Efficient 2,3-butanediol production from whey powder using metabolically engineered Klebsiella oxytoca

Background Whey is a major pollutant generated by the dairy industry. To decrease environmental pollution caused by the industrial release of whey, new prospects for its utilization need to be urgently explored. Here, we investigated the possibility of using whey powder to produce 2,3-butanediol (BDO), an important platform chemical. Results Klebsiella oxytoca strain PDL-0 was selected because of its ability to efficiently produce BDO from lactose, the major fermentable sugar in whey. After deleting genes pox, pta, frdA, ldhD, and pflB responding for the production of by-products acetate, succinate, lactate, and formate, a recombinant strain K. oxytoca PDL-K5 was constructed. Fed-batch fermentation using K. oxytoca PDL-K5 produced 74.9 g/L BDO with a productivity of 2.27 g/L/h and a yield of 0.43 g/g from lactose. In addition, when whey powder was used as the substrate, 65.5 g/L BDO was produced within 24 h with a productivity of 2.73 g/L/h and a yield of 0.44 g/g. Conclusion This study demonstrated the efficiency of K. oxytoca PDL-0 for BDO production from whey. Due to its non-pathogenicity and efficient lactose utilization, K. oxytoca PDL-0 might also be used in the production of other important chemicals using whey as the substrate.


Background
Whey, a liquid by-product generated during cheese production, contains most of the water-soluble components in milk [1,2]. Despite annual production of 145 million tons worldwide, only a little over one-half of the whey produced is utilized [3]. Whey is regarded as a serious pollutant because of its high biochemical oxygen demand (BOD, 30,000-50,000 mg/L) and chemical oxygen demand (COD, 60,000-80,000 mg/L) [3]. Economic disposal of whey has become a worldwide problem for the dairy industry. Lactose, a utilizable disaccharide for many microbial strains, is the major contributor to BOD and COD of whey [4,5]. Using the lactose in whey as a substrate for industrial microbial fermentation may transform a potential pollutant into a value-added product and this prospect deserves an intensive study.
2,3-Butanediol (BDO) is an important platform chemical that can be applied in many industrial fields [6][7][8]. Derivatives of BDO are estimated to have a potential global market of around 32 million tons per year. One common method for BDO synthesis is performed under harsh conditions (160-220 °C, 50 bar) with a C 4 hydrocarbon fraction of cracked gases as the substrate [9,10]. However, due to shortage of fossil fuels and increasing global environmental concerns, green production of BDO through microbial fermentation is desirable [11][12][13][14][15][16]. Renewable resources such as rice waste biomass,

Open Access
Microbial Cell Factories *Correspondence: chuanjuanlv@mail.sdu.edu.cn; macq@sdu.edu.cn 1 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, People's Republic of China Full list of author information is available at the end of the article sugarcane bagasse hydrolysate, and kenaf core biomass have been used in fermentative production of BDO [17][18][19].
Several BDO-producing microorganisms can use fermentable sugars, including glucose, xylose, fructose, and lactose as the sole carbon source for growth [20][21][22][23]. However, these strains exhibit unsatisfactory fermentative performance in BDO production when lactose is used as the carbon source. For example, Klebsiella oxytoca NRRL-B199 can use the mixture of glucose and galactose as substrate for growth and produce BDO as its main product. Nevertheless, BDO was present in a low concentration and the strain produced acetate as the major product in the fermentation broth with lactose [24,25].
Production of BDO using whey as the substrate can enhance the economic feasibility of BDO fermentation and facilitate resource utilization of the pollutant whey. Therefore, it is critical to identify a suitable microbial strain with BDO production potential using lactose and whey. In this study, we cultured Klebsiella pneumonia ATCC 15380, Enterobacter cloacae SDM, Bacillus licheniformis DSM13, K. oxytoca PDL-0, and Escherichia coli BL21-pETRABC in fermentation broths with lactose as the carbon source. K. oxytoca PDL-0 exhibited the best performance in lactose utilization and BDO production. Next, byproduct-producing genes in K. oxytoca PDL-0, including pox, pta, frdA, ldhD, and pflB, were knocked out to improve the efficiency of BDO production from lactose. Finally, high production of BDO from whey powder was achieved through fed-batch fermentation using the recombinant strain ( Fig. 1).

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.

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, b, K. oxytoca PDL-0 consumed 42.75 g/L lactose and produced 15.26 g/L 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 Fig. 3 Effects of by-product pathway genes knockout when using lactose as the carbon source. Biomass (a), consumption of lactose (b), by-products (c), concentration (d) and yield (e) of BDO by K. oxytoca PDL-0 and its derivatives were assayed. The experiments were conducted in a 300-mL flask containing 50 mL of M9 minimal medium supplemented with 5 g/L yeast extract and 40 g/L lactose with shaking at 180 rpm for 24 h. The culture temperature was 37 °C. Error bars indicate the standard deviations from three independent cultures 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 byproduct 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].

