Selection of K. oxytoca PDL-0 for BDO production from lactose
To select a strain for efficient BDO production from whey, we first assessed strains that can utilize lactose and produce BDO. K. pneumonia, E. cloacae, B. licheniformis, and K. oxytoca can produce BDO from glucose [16]. E. coli BL21-pETRABC carrying the BDO pathway gene cluster from E. cloacae can also efficiently bio-transform glucose into BDO [26]. In the present study, we first compared the ability of K. pneumonia ATCC 15380, E. cloacae SDM, B. licheniformis DSM13, K. oxytoca PDL-0, and E. coli BL21-pETRABC to produce BDO from lactose; results are shown in Fig. 2.
All five strains were cultured in M9 medium supplemented with 5 g/L yeast extract and ~40 g/L lactose for 48 h. B. licheniformis DSM13 is the only strain that cannot consume lactose. E. cloacae SDM and E. coli BL21-pETRABC could grow well and utilize ~30 g/L lactose within 48 h, but only accumulated about 2 g/L BDO (Additional file 1: Fig. S1, Fig. 2a-c). K. pneumonia ATCC 15380 and K. oxytoca PDL-0 can completely consume ~40 g/L lactose within 36 h and 18 h, and produce BDO from lactose with a yield of 0.21 g/g and 0.30 g/g lactose, respectively (Additional file 1: Fig. S1 and Fig. 2d). Considering the fact that K. oxytoca PDL-0 belongs to Risk Group 1 [15] and produces BDO from lactose with a higher yield than other strains, 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 BDO as its major fermentative product during lactose fermentation in a shaking flask culture. However, only 56% of theoretical yield (0.293 vs 0.526 g/g) was observed (Fig. 3). BDO is produced by a fermentative pathway known as the mixed acid-BDO pathway in K. oxytoca [7, 15]. Acetate (1.57 g/L), succinate (1.14 g/L), lactate (1.34 g/L), and formate (0.27 g/L) were also detected as by-products in the fermentation broth (Fig. 3).
In K. oxytoca PDL-0, the formation of acetate, succinate, lactate, and formate is catalyzed by pox and pta, frdA, ldhD, and pflB, respectively [27]. To achieve higher BDO yield, these genes were successively deleted in strain K. oxytoca PDL-0 (Fig. 1). Effects of these gene deletions on growth, lactose consumption, by-product accumulation, and BDO production were studied in M9 medium supplemented with 5 g/L yeast extract and ~40 g/L lactose. As shown in Fig. 3a and 3b, deletion of these by-product pathways in K. oxytoca PDL-0 had no effect on lactose consumption but did slightly increase growth. Accumulation of by-products, including acetate (0.23 g/L), succinate (0.70 g/L), lactate (0.11 g/L), and formate (0 g/L), was markedly decreased due to deletion of pox, pta, frdA, ldhD, and pflB (Fig. 3c). The final strain, K. oxytoca PDL-K5, exhibited higher concentration (16.0 g/L) and yield (0.36 g/g lactose) of BDO (Fig. 3d and 3e) and lower by-product generation (Fig. 3c) than other recombinant strains.
Performance of recombinant strain in 1-L batch fermentation
The effects of inactivation of by-product pathways on BDO production were further studied through batch fermentation in a 1-L fermenter. The strains K. oxytoca PDL-0 and K. oxytoca PDL-K5 were cultured in a fermentation medium containing corn steep liquor powder as a nitrogen source and ~ 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 BDO with a yield of 0.36 g/g at 12 h, while K. oxytoca PDL-K5 consumed 39.29 g/L lactose and produced 17.65 g/L BDO with a yield of 0.45 g/g. Thus, the recombinant strain K. oxytoca PDL-K5 demonstrates advantages over wild type in both concentration and yield of BDO.
