The development of microbial cell factories is fueled by aspirations to develop sustainable processes based on renewable resources. The goal is mitigation of the negative environmental consequences of production of fuels, chemicals and other materials.

The development of microbial cell factories for production of the platform chemical 3-hydroxypropanoic acid (3-HP) has attracted much attention in the last decade. 3-HP is a non-chiral (optically inactive), small three-carbon molecule and a structural isomer of lactic acid. Specifically, 3-HP is a precursor for the production of a number of valuable chemicals including acrylic acid and bioplastics (Figure 1). Bio-based production of 3-HP also has the potential to “turn waste into a resource” since several metabolic pathways exist for converting glycerol, a by-product of biodiesel production, into 3-HP.

Here, we will briefly introduce the metabolic pathways that can be used to produce 3-HP from glycerol. Based on work accumulated over the last decade, we will present current knowledge pertaining to different production hosts, enzymes, and strategies for optimization of production pathway and host metabolism as well as process engineering for attaining high-level production of 3-HP. Finally, we will discuss how recent developments in synthetic biology and metabolic engineering might form the basis for further improvements of production strains and the eventual goal of industrially viable and sustainable 3-HP production.

A number of microorganisms have been used for the production of 3-HP, both natural isolates as well as engineered microorganisms (Table 1). For the selection of an appropriate production host, there are several parameters to consider. The microorganism should demonstrate tolerance to organic acids, specifically 3-HP, as well as potentially toxic impurities in crude glycerol. As one of the widely used glycerol dehydratases is coenzyme B₁₂-dependent, the production host should preferentially be capable of synthesizing coenzyme B₁₂. Addition to the production medium instead of in situ synthesis would significantly increase the overall production cost.

Lactobacillus reuteri is capable of naturally producing 3-HP from glycerol via the CoA-dependent pathway (Luo et al., 2011). In one study, three L. reuteri strains were evaluated, and all were found to produce 3-HP (Burgé et al., 2015). It also was shown that 3-HPA is toxic at a concentration of 5 g/L while 2.5 g/L 3-HP is not (as long as pH is maintained above 5). Notably, L. reuteri is capable of synthesizing coenzyme B₁₂ (Burgé et al., 2015) and has been used for the bioconversion of biodiesel-derived glycerol into 3-HP (14 g/L) and 1,3-propanediol (1,3-PDO) (Dishisha et al., 2015). To our knowledge, there have been no attempts to engineer L. reuteri strains to improve production. Among lactic acid bacteria, production of 3-HP is not restricted to L. reuteri. Of 67 lactic acid bacteria isolates tested, 22 isolates belonging to Lactobacillus diolivorans and Lactobacillus collinoides were positive for 3-HP production from glycerol (Garai-Ibabe et al., 2008). Additionally, L. diolivorans is capable of using biodiesel-derived glycerol for the production of 3-HPA (Lindlbauer et al., 2017).

Klebsiella pneumoniae is a pathogenic bacterium that has been widely used for production of 3-HP. Along with E. coli, K. pneumoniae is one of the most frequently used hosts for strain development by genetic engineering for improved 3-HP production. In fact, the highest 3-HP titer reported, 83.8 g/L, was obtained in K. pneumoniae by the combining of optimized expression of aldehyde dehydrogenase (K. pneumoniae PuuC), blocking of lactic acid synthesis (ldh1, ldh2, and pta mutant), and optimization of the fermentation conditions (Li et al., 2016). An important asset of K. pneumoniae is its capability of producing coenzyme B₁₂ (Luo et al., 2011).

