Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation
© Wang et al. 2016
Received: 19 November 2015
Accepted: 11 January 2016
Published: 25 January 2016
In the industry, the conventional two-step fermentation method was used to produce 2-keto-l-gulonic acid (2-KGA), the precursor of vitamin C, by three strains, namely, Gluconobacter oxydans, Bacillus spp. and Ketogulonicigenium vulgare. Despite its high production efficiency, the long incubation period and an additional second sterilization process inhibit the further development. Therefore, we aimed to reorganize a synthetic consortium of G. oxydans and K. vulgare for one-step fermentation of 2-KGA and enhance the symbiotic interaction between microorganisms to perform better.
During the fermentation, competition for sorbose of G. oxydans arose when co-cultured with K. vulgare. In this study, the competition between the two microbes was alleviated and their mutualism was enhanced by deleting genes involved in sorbose metabolism of G. oxydans. In the engineered synthetic consortium (H6 + Kv), the yield of 2-KGA (mol/mol) against d-sorbitol reached 89.7 % within 36 h, increased by 29.6 %. Furthermore, metabolomic analysis was used to verify the enhancement of the symbiotic relationship and to provide us potential strategies for improving the synthetic consortium. Additionally, a significant redistribution of metabolism occurred by co-culturing the K. vulgare with the engineered G. oxydans, mainly reflected in the increased TCA cycle, purine, and fatty acid metabolism.
We reorganized and optimized a synthetic consortium of G. oxydans and K. vulgare to produce 2-KGA directly from d-sorbitol. The yield of 2-KGA was comparable to that of the conventional two-step fermentation. The metabolic interaction between the strains was further investigated by metabolomics, which verified the enhancement of the mutualism between the microbes and gave us a better understanding of the synthetic consortium.
As synthetic biology begins to address problems involved in the programing novel biological systems, engineering multicellular behavior is emerging as a key tool for building advanced synthetic systems that robustly perform complex behaviors . In natural environments, microorganisms commonly exist as communities of multiple species that are capable of fulfilling more varied and complicate tasks than clonal populations . Different members of a consortium assume different responsibilities, increasing overall productivity and allowing for more complex behavior than that with a single cell or a monoculture. During the last decade, experimental efforts have been made to build and maintain the synthetic communities [3–5]. However, those studies were mostly concerned with the well-defined ideal models (e.g., mutualism, parasitism and commensalism, etc.). Whereas in industrially-relevant circumstances, the situation is more complex since the exchange of metabolites, energy, and informative signals with the environment should be taken into consideration, and the relationship among the strains is often too diverse to analyze. Therefore, increasing attention has been paid on the development of synthetic consortia in industry. Several microbial consortia were constructed for studying the cooperation of cells, enhancing the production of biofuel [6, 7], electricity  and even complex natural products , etc.
Generally, microorganisms interact with each other by exchanging biomolecules (e.g., proteins, nucleic acids and metabolites) and information signals via contact-based or contact-independent interaction . Omics study can provide deep insights into the mechanism of metabolic crosstalk in a consortium at the global level and indicate the way to better understand the relationship between the species . Researchers have adopted this approach to investigate the co-cultured microbial systems, including some previous studies about the metabolic cooperation of Bacillus megaterium and Ketogulonicigenium vulgare in two-step vitamin C fermentation [12–16]. In this study, metabolomic analysis was used to better understand the specialization and cooperation between Gluconobacter oxydans and K. vulgare in the reorganized microbial consortium. These analyses verified the alleviation of competition and the enhancement of the symbiotic relationship, which provided us potential strategies for further construction of the microbes.
