Selection of K. oxytoca PDL-0 for 2,3-BD production from lactose
To select a strain for efficient 2,3-BD production from whey, we first looked for strains that can utilize lactose and produce 2,3-BD. K. pneumonia, E. cloacae, B. licheniformis, and K. oxytoca can produce 2,3-BD from glucose [16]. E. coli BL21-pETRABC carrying the 2,3-BD pathway gene cluster from E. cloacae can also efficiently bio-transform glucose into 2,3-BD [23]. In the present study, we first compared the abilities of K. pneumonia ATCC 15380, E. cloacae SDM, B. licheniformis DSM13, K. oxytoca PDL-0, and E. coli BL21-pETRABC to produce 2,3-BD from lactose, and the results are shown in Fig. 2.
All of the five strains can grow in M9 medium supply with 5 g/L yeast extract and about 40 g/L lactose. B. licheniformis DSM13 is the only strain that can not consume lactose (Fig. 2a and Fig. 2b). E. cloacae SDM and E. coli BL21-pETRABC could efficiently utilize lactose (about 30 g/L) but these two strains only accumulated about 2 g/L 2,3-BD (Fig. 2b and Fig. 2c). K. pneumonia ATCC 15380 and K. oxytoca PDL-0 can produce 2,3-BD from lactose, with a yield of 0.21 g/g and 0.30 g/g lactose, respectively. Considering the fact that K. oxytoca PDL-0 belongs to the Risk Group 1 [15] and produces 2,3-BD with a higher yield from lactose, this strain was selected for further study in successive experiments.
Inactivation of by-product pathways in K. oxytoca PDL-0
K. oxytoca PDL-0 produced 2,3-BD as its major fermentative product during lactose fermentation in a shake flask culture. However, only 58% of theoretical yield (0.526 g/g) was observed (Fig. 2). 2,3-BD is produced by a fermentative pathway known as the mixed acid-2,3-BD pathway in K. oxytoca. Succinate, lactate, formate, and acetate were also detected as by-products in the fermentation broth [7, 15].
In K. oxytoca PDL-0, the formation of acetate, succinate, lactate, and formate is catalyzed by pox and pta, frdA, ldhD, and pflB, respectively [24]. To achieve higher yield of 2,3-BD, these genes were successively deleted in strain K. oxytoca PDL-0 (Additional file 1: Fig. S1). Effects of these genes deletion on growth, lactose consumption, by-product accumulation, and 2,3-BD production were studied in M9 medium supply with 5 g/L yeast extract and about 40 g/L lactose. As shown in Fig. 3a and Fig. 3b, deletion of all these by-product pathways in K. oxytoca PDL-0 had no effect on lactose consumption but slightly increased its growth. Accumulation of by-products including acetate, succinate, lactate, and formate reduced remarkably due to deletion of pox, pta, frdA, ldhD, and pflB (Fig. 3c). The final strain K. oxytoca PDL-0 ΔpoxΔptaΔfrdAΔldhDΔpflB exhibited higher concentration and yield of 2,3-BD (Fig. 3d and Fig. 3e) and lower byproducts production (Fig. 3c) than other recombinant strains.
Performance of recombinant strain in 1-L batch fermentation
Then, the effects of inactivation of by-product pathways on 2,3-BD production were further studied through batch fermentation in a 1-L fermenter. The strains K. oxytoca PDL-0 and K. oxytoca PDL-0 ΔpoxΔptaΔfrdAΔldhDΔpflB were cultured in a fermentation medium containing corn steep liquor powder as a cheap nitrogen source and about 40 g/L lactose as carbon source. As shown in Fig. 4a and 4b, K. oxytoca PDL-0 consumed 42.75 g/L lactose and produced 15.26 g/L 2,3-BD with a yield of 0.36 g/g at 12 h, while K. oxytoca PDL-0 ΔpoxΔptaΔfrdAΔldhDΔpflB consumed 39.29 g/L lactose and produced 17.65 g/L 2,3-BD with a yield of 0.45 g/g. Thus, the recombinant strain K. oxytoca PDL-0 ΔpoxΔptaΔfrdAΔldhDΔpflB possesses advantages over wild type in both concentration and yield of 2,3-BD.
