Physiological investigations of the influences of byproduct pathways on 3‐hydroxypropionic acid production in Klebsiella pneumoniae

Klebsiella pneumoniae can naturally synthesize 3‐hydroxypropionic acid (3‐HP), 1,3‐propanediol (1,3‐PD), and 2,3‐butanediol (2,3‐BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3‐HP production, in this study, a total of eight byproduct‐producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from the K. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24‐h shake‐flask cultivation, the adhP‐ and pflB‐deletion mutants produced 0.41 and 0.44 g/L 3‐HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3‐HP in 24‐h shake‐flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3‐hydroxypropionaldehyde (3‐HPA) to 3‐HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC‐puuC) (pTAC‐puuC is PuuC expression vector) generated 66.91 g/L 3‐HP with a cumulative yield of 70.84% on glycerol in 60‐h bioreactor cultivation. Additionally, this strain showed 2.3‐, 5.1‐, and 0.67‐fold decrease in the concentrations of 1,3‐PD, 2,3‐BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC‐puuC). These results indicated that the byproducts exerted differential impacts on the production of 3‐HP, 1,3‐PD, and 2,3‐BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3‐propanediol oxidoreductase (DhaT) that catalyzes 3‐HPA to 1,3‐PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3‐HP production. This study provides a deep understanding of how byproducts affect the production of 3‐HP, 1,3‐PD, and 2,3‐BD in K. pneumoniae.


| INTRODUCTION
Bioproduction of chemicals has emerged as an alternative to conventional chemical synthesis due to depletion of oil reserves and deterioration of the environment. 3-Hydroxypropionic acid (3-HP) is one of 12 value-added chemicals proposed by the United States Department of Energy (DOE) in 2004 [1]. As a versatile platform chemical, 3-HP can be easily converted into a series of bulk chemicals including acrylic acid, acrylonitrile [2], 1,3-propanediol (1,3-PD), 3-hydroxypropionaldehyde , and malonic acid [3]. In addition, 3-HP as a monomer can be polymerized to form poly(3-hydroxypropionate) (P3HP), which is an unnatural polyhydroxyalkanoate exhibiting biocompatibility and biodegradation attributes [4]. To date, diverse microbes have been exploited in the synthesis of 3-HP, including Escherichia coli [5,6], Corynebacterium glutamicum [7], Saccharomyces cerevisiae [8], Methylobacterium extorquens [9], and Klebsiella pneumoniae [10]. K. pneumoniae is a promising species for production of 3-HP because of its noticeable biochemical properties such as active cell growth, high ability to metabolize glycerol as well as innate capability to synthesize 3-HP. In particular, K. pneumoniae can naturally produce vitamin B 12 , which is the cofactor of glycerol dehydrogenase, a key enzyme for biosynthesis of 3-HP and 1,3-PD [11,12]. That is, production of 3-HP and 1,3-PD by K. pneumoniae fermentation does not need additional vitamin B 12 and thus, reduces production cost.
Although massive efforts have been focused on deciphering of the dha regulon, the relationship between byproducts and 3-HP remains unclear [24]. To clarify this, we individually deleted eight byproduct-synthesizing genes, including pmd (GenBank No. KPN_01632, L-lactate dehydrogenase), poxB (KPN_00904, pyruvate oxidase), frdB (KPN_04552, fumarate reductase iron sulfur subunit), fumC (KPN_01517, fumarate hydratase, class II), dhaT (KPN_03491, 1,3-propanediol dehydrogenase), ilvH (KPN_00083, acetolactate synthase I/III small subunit), adhP (KPN_01853, alcohol dehydrogenase, propanol-preferring), and pflB (KPN_00931, formate C-acetyltransferase). Metabolic analysis of the mutants was done to systematically evaluate the impacts of each byproductproducing gene on glycerol consumption, cell growth, and 3-HP production. The objective of double deletion of genes was to determine whether combinatory knockout of byproduct-producing genes can reallocate metabolic flux. Overexpression of PuuC, an aldehyde dehydrogenase (ALDH) native to K. pneumoniae [25], was done to assess 3-HP production in this chassis strain. Overall, this study aims to offer a deeper understanding of byproduct pathways in K. pneumoniae, which may shed light on basic research and manufacturing of chemicals.