Chen et al.  declared the fundamental power of cell specialization and cooperation in the consortia. It is a powerful reminder that the communities are frequently more than the sum of their parts. In nature, microbes can form interacting communities to accomplish chemically difficult tasks through division of labor among different species . In this study, the industrial vitamin C fermentation was taken as an example. The conventional two-step fermentation method was used to produce 2-keto-l-gulonic acid (2-KGA), the precursor of vitamin C, by three strains (G. oxydans, Bacillus spp. and K. vulgare). During the second step, K. vulgare is responsible for the biosynthesis of 2-KGA from l-sorbose, and Bacillus spp., as a companion, promotes the growth and production efficiency of K. vulgare. Despite its high production efficiency, the long incubation period and an additional second sterilization process of the two-step fermentation inhibit the further development of industrial production. Hence, we demonstrated the concept of reconstituting a heterologous metabolic pathway in a microbial partnership with G. oxydans and K. vulgare, where 2-KGA was produced directly from d-sorbitol. Furthermore, two genes involved in sorbose metabolism from G. oxydans were knocked out to alleviate the competition for sorbose of G. oxydans. The yield of 2-KGA (mol/mol) against d-sorbitol reached 89.7 % (76.6 g/L) within 36 h, which enabled an 29.6 % increase compared to the original consortium 69.3 % (59.1 g/L). Additionally, simplifying metabolic pathway may remove some negative effects for the microbes and increase the metabolic efficiency, which makes up for the mismatch of the consortium and enhances the cell–cell interaction. Hence, metabolomic analysis was used to provide a clear and comprehensive description of the physiological relationship between them, which was the key issue of this one-step fermentation. Compared with the conventional two-step fermentation process, this new route of one-step fermentation can potentially revolutionize the industrial-scale production of vitamin C.
Relevant information of engineered strains in this study
Target genes deleted
B932_0664: FAD-dependent l-sorbose 1-dehydrogenase
B932_1330: NADPH-dependent l-sorbose reductase
B932_1370: PTS system transporter subunit IIA
B932_1684: NADPH-dependent l-sorbose reductase
B932_3022: NADPH-dependent l-sorbose reductase
B932_1330 and B932_1370
Medium and culture conditions
All E. coli strains were cultivated in Luria-Broth (LB) medium at 37 °C. The d-sorbitol/l-sorbose seed culture medium for the mono-culture of G. oxydans and K. vulgare was composed of 20 g/L d-sorbitol (for G. oxydans) or l-sorbose (for K. vulgare), 10 g/L peptone, 3 g/L corn-steep liquor (CSL), 3 g/L beef extract, 3 g/L yeast extract, 1 g/L urea, 1 g/L KH2PO4, 1 g/L CaCO3 and 0.2 g/L MgSO4·7H2O. The fermentation medium for the one-step co-culture contained 80 g/L d-sorbitol, 10 g/L CSL, 12 g/L urea, 1 g/L KH2PO4, 1 g/L CaCO3 and 0.2 g/L MgSO4·7H2O. The pH values of the medium were maintained at 7.0 by the addition of NaOH.
The mono-culture of G. oxydans and K. vulgare, as seed for the subsequent co-culture fermentation, were cultivated in 250 ml flasks with 50 ml d-sorbitol/l-sorbose seed cultures at 30 °C and 250 rpm for 24 h. The OD600 of G. oxydans and K. vulgare in the seed culture reached about 5.5 and 3.0, respectively. Whereafter, these two strains were simultaneously inoculated into a 5 L jar fermentor (Bailun Bio-technology Co. Ltd., Shanghai) with 3 L fermentation medium. The inoculation ratio (%, v/v) of G. oxydans and K. vulgare, agitation speed and aeration rate were optimized. The initial inoculum ratio of G. oxydans and K. vulgare was 4:1. pH value and temperature of the fed-batch fermentation were automatically controlled at 7.0 and 30 °C. And the agitation speed was controlled at 500 rpm with the aeration rate of 1.5 vvm.
Analysis of population of each species
Co- and mono-cultured community samples were collected from fermentations at 0, 4, 8, 14, 21, 28 and 32 h after inoculation. The genomic DNA was extracted from the samples with the TIANamp Bacteria DNA Kit (Tiangen Biotech, China). RealMasterMix (SYBR Green) was used and the quantitative PCR reactions were performed on Light-Cycler 480 with the primers designed based on 16S rDNA of each species 5′-CGATGTGTGCTGGATGTTGGG-3′ and 5′-TCTGAACCGGTCCTCCCCATG-3′ for G. oxydans, and 5′-AATGCCAGTCGTCAGGTTGCTT-3′ and 5′-CTAGGCCGGTCCTGTAATGTCA-3′ for K. vulgare. The amount of genome of each species was computed by comparison with a standard curve from pure cultures analyzed with the same manner.
