Co-culture system construction
In previous studies, we performed genetic manipulation on S. cerevisiae wild-type strain BY4741, wherein three codon-optimized cellulase genes encoding Talaromyces emersonii CBHI, Trichoderma reesei EGII, and Aspergillus aculeatus BGLI were integrated into the yeast chromosome by a POT1-mediated δ-integration strategy [25]. A series of recombinant strains with high cellulolytic activity to degrade different cellulosic substrates [Avicel, CMC, and phosphoric acid swollen cellulose (PASC)] were screened. To achieve the biosynthesis of high value-added products from lignocellulose, a highly efficient CMC-degrading strain, SK10-3, was chosen for further engineering.
The TAL-encoding gene from Rhodobacter capsulatus was introduced into SK10-3 to synthesis p-CA. However, the degrading pathway of CMC in SK10-3 increased its metabolic burden, and the p-CA titer of SK10-3b were 2.14 mg/L in 20 g/L glucose medium and only 0.46 mg/L in 10 g/L CMC medium, which are much lower than the control strain BY4741b (4.98 mg/L) in glucose medium.
To alleviate the metabolic stress of single strain, the S. cerevisiae strain NK-B2 without an encoding histone H2A gene HTZ1 [8] was selected to introduce multi aromatic compound synthetases and co-cultivate with SK10-3. In this co-culture system, CMC was used as the sole carbon source in the medium and was degraded by SK10-3. Following CMC degradation, glucose was released, which was absorbed by SK10-3 and NK-B2, immediately (Fig. 1). To verify whether this strategy is feasible, BY4742a and NK-B2a strains possessing synthases of betaxanthin were first co-cultured with SK10-3. Due to a longer time period was required for cellulose degradation, we adopted a sequential co-culture strategy. Herein, SK10-3 was first inoculated into the 10 g/L CMC medium for 24 h. Next, BY4742a or NK-B2a was inoculated into the medium in the ratio of 1:1 to SK10-3. After co-incubation of both strains for 48 h, the fermentation broth turned yellow (Fig. 2a), which indicated that BY4742a and NK-B2a were survived in the co-culture system.
After the above experimental confirmation, further biosynthesis of aromatic compounds from CMC was performed. Similar to the previous experiment, NK-B2b, which contained the TAL gene from R. capsulatus, was inoculated after incubation of SK10-3 for 24 h. During the co-culture process, the growth of both these strains was monitored (Fig. 2b). Although the growth of these strains was limited in the CMC medium, p-CA was detected in co-culture samples (Fig. 2c). In the co-culture system, 3.41 mg/L and 5.93 mg/L p-CA were accumulated by BY4742b and NK-B2b, respectively.
Effect of the inoculum ratio and interval time on p-CA production
Considering that cellulose saccharification by SK10-3 is influenced by incubation time, the changes in glucose content for different inoculum amounts of SK10-3 separately incubated in CMC medium were monitored. As shown in Table 1, the higher the amount of SK10-3 inoculated, the faster was the degradation of CMC. CMC was almost fully saccharified after 36 h of incubation, indicating that if the inoculation interval time exceeds 36 h, NK-B2b will not have adequate carbon source for growth. The inoculum ratio is also an important factor to maintain the balance of bacterial growth and product yield in co-culture systems. Thereby, various ratios of SK10-3 to NK-B2b (3:1, 2:1, 1:1, 1:2, and 1:3) and the interval time (0, 12, and 24 h) were investigated simultaneously. The total inoculum OD600 of the two engineered strains was 0.1. During the co-cultivation period, the growth was recorded and the production of p-CA was detected by HPLC after 120 h of co-culture fermentation (Figs. 3 and 4). Although the biomass was lowest when SK10-3 and NK-B2b were simultaneously inoculated, the highest p-CA titer (29.2 mg/L) was observed when these two strains inoculated at the same time with the ratio of 1:2. Additionally, when the ratio of SK10-3 to NK-B2b inoculated simultaneously was 1:1 or the ratio was 1:3 and the interval time was 12 h, the production of p-CA was considerable (23.4 and 24.3 mg/L, respectively). The glucose content of the co-culture systems was also monitored (Table S1).
Table 1
Glucose content during SK10-3 mono-culture in 10 g/L CMC medium with different inoculum doses.
