Identification of iturin A produced by B. amyloliquefaciens HM618
Iturin A is a mixture of several homologues (Chen et al. 2019b). As reported, the hydrogen adduct ions m/z of four isoforms of iturin A with a C14 to C17 chain length were 1043.55, 1057.55, 1071.56, and 1085.58, respectively (Chen et al. 2019b; Dang et al. 2019). Consistent with the reported results, LC-MS identification results showed that iturin A produced by B. amyloliquefaciens HM618 was composed of four homologues of C14, C15, C16, and C17 iturin A (Fig. S1). However, the tested C15 iturin A showed two liquid phase peaks, which may be of the same quality but different type. In addition, C14 iturin A occupied the largest proportion in iturin A according to the large response, but C17 iturin A was very small.
Construction of engineering B. subtilis heterologously expressed lipase and amylase
Since food waste was composed of nearly half of starch and about a quarter of lipid, the artificial consortia using food waste as a substrate were constructed to biotransform food waste into iturin A. As presented in Fig. 1, three lipase genes and two amylase genes were introduced into B. subtilis WB800N, meanwhile the promoter coupled with signal peptide in plasmid pHT43 were also replaced. After screening, the recombinant strain WB800N-AmyE with higher amylase activity and the recombinant strain WB800N-Lip with higher lipase activity were mixed-cultured B. amyloliquefaciens HM618. In the mixed-culture fermentation, engineering B. subtilis are defined as the functional strains, secreting hydrolases to convert the FW into glucose and fatty acid chains that are precursors for the synthesis of iturin A.
Verified by sequencing, twenty pHT43 expression plasmids were designed and constructed in B. subtilis WB800N for lipase and amylase production (Fig. S2, S3, S4, S5, S6, S7, and S8). Three controls, B. subtilis WB800N, WB800N+IPTG that was added inducer during fermentation, and B. amyloliquefaciens HM618, were set. As shown in Fig. S9 and S10, the extracellular enzyme activities of the five genes consistently increased from 16 h, and reached the maximum at 48 h Combined with the biomass curve, the increase in enzyme activity was caused by the accumulation of biomass. Further analysis found that under the effect of the IPTG inducible promoter Pgarc, the three lipase genes were better expressed. With the assistance of the signal peptide SPamyQ, the lipase activity of the recombinant strains was more excellent. In addition, the results showed that the signal peptide SPamyQ greatly promoted the secretion of amylase AmyEBa, and the amylase activity of the corresponding recombinant strain WB-A13 and WB-A14 was greatly higher than that of other strains. Regrettably, there was no repeated phenomenon that methyl parathion hydrolase activity was increased hundreds of times in recombinant WB800 under the control of the promoter P43 and signal peptide SPnprB (Zhang et al. 2005).
Comparing the extracellular enzyme activity of each strain at 48 h, the recombinant strains with the higher amylase and lipase activity were selected. As shown in Fig. 2, the recombinant strain WB-A13 had the highest amylase activity, 23406.4 U/mL, which is 8.25 times that of the control group WB+IPTG and 5.26 times that of HM618 strain. In addition, the amylase activity obtaining from HM618 was 1.65 times higher than the WB800N. However, because of the difficultly genetic manipulation on B. amyloliquefaciens HM618, the amyEBa was selected to transfer into other host WB800N. The recombinant strain WB-L01 had the highest lipase activity, 57.5 U/mL, which is 1.96 times that of the control strain WB+IPTG. Undoubtedly, the recombinant strain WB-A13 and WB-L01 were selected to mixed-culture with HM618, converting starch or lipid into a small molecule to synthesis iturin A. In same principle, the recombinant strains WB-A14 with outstanding amylase activity, WB-L02 and WB-L09 with higher lipase activity also were selected to constitute artificial consortia with HM618 strain.
Production ituirn A by artificial consortia of HM618 and recombinant B. subtilis
Co-culture of HM618 with recombinant B. subtilis producing amylase
In view of the fact that nearly half of the organic components in FW are starch, improving the degradation efficiency of starch is conducive to obtaining reducing sugars from the substrate. Hence, artificial consortia of HM618 and recombinant bacteria expressing expertly amylase was constructed for the production of iturin A. B. amyloliquefaciens HM618 under pure culture served as a control. The iturin A production under co-culture of HM618 and WB800N-AmyE was observed in Fig. 3A. The yield of iturin A reached to 7.66 mg/L under the co-culture of HM618 and WB-A14, which was increased 32.9% (p-value <0.05) compared to that of HM618 under pure culture. Compared with co-culture of HM618 and WB-A14, the iturin A production was slightly less under co-culture of HM618 and A13. More iturin A yielded form the consortia than the pure culture, which might be attributed to the addition of recombinant B. subtilis. In the fermentation about degrading complex substrates, it seems feasible to improve the yield of target through adding functional strains based on the characteristics of the substrates. According to the related reports, compared to the co-cultivation of B. amyloliquefaciens and amylase-producing fungi, the surfactin was accumulated more when B. amyloliquefaciens was co-cultured with lipase-producing fungi. This result is attributed to the food waste which contain more lipid than starch (Pan et al. 2021). Furthermore, some studies have reported the use of cellulose in rice straw (Mohapatra et al. 2020) and corn stover (Minty et al. 2013) for biotransformation into biofuels.