Transcriptional analysis of relevant genes in co- and mono-cultured systems
The transcriptional expression level of the genes in co- and mono-cultured systems at different sampling times was evaluated by qPCR. All the data were normalized to 16S rDNA of each species. The entire RNA was extracted from the samples with the ApexPrep RNA Miniprep Kit (APExBio). HiTaq EvaGreen qPCR MasterMix (APExBio) was used and the quantitative PCR reactions were performed on a CFX96 real time PCR system (Bio-Rad) with a total volume of 20 μL containing diluted cDNA (2 μL), qPCR MasterMix (10 μL),and forward primer and reverse primer (0.8 μL).
Sample preparation and metabolites analysis
The samples from different co- and mono-cultured fermentations were collected at 4, 8, 14, 21 and 28 h after inoculation. These five time points primarily represented the lag phase, the early exponential phase, the middle exponential phase, the late exponential phase and the stationary phase of community. The concentrations of extracellular d-sorbitol, l-sorbose and 2-KGA were analyzed by HPLC (Waters Corp., USA) with a refractive index detector. H2SO4 (5 mM) was used as the mobile phase on an Aminex HPX-87H column (BioRad, CA) at the temperature of 65 °C with a flow rate of 0.6 ml/min.
The intracellular metabolites were extracted and derivatizated according to our previous procedure . Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS, Waters Corp., USA) was applied to detect the metabolites in different samples, as described by Ding et al.  with identical chromatographic conditions. One microlitre sample was injected with a split ratio of 1:1 into GC, equipped with a fused-silica capillary column (DB-5MS, 30 m × 0.25 mm i.d., 0.25 μm, J&W Scientific, Folsom, CA, USA). After a 2 min delay at 70 °C, the oven temperature program increased to 290 °C at 5 °C/min, holding for 3 min. The temperature of the transfer line and the ion source was 280 and 250 °C, respectively. Helium (99.9995 %) was used as the carrier gas under a constant pressure of 91 kPa. The solvent delay was 5 min. Ions were generated by a 70 eV electron beam at an ionization current of 40 μA. Two spectra were recorded per second in the mass range of 50–800 m/z with DRE function.
Principal component analysis (PCA) and pathway enrichment analysis were performed by MetaboAnalyst 3.0 (http://www.metaboanalyst.ca/MetaboAnalyst/faces/home.xhtml) .
Results and discussion
Reorganization of a synthetic microbial consortium for one-step vitamin C fermentation
The titer of 2-KGA by this consortium was only 12.9 g/L within 36 h, and the yield was 15.0 %, which was much lower than that of the industrial two-step fermentation process (about 90 %). In order to improve the performance of this one-step fermentation process, many optimization attempts have been made, including modification of the inoculation ratio, agitation speed, and aeration rate. In this way, the titer of 2-KGA reached to 59.1 g/L within 28 h, which shorten the fermentation time by about 40 % (Additional file 1: Figure S2b). Whereas in the control experiment in which only K. vulgare (Fig. 1d) or G. oxydans (data not shown) was cultured, no 2-KGA was produced. These results showed that l-sorbose produced by G. oxydans diffused into K. vulgare cells and was subsequently oxidized. However, though the optimization of fermentation conditions indeed improved the titer and yield of 2-KGA, the natural limitation of l-sorbose consumption by G. oxydans in this consortium cannot be overcome without genetic modification. In this study, G. oxydans was cultivated in d-sorbitol seed culture medium and the composition of the culture broth from at different time points during the fermentation was measured by HPLC. We found that l-sorbose cannot be consumed until d-sorbitol was exhausted after 12 h in the mono-culture of G. oxydans, which matched the conclusion drawn by Soemphol et al. . Then the accumulation of an unknown byproduct was detected while l-sorbose was consumed (Fig. 1e), which would reduce the 2-KGA production and make the efficiency too low to fully meet industrial requirements. In industrial fermentation, even one percent loss of carbon source will cause a significant financial burden. Therefore, we further optimized this two-strain consortium by alleviating the metabolic competition of G. oxydans with K. vulgare for sorbose, which was helpful for establishing a better homeostasis between microbes and making them work better together.