E. coli is commonly used for metabolic engineering to produce a wide variety of compounds. It has also been widely used as a chassis for the production of 3-HP. The highest 3-HP titer reported in E. coli so far is 71.9 g/L, using a strain in which, besides introduction of glycerol dehydratase and aldehyde dehydrogenase, the central metabolism was modified to reduce by-product formation (Chu et al., 2015). It has been indicated that careful choice of the strain could be of importance. A comparison of nine E. coli strains in which a heterologous pathway for 3-HP synthesis was introduced, demonstrated differences in 3-HP production, as well as in enzyme level and activity (Sankaranarayanan et al., 2014). A drawback of using E. coli is the fact that it is not naturally capable of producing coenzyme B₁₂. However, insertion of the Pseudomonas denitrificans genes for coenzyme B₁₂ synthesis (more than 25 genes in 6 operons) on three plasmids led to the production of coenzyme B₁₂ under both anaerobic and aerobic conditions (Ko et al., 2014).

While K. pneumoniae and E. coli are by far the most widely used production hosts, a number of other bacteria have been engineered for 3-HP production as well. P. denitrificans synthesizes coenzyme B₁₂ in aerobic conditions where NAD(P)⁺ is efficiently regenerated (Zhou et al., 2013). Introduction of glycerol dehydratase and glycerol dehydratase reactivase from K. pneumoniae allowed for the production of 3.4 g/L 3-HP. By further introducing the K. pneumoniae aldehyde dehydrogenase, the yield increased to 4.9 g/L (Zhou et al., 2013).

Another bacterium able to synthesize coenzyme B₁₂, Shimwellia blattae, is a 1,3-PDO producer using a native coenzyme B₁₂-dependent glycerol dehydratase. Introduction of various genes including aldehyde dehydrogenase from Pseudomonas putida KT2442 enabled the production of 0.26 g/L Poly-3HP using crude glycerol from biodiesel production as the substrate (Heinrich et al., 2013).

Corynebacterium glutamicum was engineered to produce 3-HP from glucose and xylose. By a combination of efforts, an impressive titer of 62.6 g/L 3-HP was obtained in a fed-batch fermentation (Chen et al., 2017). The steps taken to achieve this titer included enabling efficient production of glycerol from glucose, introduction of the K. pneumoniae pduCDEGH genes encoding diol dehydratase and its reactivase and Cupriavidor necator aldehyde dehydrogenase and modification in sugar uptake and glycolytic flux. In Bacillus subtilis, introduction of glycerol dehydratase along with its reactivases, and aldehyde dehydrogenase from K. pneumoniae, enabled the production of 3-HP from glycerol. Upon inactivation of glycerol kinase and optimization of growth conditions, a 3-HP titer of 10 g/L was obtained in a shake flask culture (Kalantari et al., 2017). A drawback of both of these bacteria is their inability to synthesize coenzyme B₁₂, which thus has to be supplemented to the production medium.

In an engineered cyanobacterium Synechococcus elongatus CO₂ was converted via glycerol to 3-HP, albeit at very low titer (31.7 mg/L). Synechococcus elongatus is unable to synthesize coenzyme B₁₂, but the problem was alleviated by producing 3-HP under anaerobic conditions using the oxygen-sensitive B₁₂-independent glycerol dehydratase from Clostridium butyricum (Wang et al., 2015). The use of glycerol as an additional carbon source in a strain engineered to assimilate glycerol has been suggested for increasing productivity (Kanno and Atsumi, 2017).

As mentioned above, there are two principal pathways for production of 3-HP from glycerol. In both CoA-dependent and -independent pathways, the initial step is the conversion of glycerol to 3-HPA. In the CoA-dependent pathway, 3-HPA is converted via 3-hydroxypropanoyl-coenzyme A (3-HP-CoA) and 3-hydroxypropanoyl-phosphate (3-HP-P) to 3-HP, catalyzed by propionaldehyde dehydrogenase, phosphotransacylase, and propionate kinase, respectively (Dishisha et al., 2014). In the CoA-independent pathway, 3-HPA is converted directly to 3-HP by the action of aldehyde dehydrogenase (Kumar et al., 2013a). Both of the pathways have characteristics that are of importance for the design of cell factory and process conditions, for example, oxygen-sensitivity of enzymes and the need for co-factors such as coenzyme B₁₂, and NAD⁺. In the following, we will describe the enzymes that make up these two pathways. Where available, kinetic data for the enzymes is presented in Table 2, to facilitate comparison.