Utilization of lactose for 2,3-BD production in fed-batch fermentation
To achieve a higher product concentration, fed-batch fermentation using strain K. oxytoca PDL-0 ΔpoxΔptaΔfrdAΔldhDΔpflB with an initial lactose concentration of 100 g/L was conducted. Fermentation medium containing corn steep liquor was used in a 7.5-L fermenter. As shown in Fig. 5a, 173.2 g/L lactose was consumed and 74.9 g/L 2,3-BD was produced within 33 h. The productivity was 2.27 g/L/h, and the yield was 0.43 g/g lactose. The concentration of acetate, which was included in the medium, was 0.59 g/L at the end of the fermentation. The concentration of lactate, which was also included in the medium, decreased to 0.13 g/L at 33 h. The final concentration of succinate was 0.82 g/L and there was no formate production throughout the fermentation process (Additional file 1: Fig. S2a).
Utilization of whey powder for 2,3-BD production in fed-batch fermentation
Fed-batch fermentation using whey powder as the carbon source by strain K. oxytoca PDL-0 ΔpoxΔptaΔfrdAΔldhDΔpflB was also carried out. After 24 h of fermentation, 65.5 g/L 2,3-BD was obtained from 148.3 g/L lactose (Fig. 5b). The productivity and yield of 2,3-BD were 2.73 g/L/h and 0.44 g/g, respectively. The major by-products in final fermentation broth were acetate and lactate, which were found at concentrations of 3.24 g/L and 0.38 g/L, respectively (Additional file 1: Fig. S2b).
Several microbial strains have been screened to produce 2,3-BD from whey or lactose. However, as shown in Table 1, the final concentration and yield of 2,3-BD produced by wild type isolates were relatively low. For example, Vishwakarma tried to use strain K. oxytoca NRRL-13-199 for 2,3-BD production from whey. After the addition of 50 mM acetate, a concentration of 8.4 g/L 2,3-BD was acquied with a yield of 0.365 g/g lactose [25]. Barrett et al studied production of 2,3-BD from whey by K. pneumoniae ATCC 13882 [20]. After 60 h of fermentation, 19.3 g/L 2,3-BD was produced from whey with a productivity of 0.32 g/L/h. Ramachandran et al got a concentration of 32.49 g/L 2,3-BD from lactose by using K. oxytoca (formerly known as Aerobacter aerogenes or K. pneumoniae ATCC 8724), however, the yield (0.207 g/g lactose) and productivity (0.861 g/L/h) of 2,3-BD were still unsatisfactory [26]. In a previous work, Lactococcus lactis MG1363 was metabolic engineered to produce 2,3-BD from residual whey permeate and the final titer of 51 g/L was acquired [27]. Exogenous antibiotics was needed for the maintenance of two plasmids pJM001 and pLP712, which respectively carries the genes needed for 2,3-BD production and metabolism of lactose. To make bio-based 2,3-BD production from whey more economically efficient and environment-friendly, 2,3-BD production without antibiotic addition in the fermentation system for the maintenance of plasmid should be initiated. In this work, K. oxytoca PDL-0 was metabolic engineered to efficiently produce 2,3-BD from lactose through deleting pox, pta, frdA, ldhD, and pflB. Using whey powder as the carbon source, the recombinant strain can produce 65.5 g/L 2,3-BD (Table 1). Compared with other strains used for 2,3-BD production from whey, the engineered strain has significant production advantages such as high product concentration (65.5 g/L), high productivity (2.73 g/L/h), and unnecessary exogenous antibiotics.
Table 1
Comparison of 2,3-BD production using whey/lactose as substrate by different microorganisms.