| Strains, vectors, and chemicals
The strain of E. coli Top10 was purchased from the China General Microbiological Culture Collection Center (CGMCCC). The strain of K. pneumoniae DSM 2026 was purchased from DSMZ GmbH, Germany. Vector pET-28a is a product of Novagen. The original T7 promoter in pET-28a was replaced by the tac promoter, leading to the vector pTAC. Vector pTAC-puuC was previously constructed, where puuC is an ALDH native to K. pneumoniae, and its expression is controlled by the tac promoter [10]. The E. coli strain was cultivated in F I G U R E 1 Glycerol pathways in Klebsiella pneumoniae. Red crosses indicate byproduct-producing enzyme genes to be knocked out. 1,3-PD, 1,3-propanediol; 2,3-BD, 2,3-butanediol; 3-HP, 3-hydroxypropionic acid; 3-HPA, 3

| Construction of the recombinants
To investigate the impacts of byproducts on 3-HP production, a total of eight genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually disrupted from the K. pneumoniae genome. To achieve this, their upstream and downstream homologous arms of 1,000 bp each were cloned by polymerase chain reaction (PCR) from the genomic DNA of K. pneumoniae. The PCR protocol used was: 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 55°C for 45 s, 72°C for X min, where X depends on the length of the amplified gene. The upstream and downstream homologous arms as well as chloramphenicol resistance gene were ligated and cloned into the vector pTAC, resulting in the vectors pET-pmdUD, pET-poxBUD, pET-frdBUD, pET-fumCUD, pET-dhaTUD, pET-ilv-HUD, pET-adhPUD, and pET-pflBUD, respectively ("UD" indicates upstream and downstream homologous arms). These vectors were separately transformed into K. pneumoniae and cultivated in an LB plate containing 170 μg/ml chloramphenicol for screening mutants. The mutants were further confirmed by colony PCR and sequencing. To dispel the vectors out of cells, the K. pneumoniae mutants were grown in LB medium containing 50 mM CaCl 2 for 3 days. The strains unable Not I to survive in LB chloramphenicol plates were the strains devoid of vectors and named K. pneumoniaeΔpmd, K. pneumoniaeΔpoxB, K. pneumoniaeΔfrdB, K. pneumo-niaeΔfumC, K. pneumoniaeΔdhaT, K. pneumoniaeΔilvH, K. pneumoniaeΔadhP, and K. pneumoniaeΔpflB, respectively. These strains, devoid of vectors, were further confirmed by plasmid extraction and agarose gel electrophoresis. Double deletion of genes followed the protocol of RecA homologous recombination. The recombinants were confirmed by colony PCR and sequencing.

| Shake-flask and bioreactor cultivation
Before shake-flask cultivation, the strains were precultivated in LB medium containing the following components per liter: 10 g peptone, 10 g NaCl, 5 g yeast extract, and 170 μg/ml chloramphenicol. One percent of overnight broth with OD 600 of 4.0 was inoculated to a 250 ml Erlenmeyer flask containing 100 ml medium for producing 3-HP (see aforementioned) and antibiotics as appropriate at 37°C and 150-rpm shaking. To maintain microaerobic conditions, the flasks were plugged with an O 2 -permeable cotton stopper and incubated in an orbital shaker at 180 rpm and 37°C. Samples were collected every 3 h to measure residual glycerol, biomass, and metabolites.
Fed-batch cultivation of the strains was carried out in a 5 L bioreactor (Baoxing, China) containing 3-L fermentation medium and 0.5 mM IPTG (isopropyl-β-D-thiogalactoside). The fermentation conditions were followed according to the previously reported method [10]. The strain was precultivated in 100 ml fermentation medium overnight at 37°C and then transferred to a bioreactor. The agitation speed was 400 rpm and air was supplied at 1.5 vvm. The temperature was 37°C and pH value was maintained at 7.0 by adding 5 M NaOH. Dissolved oxygen was monitored automatically. Samples were taken out every 3 h to examine cell concentration, residual glycerol, and metabolites.
2.4 | Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis K. pneumoniae strain was precultured in a test tube containing 4 ml LB medium at 37°C and rotated at 180 rpm for 12 h. One percent of overnight broth with OD 600 of 4.0 was inoculated in a 250-ml shake-flask containing 100 ml medium (for production of 3-HP), 170 mg/L chloramphenicol and 0.5 mM IPTG. After 16-h cultivation, the cell samples were harvested by centrifugation at 10,000 rpm for 5 min, and the supernatant was discarded, as the target protein was intracellularly expressed. The cells were mixed with loading buffer (Takara, China) and boiled for 5 min. Protein expression was analyzed by 12% (v/w) SDS-PAGE with cell-free extract under denaturing conditions. The pH values of stacking gel and resolving gel were 6.8 and 8.8, respectively. A Mini-Protein III Electrophoresis System (Bio-Rad) was employed to perform the operation. Coomassie Brilliant Blue R-250 (0.2%, w/v) was utilized to stain proteins on the gel. Upon decolorization, the protein concentration was determined by the Bradford method with bovine serum albumin as standard protein.