There are several parameters to consider when engineering bacteria to produce 3-HP. As covered in sections Production Hosts and The Suite of Enzymes Used in Heterologous 3-HP Synthesis Pathways, a suitable host should be established and in most cases some or all of the enzymes needed for converting glycerol to 3-HP should be introduced. This provides a basis for 3-HP production, but is not enough to ensure the desired high titers. There are several hurdles that need to be overcome, and these include, but are not limited to, proper balancing of enzyme activities to prevent buildup of the toxic intermediate 3-HPA, efficient channeling of substrate into product, co-factor regeneration, prevention of by-product formation and countering of stress. In the following, we will explore challenges and strategies that have been applied in the engineering of strains for 3-HP production.

In a majority of the studies reviewed for this paper, the ability of the recombinant strains to produce 3-HP was tested in 1.5–5 L bioreactors operated in fed-batch mode (Table 3; Supplementary Table 1). A fed-batch operation typically entails growing the cells until the end of the exponential phase. This is followed by the addition of feed medium containing the substrates, thus keeping the growth rate of the microorganism at a desired level (Liu, 2017). A fed-batch mode of operation has certain advantages over batch fermentation. For instance, a fed-batch operation has the ability to prevent overflow metabolism, that is, the phenomenon where fast-growing cells, in the presence of oxygen, utilize fermentation instead of respiration to produce energy (Basan et al., 2015). Another advantage of a fed-batch operation is its ability to maintain the substrate concentration at a low level (de Fouchécour et al., 2018). This is particularly important when the substrate is glycerol. It was reported that no growth of K. pneumoniae cells could be seen above 110 and 133 g/L glycerol (extrapolated values) in aerobic and anaerobic conditions, respectively, with glycerol concentrations higher than 40 g/L showing significant growth inhibition (Cheng et al., 2005). Consequently, high titers of 3-HP cannot be obtained from glycerol in a batch fermentation. In L. reuteri, the conversion of glycerol to 3-HPA was reported to be about 10 times faster than the subsequent conversion of 3-HPA to 3-HP and 1,3-PDO, resulting in the accumulation of the toxic intermediate 3-HPA. To circumvent this problem, a fed-batch fermentation process was established. It produced 3-HP and 1,3-PDO without any accumulation of 3-HPA. This was achieved by identifying a maximum value of specific glycerol consumption rate at which only 1,3-PDO and 3-HP are produced, and no accumulation of 3-HPA takes place. By maintaining the glycerol amount in the bioreactor such that its specific consumption rate stayed under this maximum value, a 3-HP titer of 14 g/L with a productivity of 0.25 g/L.h was obtained (Dishisha et al., 2014, 2015). This underlines how the knowledge of cell metabolism can be used to solve production problems by means of process engineering.

The challenge concerning the requirement of anaerobic conditions for coenzyme B₁₂ generation and aerobic conditions for efficient NAD⁺ regeneration for production of 3-HP from glycerol was tackled by electro-fermentation in a recently published study (Kim et al., 2017). Certain microorganisms possess the ability to deliver electrons to solid electrodes when grown under anaerobic conditions. These exoelectrogens, as these microorganisms are called, can be used to control the redox state in the cells independent of the electron transport chain or fermentation. Electro-fermentation utilizes this property of exoelectrogens to drive the unbalanced fermentations. Anodic electro-fermentation has been used for bioconversion of glycerol to 3-HP using recombinant K. pneumoniae over-expressing KGSADH from A. brasilense. An electrical potential of + 0.5 V vs. Ag/AgCl was applied to the anode, with 2-hydroxy-1,4-naphthoquinone used for shuttling the electrons between the bacteria and the anode. The transfer of electrons from the bacteria to the anode during anaerobic fermentation led to a decrease in NADH/NAD⁺ ratio in the cells, and hence to an enhanced 3-HP production as compared to the fermentative control (Kim et al., 2017). Although the 3-HP titer reached was low, the study nevertheless demonstrated that it is possible to improve NAD⁺ regeneration under anaerobic conditions. With further optimization, this novel strategy might pave the way for providing new opportunities to enhance 3-HP production from glycerol. It might similarly enhance other bioconversions where there is a need to control the intracellular redox state of a cell factory (Kim et al., 2017; de Fouchécour et al., 2018).