The activity of amylase in the co-culture was detected every 24 hours. As observed in Fig. 3B, the changes of amylase activity in co-culture fermentation were almost the same as those of HM618 under pure culture, but they were obviously different from those of recombinant B. subtilis under pure culture. In specifical, the amylase activity in pure-culture fermentation of the recombinant strains reached more than 6000 U/mL at 24 h, and it remained until the late fermentation stage. However, the amylase activity in co-culture fermentation were lower, less than 3000 U/mL, which was almost equivalent to the amylase activity of HM618 under pure culture. Combined with the curves in Fig. S10, the phenomenon that the consumption rate of glucose by HM618 is faster than that of B. subtilis was obviously observed. Theoretically, HM618 could prioritize the use of energy materials to gain an advantage in quantity, causing the limited growth of recombinant B. subtilis in the co-culture fermentation. Thus, the amylase activity of co-culture was similar as the pure culture.
Co-culture of HM618 with recombinant B. subtilis producing lipase
Lipid is the second most abundant nutrient in FW, and the fatty acid chain, which is the degradation product of lipid, is also an important precursor for the synthesis of iturin A, making it promising to construct artificial consortia combining the recombinant B. subtilis producing lipase. However, the effect of co-culture of HM618 and WB800N-Lip was even less satisfactory for iturin A production (Fig. 3D). Although the yield of iturin A was 10.51 mg/L in co-cultivation of HM618 and WB-L02, which was the highest yield of iturin A in co-cultivation fermentation, it was still less than the yield of itruin A in the control group. An interesting phenomenon was that the yield of iturin A was different in the pure culture of HM618. This difference may be mainly due to the instability of the food waste components. In general, the iturin A was produced relatively more under co-culture of HM618 and WB800N-AmyE, which may be related to the more starch content in food waste. Furthermore, the changes of lipase activity were same as the amylase changes (Fig. 3E). This phenomenon possibly reflects the relationship of the microorganisms in mixed-culture fermentation. The fact in fermentation was inferred that HM618 occupied a dominant position in mixed-culture fermentation by virtue of its stronger substrate utilization capacity. Due to the poor substrate utilization, recombinant B. subtilis strains could not accumulate biomass for the secretion hydrolase. This might be the other reason why artificial consortia of HM618 and WB800N-Lip produced less iturin A.
In order to make full use of food waste, the recombinant strain WB-A14 and WB-L02 were selected to mixed-culture with HM618 strain together based on above results. However, in mixed-culture fermentation, the interaction between microorganisms inevitably occurs, including competition for resources and sharing of metabolism (Mee and Wang, 2012). Therefore, a reasonable distribution of substrates and metabolic intermediates in the mixed culture is of great significance for increasing the yield of iturin A. According to reports, by optimizing the inoculation amount of Bacillus polymyxa which could synthesis acetoin and the inoculation time of E coli that produces vitamin B2, the artificial consortium can produce more acetoin (Liu et al. 2017b). Similarly, in the co-culture fermentation with distiller’s grains, optimizing the inoculum of two B. amyloliquefaciens resulted in an increase in the yield of surfactant to 2.54 g/L (Zhi et al. 2017). Therefore, from the perspective of optimizing the inoculation methods, the energy distribution between B. amyloliquefaciens HM618 and recombinant B. subtilis strains should be balanced as much as possible.
Effect of inoculation method of mixed-culture fermentation of HM618, WB-L02, and WB-A14 on iturin A production
The recombinant strains that degrade the food waste were added into the FW medium earlier, to obtain more sufficient small molecules when the strain HM618 was inoculated. Taking into account the higher biomass of recombinant B. subtilis strains after 24 h, different experimental groups that increase the inoculate volume of HM618 were set up. As illustrated in Fig. 4A, when the strain HM618 was directly inoculated with other two recombinant strain, the yield of iturin A reached 8.12 mg/L, which was an increase of 38.8% compared with the control group (p-value <0.01). In contrast, in the group d which even was set to inoculate HM618 for maximum OD600 0.6, the yield of iturin A only ranged between 2.05 to 2.52 mg/L under the delay of HM618 inoculation for 24 hours, greatly less than the control group with the yield of 5.86 mg/L (p-value <0.01). It is not achieved the expectation that the titer of iturin A was dramatically decreased when HM618 was postponed 24 h. The reason might be explained by analyzing other results in mixed-fermentation. As shown in Fig. 4B, the amylase activity in groups b-d at 24 h was higher than that in group a, and this advantage lasted until the later stage of fermentation. Unanimously, the glucose concentration in groups b-d at 24 h were almost 4 times higher than that in group a, and this difference gradually increased with the increase of fermentation time (Fig. 4D). The above results indicated that the amount of B. subtilis was fully accumulated when HM618 was inoculated at 24 h. Based on this speculation, the growth of HM618 was affected in a certain extent in the mixed-fermentation, causing the decreased yield of iturin A in the mixed-fermentation. In addition, the changes of lipase activity shown in Fig. 4C were also embodied that HM618 strain no longer had the advantage of growth in the three-strains consortia when HM618 was inoculated after 24 hours. Although three-strains consortium containing HM618, WB-L02 and WB-A14 showed certain advantages in the production of iturin A compared with other fermentation methods, the energy distribution between the three strains required greater efforts to regulate.