Strain
|
Substrate
|
Method
|
Concentration (g/L)
|
Yield (g/g)
|
Productivity (g/L/h)
|
Reference
|
Bacillus. polymyxa ATCC 1232
|
Cheese whey
|
Wild-type
|
5.5
|
0.25
|
0.03
|
[19]
|
K. pneumoniae NCIB 8017
|
Rennet whey permeate
|
Wild-type
|
7.5
|
0.46
|
0.08
|
[36]
|
K. oxytoca NRRL-13-199
|
Whey
|
Wild-type
|
8.4
|
0.37
|
–
|
[25]
|
Enterobacter aerogenes 3889
|
Whey
|
Wild-type, using neutralized acid whey with 50 mM acetate
|
15.1
|
–
|
0.24
|
[20]
|
K. pneumoniae ATCC 13882
|
Whey
|
Wild-type, using unsterilized acid whey and adjusting pH to 6.5
|
19.3
|
–
|
0.32
|
[20]
|
Lactococcus lactis MG1363
|
Residual whey permeate (lactose)
|
Deletion of ldh, ldhB, ldhX, pta, adhE, butBA, overexpression of bdh and lactose utilizing pathway
|
51
|
0.47
|
1.46
|
[27]
|
K. oxytoca PDL-0
|
Whey powder
|
Deletion of pox, pta, frdA, ldhD, pflB
|
65.5
|
0.44
|
2.73
|
This study
|
K. pneumoniae KG1
|
Lactose
|
Wild-type
|
4.4
|
0.33
|
0.37
|
[18]
|
K. oxytoca NRRL-B199 with Nonviable Cells of Kluyveromyces lactis CBS 683
|
Lactose
|
Wild-type, co-immobilization by adhesion of β-galactosidase in nonviable cells of K. lactis with K. oxytoca
|
14.3
|
0.29
|
0.80
|
[22]
|
K. oxytoca (K. pneumoniae ATCC 8724)
|
Lactose
|
Wild-type
|
32.49
|
0.21
|
0.86
|
[26]
|
K. oxytoca PDL-0
|
Lactose
|
Deletion of pox, pta, frdA, ldhD, pflB
|
74.9
|
0.43
|
2.27
|
This study
|
Recently, lactose or whey have been used to produce various biochemicals, e.g., ethanol [28], butanol [29], lactic acid [30], citric acid [31], poly(3-hydroxybutyrate) (PHB) [32], and gluconic acid [33], through endogenous or exogenous biosynthetic pathways (Table 2). However, because of the low utilization efficiency of lactose in these chassis cell, it is difficult to produce the target chemicals with high productivity and high yield [29, 31]. Ahn et al constructed a fermentation strategy with cell recycle membrane system for the production of PHB from whey [32]. High consumption rate of lactose (7.67 g/L/h) was acquired using this complicated fermentation strategy. In this work, the engineered K. oxytoca PDL-0 was confirmed to have the ability to efficiently transform lactose into 2,3-BD with relatively high yield (0.44 g/g) and high consumption rate of lactose (6.18 g/L/h) (Table 1 and Table 2). Considering its excellent characteristics of non-pathogenicity (Risk Group 1) and efficient lactose utilization, K. oxytoca PDL-0 might be a promising chassis for production of various chemicals from whey through metabolic engineering.
Table 2
Other products using whey/lactose as substrate by different strains.
Product
|
Strain
|
Substrate
|
Strategies
|
Concentration (g/L)
|
Yield (%)a
|
Lactose consumption rate (g/L/h)
|
Reference
|
Ethanol
|
Saccharomyces cerevisiae STX 23-5B and Kluyveromyces fragilis 55–55
|
Lactose
|
Constructing hybrids between S. cerevisiae and K. fragilis through protoplast fusion
|
105
|
–
|
–
|
[28]
|
Butanol
|
Clostridium saccharobutylicum P262
|
Lactose
|
Using pervaporation membrane to recover and concentrate product
|
72.4
|
79
|
1.14
|
[29]
|
Lactic acid
|
Lactobacillus casei SU No 22 and L. lactis WS 1042
|
Deproteinized whey
|
Coimmobilization of L. casei and L. lactis cells
|
47
|
61
|
3.04
|
[30]
|
Citric acid
|
Yarrowia lipolytica B9
|
Partly deproteinized whey
|
Using immobilized cells of Y. lipolytica
|
33.3
|
47
|
0.53
|
[31]
|
PHB
|
E. coli CGSC 4401
|
Whey
|
Using cell recycle membrane system by E. coli expressing pha genes
|
168
|
–
|
7.67
|
[32]
|
Gluconic acid
|
Aspergillus niger NCIM 548
|
Lactose and glucose
|
Using A. niger immobilized in polyurethane foam
|
92
|
80
|
1.98
|
[33]
|
2,3-BD
|
K. oxytoca PDL-0
|
Lactose
|
Deletion of pox, pta, frdA, ldhD, pflB
|
74.9
|
82
|
5.25
|
This study
|
2,3-BD
|
K. oxytoca PDL-0
|
Whey powder
|
Deletion of pox, pta, frdA, ldhD, pflB
|
65.5
|
84
|
6.18
|
This study
|
aRatio between actual yield and theoretical yield of each product. |