In a fermentation operation, the process parameters such as aeration rate, medium composition, etc. play an important role in determining the final outcome. While there are ample reports on strain development, generally less focus has been put on the optimization of the production conditions. Conversion of glycerol to 3-HP being an oxidative reaction, the aeration rate does affect the final yield of the product (de Fouchécour et al., 2018). In one study, aerobic, micro-aerobic and anaerobic conditions were tested in batch fermentations performed to convert glycerol to 3-HP using recombinant K. pneumoniae. They reported that amongst the three conditions tested, the micro-aerobic condition yielded the highest 3-HP titer (2.2 g/L; Zhu et al., 2009). In a more recent study, the transcript levels of the genes encoding the enzymes of the CoA-independent pathway of glycerol conversion to 3-HP and the genes coding for the enzymes of the formate hydrogen lyase pathway were analyzed to understand the intracellular response of K. pneumoniae under aerobic, micro-aerobic and anaerobic growth conditions (Huang et al., 2016). The transcription of the glycerol dehydratase operon was downregulated in the presence of oxygen, while aldehyde dehydrogenase-, hydrogenase- and formate dehydrogenase-coding genes were found to be upregulated. The authors suggested that in the presence of oxygen, the formate hydrogen lyase pathway consumed the excess NADH generated due to the over-expression of aldH (Huang et al., 2016). In another study aimed at enhancing the coproduction of 3-HP and 1,3-PDO in K. pneumoniae, the production of by-products such as lactate, ethanol, succinate, and acetate was reduced by disrupting their synthesis (Ko et al., 2017). However, the disruption of pta-ackA gene of the acetate pathway led to a reduction in cell growth, glycerol uptake, and 3-HP and 1,3-PDO production. The authors bypassed the disruption of this gene for reducing acetate production by testing various agitation speeds between 200 and 600 rpm. They reported an increase in 3-HP yield from 0.18 to 0.38 mol_3−HP/mol_glycerol, respectively, which could be attributed to an enhanced cell growth rate because of better oxygen transfer at 600 rpm. Although acetate production was not completely abolished, they did manage to reduce it from 163 mM (at 400 rpm) to only 80 mM (at 600 rpm; Ko et al., 2017). Recently, a production of 61.9 g/L 3-HP with a yield of 0.58 mol_3−HP/mol_glycerol in 38 h in a 5 L bioreactor operated in fed-batch mode by using an engineered K. pneumoniae strain was reported (Jiang et al., 2018). The aeration rate was reduced to half of the initial rate once the cell biomass OD reached close to the maximum value. Furthermore, using the same strategy, they were able to scale-up the process in a 300 L bioreactor and reported a titer of 54.5 g/L for 3-HP with a yield of 0.58 mol_3−HP/mol_glycerol in 51 h.

The growth medium used for cultivating a microorganism plays an important role in influencing the production process. The current record holder for the highest 3-HP titer, addressed this issue by testing different media for the production of 3-HP in K. pneumoniae in shake flasks (Li et al., 2016). A gradient concentration of each component of the media (except CaCl₂) was individually analyzed. The ameliorated medium enabled an increase of 80.5% in the production of 3-HP. They also tested the effect of pH on 3-HP production and found pH 7.0 to be the most appropriate for 3-HP production (Li et al., 2016). To optimize the growth medium for 3-HP production using L. reuteri, 30 different media with variable amounts of sugar beet and wheat processing coproducts (used as the carbon source), yeast extract, tween 80 and vitamin B₁₂ were tested. The authors reported an increase in 3-HP production yield by 70 %, accompanied with a decreased 3-HPA titer (Couvreur et al., 2017).