Inoculation amount of SK10-3 (OD600) | Glucose content (mg/L) during mono-cultivation |
0 h | 12 h | 24 h | 36 h | 48 h |
0.075 | - | 31.98 ± 1.28 | 6.48 ± 2.04 | - | - |
0.067 | - | 29.10 ± 2.40 | 22.32 ± 1.54 | 4.50 ± 1.44 | - |
0.050 | - | 25.38 ± 1.43 | 25.56 ± 1.88 | 2.46 ± 0.91 | 1.38 ± 0.85 |
0.033 | - | 15.54 ± 0.75 | 26.40 ± 0.81 | 3.36 ± 1.33 | 3.72 ± 0.81 |
0.025 | - | 13.14 ± 1.18 | 29.10 ± 2.15 | 10.02 ± 1.26 | 4.11 ± 0.75 |
-, indicates no glucose was detected |
Optimization Of The Total Supply Of The Carbon Source
To further increase the production of p-CA in the co-cultivation system, the CMC content in the medium was increased. We increased the final concentration of CMC to 20 and 30 g/L, as adding too much CMC will make the medium thick and almost gelatinous, which is not conducive to medium preparation and fermentation. Subsequently, the three best conditions in the previous result (Fig. 4) were selected to conduct the co-cultivation experiment in the high-carbon source medium, i.e., SK10-3 and NK-B2b inoculated simultaneously at the ratio of 1:1 or 1:2 or inoculated at an interval time of 12 h at the ratio of 1:3. The growth of these strains and the titer of p-CA were measured simultaneously (Fig. 5).
After 120 h of co-cultivation, the p-CA titer was 46.55 mg/L in 30 g/L CMC medium when SK10-3 and NK-B2b were inoculated simultaneously in the ratio of 1:2 (Fig. 5c), although the lowest level of growth was recorded. This might be probably due to the high consistency of the medium with high CMC content, which limited the efficiency of SK10-3 to degrade CMC. High concentrations of glucose were detected in these co-culture samples after 96 h of culture, and CMC was almost completely decomposed and utilized after 168 h of co-cultivation (Table S2). The p-CA titer after 168 h of fermentation was also monitored, and the results were very optimistic. The highest p-CA titer was detected in the sample of SK10-3 and NK-B2b simultaneously inoculated at the ratio of 1:2. The production of p-CA was increased by 54% (71.71 mg/L) as compared to the titer at 120 h (Fig. 5d). Moreover, to determine the ratio of these two strains after co-culture fermentation, the spotting plate experiment was performed because NK-B2b alone cannot survive in CMC medium. In the optimum co-culture condition, the proportion of NK-B2b was 62% after fermentation.
De novo biosynthesis of caffeic acid from CMC
To confirm whether the bioconversion of CMC to more high value-added compounds could be achieved under this co-culture strategy, the production of caffeic acid was assessed; caffeic acid has a high medicinal value and is biosynthesized with p-CA as a precursor. The strain NK-B2c, which possesses the codon-optimized caffeic acid synthase gene HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica [35, 37] on the basis of NK-B2b, was co-cultured with SK10-3 under the optimum co-culture condition we screened before. After 168 h of fermentation, 8.33 mg/L caffeic acid was detected by HPLC (Fig. 6) from 30 g/L CMC medium without any precursor addition. Thus, the de novo biosynthesis of caffeic acid from lignocellulose was achieved.
Improving the production of caffeic acid by a multi-strain co-culture system.
It has been confirmed that the caffeic acid synthases, PaHpaB and SeHpaC, are highly catalytically efficient when expressed in S. cerevisiae [35, 37]. However, in the co-culture system we studied, the titer of caffeic acid was only 8.33 mg/L, and there was a large amount of p-CA residues (Fig. 6c). Accordingly, we speculated that under such low-sugar, unfavorable growth conditions, the caffeic acid biosynthesis pathway increases the growth pressure of the NK-B2c strain, whereby limiting the expression of PaHpaB and SeHpaC. Therefore, we also split the caffeic acid biosynthesis pathway into two strains, one is the NK-B2b strain, which only expresses the RcTAL gene, and the other is the NK-B2d strain, which expresses PaHpaB and SeHpaC genes. To alleviate the metabolic stress of NK-B2c, a multi-strain co-culture system was constructed (Fig. 7a). In this system, SK10-3 still accounted for one-third of the total biomass to meet the glucose supply. To find a balance between the other two strains to maximize the caffeic acid production, we set different inoculation ratios of NK-B2b to NK-B2d (3:1, 2:1, 1:1, 1:2 and 1:3), and detected the final caffeic acid titers (Fig. 7b). After 168 h fermentation, 16.91 mg/L caffeic acid was accumulated while NK-B2b and NK-B2d was inoculated equally, and the residual amount of p-CA was considerably reduced.