP production and its accumulation should be avoided. The accumulation of 3-HPA at 15–30 mM often completely stops 3-HP production. Although several hypotheses have been proposed for its bactericidal action, the exact mechanism is unclear (Bar- birato et al., 1996; Rasch, 2002). 3-HPA accumulation is caused by an imbalance between the activities of DhaB and AldH and/or PDOR. In addition, PDO buildup can contribute to the accumulation of 3-HPA since PDOR can catalyze the reverse reaction (PDO→3-HPA) under high PDO concentrations. During 3-HP production from glycerol, 3-HPA accumulation can be avoided by balancing the rates of its production and consumption. This can be accomplished by either reducing DhaB activity or enhancing the activity of AldH (Celi´nska, 2011). The former strategy would reduce the overall rate of production; therefore, the latter is considered to be more desirable.
14.5.2 Redox Balance and By-products Formation
Regeneration of NAD+is a challenging issue in glycerol-based 3-HP production.
Redox balance affects a broad range of genes, cellular functions, and metabolite profiles. Continuous regeneration of NAD+is necessary for high 3-HP production, and the easiest way to accomplish this without wasting carbons is to conduct the cultivation under fully aerobic conditions. However, the maintenance of a high oxygen concentration suppresses the expression of the dha operon (Celi´nska, 2011) and synthesis of vitamin B12. If oxygen levels are reduced to microaerobic/anaerobic
conditions, a large amount of by-products (lactic acid, acetic acid, and ethanol) are accumulated and 3-HP yield is decreased. It is challenging to determine and maintain optimal oxygen levels that allow continuous regeneration of NAD+while not interfering with the synthesis of vitamin B12or proper carbon flux toward 3-HP production. Anaerobic 3-HP production in the presence of nitrate is one strategy to deal with this problem. The potential of this approach was demonstrated by the high 3- HP final titer,∼22 g/L, in one recombinantK. pneumoniae(Ashok et al., 2013). In this case, it is essential to maintain high nitrogenase activity to actively regenerate NAD+ and prevent accumulation of the toxic nitrite. The NADH accumulation can also be alleviated by introducing NADH oxidase, which converts NADH into NAD+using molecular oxygen (Auzat et al., 1999; Hummel and Riebel, 2003). This method will potentially be useful when the rate of NADH oxidation by NADH dehydrogenase(s) in the ETC is limited by a low ATP synthesis rate due to low ATP synthetase activity.
NADH oxidation can also be limited when intracellular ATP levels are high due to the rapid production of ATP by oxidative phosphorylation. Overexpression of transhydrogenase and interconversion of NADH and NADPH will also help to some extent, although these methods have never been tested.
14.5.3 Vitamin B12Supply
Glycerol dehydratase (DhaB) is a vitamin B12-dependent enzyme that catalyzes the free radical–mediated conversion of glycerol into 3-HPA. Coenzyme B12is often inac- tivated during this reaction and should therefore be supplied continuously (Toraya, 2002). In many microorganisms, includingK. pneumoniae, coenzyme B12is not syn- thesized well under aerobic conditions, which are needed for active regeneration of NAD+.Pseudomonas denitrificansis known to produce vitamin B12under aerobic conditions. The use ofP. denitrificansas a host can be an effective strategy for solv- ing the problems associated with aerobic vitamin B12synthesis. However, glycerol metabolism has not been reported inP. denitrificans, and this organism has not been studied as a recombinant host and no genetic tool box is currently available for this strain. The other option is introduction of the complete vitamin B12biosynthetic path- way ofP. denitrificansinE. coli. However, this will also be challenging since more than 20 genes of the vitamin B12biosynthetic pathway should be properly expressed inE. coli.