Utilization of lactose for BDO production in fed-batch fermentation
To achieve higher product concentration, we performed fed-batch fermentation using strain K. oxytoca PDL-K5 with initial lactose concentration of ~100 g/L. 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 BDO was produced within 33 h. The productivity was 2.27 g/L/h and the yield was 0.43 g/g lactose. The final concentration of the major by-product 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 BDO production in fed-batch fermentation
Fed-batch fermentation using K. oxytoca PDL-K5 with whey powder as the carbon source was also conducted. After 24 h of fermentation, 65.5 g/L BDO was obtained from 148.3 g/L lactose (Fig. 5b). The productivity and yield of BDO were 2.73 g/L/h and 0.44 g/g, respectively. The major by-products in the 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). During fermentation, agitation and airflow were set at 400 rpm and 1 vvm, respectively, and dissolved oxygen was uncontrolled. Acetoin started to accumulate at the end of fermentation and feeding more whey powder into the fermentation system did not increase BDO production. Dissolved oxygen has a profound impact on the distribution of BDO and its dehydrogenation product, acetoin. Since BDO biosynthesis occurs under microaerobic conditions [28, 29], fine-tuning the dissolved oxygen through an automatic control system might provide the optimal microaerobic condition to further increase BDO production.
Several microbial strains have been screened to produce BDO from whey or lactose. However, as shown in Table 1, the final concentration and yield of BDO produced by wildtype isolates were relatively low. For example, Vishwakarma tried to use strain K. oxytoca NRRL-13-199 for BDO production from whey. After the addition of 50 mM acetate, 8.4 g/L BDO was acquired with a yield of 0.365 g/g lactose [30]. Barrett et al studied production of BDO from whey by K. pneumoniae ATCC 13882 [23]. After 60 h of fermentation, 19.3 g/L BDO was produced from whey with a productivity of 0.32 g/L/h. Ramachandran et al obtained a concentration of 32.49 g/L BDO from lactose by using K. oxytoca ATCC 8724; however, the yield (0.207 g/g lactose) and productivity (0.861 g/L/h) of BDO were still unsatisfactory [31]. In a previous work, Lactococcus lactis MG1363 was metabolically engineered to produce BDO from residual whey permeate, and a final titer of 51 g/L BDO was acquired [32]. Exogenous antibiotics were needed for the maintenance of two plasmids, pJM001 and pLP712, which carry the genes needed for BDO production and metabolism of lactose, respectively. To make bio-based BDO production from whey more economically efficient and environment-friendly, BDO production without antibiotic addition to the fermentation system for the maintenance of plasmids should be initiated. In this work, K. oxytoca PDL-0 was metabolically engineered to efficiently produce BDO from lactose in whey powder through deleting pox, pta, frdA, ldhD, and pflB. Using whey powder as the carbon source, the recombinant strain K. oxytoca PDL-K5 can produce 65.5 g/L BDO (Table 1). Compared with other strains used for BDO 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 lack of a need for unnecessary exogenous antibiotics.
Recently, lactose or whey have been used to produce various biochemicals, e.g., ethanol [33], butanol [34], lactic acid [35], citric acid [36], poly(3-hydroxybutyrate) (PHB) [37], and gluconic acid [38], through endogenous or exogenous biosynthetic pathways. However, because of the low utilization efficiency of lactose in these chassis cells, it is difficult to produce the target chemicals with high productivity and high yield [34, 36]. Ahn et al constructed a fermentation strategy with a cell-recycle membrane system for the production of PHB from whey [37]. A high consumption rate of lactose (7.67 g/L/h) was acquired using this complicated fermentation strategy. The engineered strain K. oxytoca PDL-K5 in this study had the ability to efficiently transform lactose in whey powder into BDO with relatively high yield (0.44 g/g) and high consumption rate of lactose (6.18 g/L/h). This work provides a suitable method for BDO production as well as whey utilization (Fig. 6). Considering its excellent characteristics of non-pathogenicity (Risk Group 1) and efficient lactose utilization, K. oxytoca PDL-0 may be a promising chassis for production of various chemicals from whey through metabolic engineering. For example, acetoin, the oxidized precursor of BDO, might be produced through increasing dissolved oxygen levels and deleting 2,3-butanediol dehydrogenases responsible for BDO production from acetoin [39].