| Analytical methods
Cell density was measured by using a microplate reader at 600 nm with 200 µl fermentation broth added in a cuvette. Residual glycerol was measured by the titration method with NaIO 4 (for control of glycerol). Metabolites 3-HP, lactic acid and acetic acid were measured by using a HPLC system (Shimazu, Kyoto, Japan) equipped with a C 18 column and a SPD-20A UV detector at 210 nm. The mobile phase was 0.05% phosphoric acid at 0.8 ml/min. Column temperature was 25°C. 1,3-PD was determined by gas chromatography (GC; PerSee, China) equipped with a capillary column of TR-WAX (30 m × 0.25 mm ID, 0.25 µm). Nitrogen was used as carrier gas, and the column flow rate was maintained at 0.5 ml/min. Injector and detector temperatures were maintained at 230°C. The initial GC oven temperature was maintained at 60°C for 3 min, and increased to 180°C at a rate of 10°C/min and maintained for 5 min. All samples were filtered through a 0.22 μm membrane filter.

| Characterization of recombinants
To dissect the influences of byproducts on 3-HP production, a total of eight genes pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were independently deleted through the RecA homologous recombination technique. Colony PCR analysis showed that the above eight genes were independently but not simultaneously deleted from the K. pneumoniae genome, and the eight mutants were designated as K. pneumoniaeΔpmd, K. pneumoniaeΔpoxB, K. pneumo-niaeΔfrdB, K. pneumoniaeΔfumC, K. pneumoniaeΔdhaT, K. pneumoniaeΔilvH, K. pneumoniaeΔadhP, and K. pneu-moniaeΔpflB. In addition, the two genes adhP and pflB were shown to be double-disrupted from the K. pneumoniae genome, and the recombinant strain was named as K. pneumoniaeΔadhPΔpflB. Subsequently, this strain was transformed with vector pTAC-puuC to overproduce 3-HP, and the resulting strain was designated as K. pneumo-niaeΔadhPΔpflB(pTAC-puuC). Plasmid extraction, restriction digestion and DNA sequencing showed that the vector pTAC-puuC was correctly constructed and transformed into K. pneumoniaeΔadhPΔpflB.

| SDS-PAGE analysis
To investigate the influences of gene deletion on protein expression, K. pneumoniaeΔadhPΔpflB(pTAC-puuC) was analyzed by SDS-PAGE, and wild-type K. pneumoniae, K. pneumoniae(pET-28a), and K. pneumoniae(pTAC-puuC) were used as reference strains. As shown in Figure 2, lane 1-4 indicate wild-type K. pneumoniae, K.
pneumoniae(pET-28a), K. pneumoniae(pTAC-puuC), and K. pneumoniaeΔadhPΔpflB(tac-puuC), respectively. Due to overexpression of PuuC, a 53 kDa band was observed in K. pneumoniae(pTAC-puuC) and K. pneumoniaeΔadhPΔpflB(pTAC-puuC) strains, which was much stronger than those in wild-type and K. pneumoniae(pET-28a) strains. Moreover, the 38 KDa protein in K. pneumoniaeΔadhPΔpflB(pTAC-puuC) was significantly weaker than those in wild-type K. pneumoniae (lane 1) and K. pneumoniae(pTAC-puuC) (lane 2), indicating that the adhP gene was successfully disrupted. Interestingly, no difference was observed for the 85 kDa band (pflB) in all four strains. This may be explained by the innate low expression of pflB and probable existence of proteins having a molecular weight similar to PlfB. Colony PCR validated the fact that the pflB gene was deleted from K. pneumoniae genome. Overall, the above results suggested that the recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) was successfully engineered and the two genes adhP and pflB were double-deleted from the K. pneumoniae genome.