Conclusions
In this study, the ability of K. oxytoca PDL-0 to metabolize lactose and produce BDO was identified. Then, byproduct pathways encoding genes in K. oxytoca PDL-0 were knocked out to improve the yield of BDO. The engineered strain K. oxytoca PDL-K5 was able to utilize whey powder as the substrate for high production of BDO. The fermentative process developed here is a promising alternative method for both biotechnological production of BDO and whey utilization. In addition, other important chemicals may also be produced from whey using metabolically engineered K. oxytoca PDL-0, which has the characteristics of efficient lactose utilization.

Bacterial strains, plasmids and culture medium
The strains and plasmids used in this study are listed in Table 2. All engineered strains used in this work are based on K. oxytoca PDL-0 and its derivatives. E. coli S17-1 was used to hold and amplify plasmids as well as for conjugation with K. oxytoca. The plasmid pKR6K Cm was used for gene knockout in K. oxytoca [27]. Luria-Bertani (LB) medium was used for the cultivation of all the strains used. The M9 minimal medium [40] supplemented with 5 g/L yeast extract and 40 g/L lactose was used in shake flasks experiments for selection of the efficient BDO producing strain. The selection medium for single exchange strains of K. oxytoca was M9 minimal medium supplemented with 20 g/L sodium citrate and 40 µg/mL chloramphenicol. The selection medium for double exchange strains of K. oxytoca was solid LB medium supplemented with 15% sucrose.

Knockout the genes of K. oxytoca PDL-0
The primers used for knockout of byproduct-producing genes in K. oxytoca PDL-0 are listed in Additional file 1: Table S1. Vector isolation, restriction enzyme digestion, agarose gel electrophoresis, and other DNA manipulations were carried out using standard protocols [41]. Knockout mutants of K. oxytoca PDL-0 were generated via allele exchange using the suicide plasmid pKR6K Cm [27]. The left and right flanking sequences were amplified from K. oxytoca PDL-0 and then ligated through Scheme of BDO production from whey using metabolic engineered K. oxytoca. G6P glucose-6-phosphate PCR to get Δpox fragment using primer pairs PΔpox.f (EcoRI)/PΔpox.r (overlap) and PΔpox.f (overlap)/PΔpox.r (BamHI), respectively. The gel-purified Δpox fragments were ligated to the pKR6K Cm digested with EcoRI and BamHI. The resulting plasmid was designated pKDΔpox and introduced into E. coli S17-1. Then, a three-step deletion procedure was applied to select the Δpox mutant after conjugating the pKDΔpox in K. oxytoca PDL-0 as described previously [27]. The pta, frdA, ldhD, and pflB mutants of strain K. oxytoca PDL-0 were generated by using the same procedure and primers listed in Additional file 1: Table S1.

Batch and fed-batch fermentations
Batch fermentations were conducted in a 1-L bioreactor (Multifors 2, Infors AG, Switzerland) with 0.8 L of medium. The seed culture was inoculated (10%, v/v) into the fermentation medium containing 8.27 g/L corn steep liquor powder (CSLP); 4.91 g/L (NH 4 ) 2 HPO 4 ; 3 g/L sodium acetate; 0.4 g/L KCl; 0.1 g/L MgSO 4 ; 0.02 g/L FeSO 4 ·7H 2 O; 0.01 g/L MnSO 4 ·7H 2 O and 40 g/L lactose. The cultivation was carried out at 37 °C, stirring at 400 rpm, airflow at 1.0 vvm and initial pH of 7.0. When pH dropped to 6.0, it was maintained at this level by automatic addition of 4 M H 3 PO 4 or 5 M NaOH. Fedbatch fermentation was carried out in a 7.5-L fermenter (BioFlo 310, NBS, USA) containing 5 L of medium and the cultivation condition was the same as 1-L fermenter except that the initial concentration of lactose was about 100 g/L. Alternatively, 130 g/L whey powder was fed into the fermentation broth to make the initial concentration of lactose at about 100 g/L. Solid lactose or whey powder was fed in the fermenter when residual lactose concentration was reduced to about 20 g/L.

Analytical methods
The optical density (OD) was measured at 600 nm using a spectrophotometer (V5100H, Shanghai Metash Instruments Co., Ltd, China) after an appropriate dilution. The concentrations of lactose and other by-products were detected by high performance liquid chromatography (HPLC) in an Agilent 1100 series, equipped with a Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, USA) and a refractive index detector [40]. The mobile phase was 10 mM H 2 SO 4 at a flow rate of 0.4 mL/min at 55 °C. The concentrations of acetoin and BDO were analyzed by gas chromatography (GC) (Shimadzu, GC2014c) using a capillary GC column (AT. SE-54, inside diameter, 0.32 mm; length, 30 m, Chromatographic Technology Center, Lanzhou Institute of Chemical Physics, China). Prior to GC analysis, the sample was extracted by ethyl acetate with isoamyl alcohol as the internal standard. Nitrogen was used as the carrier gas for GC analysis. The temperature of both the injector and the detector was 280 °C, the column oven was maintained at 80 °C for