Another way to solve the earlier-mentioned challenge of conflicting requirements for coenzyme B₁₂ generation and NAD⁺ regeneration, is to divide the conversion of glycerol to 3-HP into 2 steps. First, glycerol is converted into either 3-HPA or a mixture of 3-HP and 1,3-PDO. The second step involves the conversion of 3-HPA and 1,3-PDO to 3-HP. This two-step strategy was employed in a study where wild-type L. reuteri resting cells first converted glycerol to 1,3-PDO and 3-HP in equimolar amounts. In the subsequent step, the supernatant was fed to the resting cells of Gluconobacter oxydans which oxidized 1,3-PDO to 3-HP, yielding a 3-HP titer of 23.6 g/L (Dishisha et al., 2015). In a similar study, Zhao et al. employed K. pneumoniae in the first step to convert glycerol into 1,3-PDO. In the second step, the resting cells of G. oxydans were introduced in the same bioreactor after heat inactivating K. pneumoniae. This led to the conversion of 1,3-PDO to 3-HP with a yield of 0.52 mol/mol on glycerol and 0.94 mol/mol on 1,3-PDO, and a 3-HP titer of 60.5 g/L (Zhao et al., 2015). The advantage of using two bioconversion steps is that each uncoupled step can be optimized separately. As attractive as it may appear because of high yields, the industrial implementation of this two-step strategy may not be as cost-effective and economical as a single step fermentation because of higher associated costs. Nevertheless, this strategy could well be a first step for achieving an integrated continuous process for the production of 3-HP, which in addition would be highly desirable because of its low operating costs (de Fouchécour et al., 2018).

As outlined in this review considering the case of 3-HP production in bacterial cell factories, there are many elements to the successful design of a suitable production strain, ranging from the choice of organism, pathway, and enzymes to engineering of the cell for improved flux through the production pathway and the ability to tolerate the stress imposed by intermediate, by-, and final products (Figure 4). Most of the studies described in this review have targeted some of these elements. The highest titer recorded so far is 83.8 g/L which was achieved in K. pneumoniae by optimizing the expression of aldehyde dehydrogenase in the production pathway and increasing flux through the production pathway by blocking synthesis of the by-products lactic acid and acetic acid (Li et al., 2016). This impressive feat was based on rational design and accomplished by combining some of the successful approaches outlined above in very few engineering steps.

For bacterial 3-HP production to reach a level of economic viability, further improvements in titer, and productivity will be necessary. By now, many different enzyme variants have been used for constructing 3-HP production pathways but in many cases the enzymes have not been characterized biochemically. A better understanding of the properties of the enzymes with respect to catalytic properties, pH range, and stability could prove instrumental in identifying the optimal enzymes and would also help to identify enzyme qualities to improve by enzyme engineering.

The impressive body of research on strain engineering has identified numerous challenges and solutions such as balancing enzyme activities, preventing by-product formation and alleviating stresses. With this accumulating knowledge base pertaining to 3-HP cell factory design, it seems likely that it will be possible to further tweak production by combining all of the successful approaches described herein. To facilitate this work, powerful engineering techniques are continually emerging that allow the design of large combinatorial libraries of targeted mutations. Notably, the recent development of a 3-HP biosensor can be used in combination with adaptive laboratory evolution for identifying as-of-yet unknown mechanisms for further fine-tuning of the production strains. While it seems reasonable that further improvements in titer will be achieved in the coming years, there should be an increased focus to also develop industrially relevant process designs and subsequent upscaling in order to provide proof of concept.

In conclusion, the massive developments in synthetic biology and metabolic engineering could be expected to soon deliver the long-sought goal of converting the abundant by-product glycerol into the much desired value-added chemical 3-HP.

CJ, AG, and AK performed literature search and drafted the manuscript. All authors revised the manuscript and approved the final version.