| Shake-flask cultivation of single gene deletion mutants
To clarify the influences of byproduct-producing enzymes on 3-HP production, a total of eight genes were individually deleted, resulting in eight mutants K. pneumoniaeΔpmd, K. pneumoniaeΔpoxB, K. pneumo-niaeΔfrdB, K. pneumoniaeΔfumC, K. pneumoniaeΔdhaT, K. pneumoniaeΔilvH, K. pneumoniaeΔadhP, and K. pneumoniaeΔpflB (Figure 3). Considering K. pneumoniae cells had vectors dispelled from them, the wild-type strain instead of the recombinant strain-harboring the empty vector was used as the control. Compared with the wild-type strain, all mutants showed increased growth in the first 12 h, however, this situation changed in the next 12 h (Figure 3). The mutants K. pneumo-niaeΔpmd, K. pneumoniaeΔpoxB, K. pneumoniaeΔfrdB, and K. pneumoniaeΔfumC displayed expedited growth, while the mutants K. pneumoniaeΔdhaT, K. pneumo-niaeΔilvH, K. pneumoniaeΔadhP, and K. pneumo-niaeΔpflB demonstrated retarded growth. Consistent with cell growth, all strains consumed glycerol and produced 3-HP in the first 12-h fermentation. Interestingly, compared with the wild-type strain, all eight mutants produced less 1,3-PD and 2,3-BD in the first 12 h, except the strain K. pneumoniaeΔpmd which produced more 2,3-BD during the entire 24-h fermentation. More importantly, the mutants K. pneumo-niaeΔadhP and K. pneumoniaeΔpflB produced higher levels of 3-HP (0.40 and 0.44 g/L, respectively) than other mutants in the first 12 h. During the entire fermentation process, K. pneumoniaeΔadhP and K. pneumoniaeΔpflB were the best-acting strains for production of 3-HP. Hence, the genes adhP and ΔpflB were double-deleted, and the resulting strain

| Shake-flask cultivation of double deletion gene mutants
Now that PCR and SDS-PAGE analysis had experimentally validated the double deletion of adhP and pflB genes from the K. pneumoniae genome, the mutant K. pneumoniaeΔadhPΔpflB(pTAC-puuC) was grown in shake-flasks to investigate 3-HP production and cell growth. To do so, wild-type K. pneumoniae, K. pneumoniae(pET-28a), and K. pneumoniae(pTAC-puuC) were used as control strains. As shown in Figure 4a, the strain K. pneumoniaeΔadhPΔpflB presented similar levels of biomass and glycerol consumption to the wild-type strain, indicating that double deletion of adhP and pflB had no significant impacts on cell growth. In contrast, the three strains K. pneumoniae(pET-28a), K.pneumoniae(pTAC-puuC), and K. pneumoniaeΔadhPΔpflB(pTAC-puuC) showed retarded growth. This may be explained by the metabolic burden imposed by the vector pET-28a or pTAC-puuC. For 3-HP production, no significant difference was observed between K. pneumoniae and K. pneumoniaeΔadhPΔpflB. However, K. pneumo-niaeΔadhPΔpflB(pTAC-puuC) produced 2.09 g/L 3-HP in 24 h, which was 1.2-fold of that produced by K.pneumoniae(pTAC-puuC). It should be noted that the lactic acid level showed irregular fluctuation, and no difference was observed for the strains K. pneumo-niaeΔadhPΔpflB and K. pneumoniaeΔadhPΔpflB (pTAC-puuC). Notably, although PflB catalyzes the formation of formic acid, the pflB-deletion mutant produced a similar level of formic acid to the wildtype strain. This may be due to the existence of multiple formic acid pathways. In view of 24-h shake-flask fermentation, we found that double deletion of adhP and pflB imposed no significant influences on cell growth.

| Bioreactor cultivation
To further elucidate the influences of double deletion of genes adhP and pflB on 3-HP production, the strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) was cultivated in a 5-L bioreactor, where the pH value was maintained at 7.0 by adding NaOH. The wild-type K. pneumoniae, K. pneumoniaeΔadhPΔpflB and K. pneumoniae(pTAC-puuC) were referenced as control. The strain K. pneumoniae(pTAC-puuC) presented a cumulative 3-HP titer in 54 h with a titer of 67.47 g/L and 65.66% yield on glycerol (Figure 5a; Table 2), while the strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) presented a 3-HP peak in 60 h with a titer of 66.91 g/L and 70.84% yield on glycerol (Figure 5b; Table 2). Importantly, the strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) produced much less lactic acid, formic acid, 1,3-PD and 2,3-BD | 1203 compared with K. pneumoniae(pTAC-puuC). These results indicated that although double deletion of adhP and pflB has little influences on 3-HP production, most byproducts were largely attenuated.