The relationship optimization of G. oxydans–K. vulgare consortium
The optimization of the relationship between the two microbes, G. oxydans and K. vulgare, was further studied. The consortia population compositions throughout the process were analyzed to validate the variation of the relationship. Figure 2f and g showed the relative density of different microbes in co- and mono-cultured systems. We found that the K. vulgare in the engineered consortium H2 + Kv also showed a better growth than that in the primary consortium Go + Kv (Fig. 2f), coupled with the higher production of 2-KGA in H2 + Kv. Meanwhile, the growth levels of engineered H2 and G. oxydans were similar in mono-culture. While after the introduction of K. vulgare, the engineered H2 grew much faster than the wild type since 8 h after inoculation and reached almost twice of the wild type after 28 h (Fig. 2g). In the present study, another interesting phenomenon about the initial inoculum ratio of G. oxydans to K. vulgare has been found. The inoculum ratio (%, v/v) of G. oxydans to K. vulgare was firstly set as 1:4 because of the growth defect of K. vulgare. Due to a low yield of 2-KGA, we then adjusted it to 4:1, which led to a great improvement in 2-KGA productivity (data not shown). This appears to be a counterintuitive finding that high ratio of inoculated G. oxydans was beneficial for the synthetic consortium. We speculated that because of the survival traits of K. vulgare, more G. oxydans were needed to provide more nutrients for the growth and productivity of K. vulgare. It was found that G. oxydans was the most populous consortium member throughout the whole process. However the ratio of G. oxydans to K. vulgare decreased during the fermentation in the engineered consortium, which was contrary to the original consortium Go + Kv. From this point of view, the engineered H2 promoted the growth and productivity of K. vulgare and the latter stimulated the growth of H2 in return. We hypothesized that there was more interaction of biomolecules or information signals between the two microbes in this mutualistic G. oxydans–K. vulgare consortium (Fig. 2h) compared with the primary competitive consortium. Hence, metabolomic analysis of the different consortium should be done for a comprehensive description of the relationship optimization between the members.
Metabolomic analysis on the relationship optimization of G. oxydan s–K. vulgare consortium
Improved amino acids metabolism in G. oxydans–K. vulgare consortium
Improved purines metabolism in G. oxydans–K. vulgare consortium
Improved fatty acids metabolism in G. oxydans–K. vulgare consortium
In this study, all the detected free fatty acids represented higher levels in consortium samples than that in mono-cultured G. oxydans samples, especially the unsaturated fatty acids including oleic acid (18:1), elaidic acid (18:1), and palmitelaidic acid (16:1). All of these three fatty acids were presented at over 20-fold higher levels in both consortia compared to monoculture. Additionally, their levels in consortium Go + Kv were higher than those in H2 + Kv, respectively (Fig. 3e). It was reported that the increased unsaturated fatty acid level facilitated the stress defense . Thus, we speculated that G. oxydans might be subjected to several stresses after co-cultured with K. vulgare, such as the changed growth environment caused by the metabolites secreted by K. vulgare. More unsaturated fatty acids were synthesized by G. oxydans to respond to the pressure of the co-culture conditions. Compared to Go + Kv, the lower levels of unsaturated fatty acids in consortium H2 + Kv suggested that the engineered H2 possessed preferable adaptability to the environment co-culture with K. vulgare. On the other hand, more unsaturated fatty acids would increase the cell membrane fluidity and permeability under unfavorable conditions by affecting the plasma membrane integrity, fluidity and function . The dramatic increase in the levels of these unsaturated fatty acids may indicate that cells in this consortium increased their membrane permeability for exchanging more nutrients, which would promote the interaction between two strains.
In this study, a synthetic consortium for one-step vitamin C fermentation was reorganized with G. oxydans and K. vulgare. Further optimization was carried out to alleviate the competition for sorbose of G. oxydans with K. vulgare. The yield of 2-KGA of this consortium reached 89.7 % within 36 h, which is comparable to the conventional two-step fermentation. The metabolic interaction between the strains was further investigated by metabolomics, which verified the enhancement of the mutualism between the microbes and gave us a better understanding of the synthetic consortium.
EXW, MZD and YJY designed the study and drafted the manuscript. EXW and MZD carried out the molecular genetic studies and metabolomics analysis. QM and XTD assisted in the fermentation and metabolomics analysis. YJY supervised the whole research and revised the manuscript. All authors read and approved the final manuscript.
There is no conflict of interest among the authors. This work was funded by the Ministry of Science and Technology of China (“973” Program: 2014CB745100, “863” Program: 2012AA02A701), and the National Natural Science Foundation of China (Major Program: 21390203).
The authors declares that they have no competing interests.
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