| DISCUSSION
Biosynthesis of 3-HP, 1,3-PD, and 2,3-BD in K. pneumoniae encounters buildup of byproducts, which not only take up the carbon source but also entangle downstream separation [10,16]. To decipher the influences of byproducts on 3-HP production, here, a total of eight byproduct-synthesizing genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from K. pneumoniae genome, and the resultant eight mutants were cultivated in shake-flasks for metabolic analysis. Results showed that the eight mutants demonstrated different levels of cell growth, glycerol consumption, and the formation of 3-HP, 1,3-PD, and 2,3-BD. Although all mutants produced more 3-HP than wild-type K. pneumoniae in the first 12 h, this situation changed in the next 12 h. As shown in Figure 3, compared to wild-type K. pneumoniae, the mutants K. pneumoniaeΔfrdB, K. pneumoniaeΔilvH, K. pneumo-niaeΔadhP, and K. pneumoniaeΔpflB produced more 3-HP, while the other mutants produced less 3-HP ( Figure 3). Notably, the mutants K. pneumoniaeΔadhP and K. pneumoniaeΔpflB generated 0.42 and 0.45 g/L 3-HP, respectively, which were higher than that of other mutants. To further enhance 3-HP production, the two genes adhP and pflB were double-deleted. The resulting mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3-HP in 24-h shake-flask cultivation. When this strain was transformed with vector pTAC-puuC that catalyzes the formation of 3-HP [10], the recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC-puuC) was engineered. In a 5-L bioreactor, this strain produced 66.91 g/L 3-HP in 60 h with 70.84% yield on glycerol ( Figure 5b and Table 2). Although this 3-HP titer (66.91 g/L) was lower than that produced by K. pneumoniae(pTAC-puuC) (67.47 g/L; Figure 5a and Table 2), the byproducts acetic acid, 1,3-PD, and 2,3-BD in K. pneumoniaeΔadhPΔpflB(pTAC-puuC) were significantly attenuated, indicating that double disruption of adhP and pflB remarkably altered metabolic flux. Overall, these results indicated that byproduct-producing genes exhibited differential influences on 3-HP production. For glycerol pathways (Figure 1), the enzyme genes far away from 3-HP imposed minimal impacts on glycerol consumption and 3-HP formation, while the enzyme genes in the vicinity of 3-HP such as dhaT and puuC substantially affected 3-HP production. To the best of our knowledge, this is the first systematic exploration on how byproducts influence glycerol metabolism in K. pneumoniae.
Deletion of competing pathways is a common strategy to minimize byproducts [17]. However, this strategy in most cases compromises cell viability and thereby hinders biosynthesis of desired metabolites [26]. To date, 3-HP production remains to be improved. This is mainly due to incomplete understanding of the dha regulon that governs 3-HP biosynthesis. In view of our study and the work of other groups, we realize that the dha regulon in K. pneumoniae manifests both structural "plasticity" and "rigidity". The "plasticity" indicates that disruption of partial pathways has no significant impacts on cell growth. This viewpoint is evidenced by the pflB-and pmd-deletion mutants which showed similar levels of biomass to wild-type strain in 12-h cultivation (Figure 3). This phenomenon may be attributed to tailored metabolic compensation. For instance, although DhaT catalyzes the formation of 1,3-PD, the dhaT-deletion mutant still produced 1,3-PD because of the expression of NAD (P)H-dependent hypothetical oxidoreductase, an isoenzyme of DhaT [17]. In contrast to "plasticity", the structural "rigidity" of the dha regulon indicates that some pathways are essential for cell viability, and deleting them may substantially halt cell growth or even trigger cell death. For instance, compared with wild-type strain, the dhaT-deletion mutant presented slower growth and produced less 1,3-PD and 3-HP in 24-h fermentation ( Figure 3). The reason behind this is that DhaT catalyzes 3-HPA into 1,3-PD, and this reaction is a central metabolism in K. pneumoniae when glycerol is the sole carbon source (Figure 1). In addition, this reaction converts NADH to NAD + , and NAD + is the cofactor of ALDH that catalyzes 3-HPA into 3-HP. Presumably, 1,3-PD biosynthesis is coupled with 3-HP production via cofactor recycling [27], implying the feasible coproduction of 1,3-PD and 3-HP [20,21]. In addition to supplying cofactor, byproduct pathways F I G U R E 4 Shake-flask cultivation of Klebsiella pneumoniae strains for production of 3-hydroxypropionic acid. K. pneumoniae WT, wild-type K. pneumoniae; K. pneumoniae(pET-28a), recombinant K. pneumoniae-harboring empty vector pET-28a; K. pneumoniae(pTAC-puuC), recombinant K. pneumoniae-harboring PuuC expression vector pTAC-puuC; K. pneumoniaeΔadhPΔpflB(pTAC-puuC), the mutant K. pneumoniaeΔadhPΔpflB-harboring PuuC expression vector pTAC-puuC. PuuC, an aldehyde dehydrogenase native to K. pneumoniae. 3-HP, 3-hydroxypropionic acid; 1,3-PD, 1,3-propanediol; 2,3-BD, 2,3-butanediol provide ATP for the cells. For example, the enzyme genes pmd, poxB, adhP, fumC, and frdB participate in glycolysis and tricarboxylic acid cycle and thus supply energy for the cells (Figure 1). In summary, glycerol metabolism relies on both core and subsidiary pathways. Although all pathways affect 3-HP production, only a small part of them are pivotal.
From the viewpoint of genetics, the fermentation titer is a quantitative trait that depends on multiple factors. Of the myriad factors affecting 3-HP production, ALDH is most influential because it directly catalyzes 3-HPA to 3-HP. Previously, PuuC as a native ALDH was overexpressed in K. pneumoniae, and 83.8 g/L 3-HP was produced in a 5-L bioreactor [10]. This study suggests that optimizing key enzymes instead of multiple enzymes also enables overproduction of desired metabolites. This "pathway-focused approach" is popular in most labs, even though rational design-dependent systems metabolic engineering may be more powerful in strain engineering. In fact, systems metabolic engineering is time-consuming due to its reliance on global reprograming of cell metabolism and subsequent high throughput screening. Hence, the "pathway-focused strategy" is more applicable in most cases. Following this thinking, three strategies might be feasible for boosting 3-HP production in K. pneumoniae. The first is directed evolution of ALDH [15], aiming to improve its specificity toward 3-HPA which is toxic to cells. A research team in Korea altered the substrate specificity of α-ketoglutaric semialdehyde dehydrogenase (KGSADH), an enzyme from Azospirillum brasilense catalyzing 3-HPA to 3-HP [28]. The improved KGSADHs exhibited lower K m values for both 3-HPA and NAD + . The enzymes also displayed higher substrate specificities for aldehyde and NAD + and weaker inhibition by NADH. Furthermore, the recombinant Pseudomonas denitrificans strain carrying one of KGSADH variants exhibited less 3-HPA and higher cell growth compared with the wild-type KGSADH [29]. The second approach for improving 3-HP production may be promoter engineering, by which sufficient RNA polymerases (RNAPs) can be recruited and thus facilitate PuuC expression and boost 3-HP F I G U R E 5 Bioreactor cultivation of recombinant Klebsiella pneumoniae strains for production of 3-hydroxypropionic acid.  Abbreviations: 1,3-PD, 1,3-propanediol; 2,3-BD, 2,3-butanediol; 3-HP, 3-hydroxypropionic acid; AA, acetic acid; GCR, glycerol conversion rate. K. pneumoniae (pTAC-puuC), recombinant K. pneumoniae strain-harboring vector pTAC-puuC, where puuC is controlled by the tac promoter; K. pneumoniaeΔadhPΔpflB (pTAC-puuC), mutant K. pneumoniaeΔadhPΔpflB-harboring PuuC expression vector pTAC-puuC.
production [30]. It has been shown that RNAP as an intracellular "resource allocator" significantly affects metabolic flux [31,32]. The third strategy is mitigation of metabolite inhibition on the host cell. It has been shown that adjusting the pH value with NaOH benefits cell growth and thus facilitates 3-HP production [10]. This may be ascribed to the close coupling between 3-HP production and cell growth especially in the exponential phase [27]. Judging from this, measures facilitating cell growth might benefit 3-HP production.
In addition to cell growth, many other factors also contribute to 3-HP production, such as substrate provision, cofactor availability, redox balance, and cell tolerance to substrate and metabolites. In this study, we systematically investigated the influences of byproduct pathways on the production of 3-HP, 1,3-PD, and 2,3-BD. We believe that this study offers valuable insights for basic research and metabolic engineering of K. pneumoniae.