**3. Conclusions**

Marine microorganisms have attracted more attention as natural producers of lead compounds. Marine microbes especially are considered as a renewable and reproducible source that can be easily cultured [98,99]. However, the speed of new lead compound discovery is slowing down. Thus, marine microbial co-culturing represents a powerful strategy for the production of novel bio-substances. The strategy can induce the biosynthesis of novel compounds and various NPs coded by corresponding genomes through the activation of the silent gene clusters or previously unexpressed biosynthetic routes.

In the last ten years, the overall statistical studies showed that 156 metabolites were discovered from the co-culture of di fferent marine microbes. Figure 2 and Table 1 illustrated that 59 compounds were isolated from the co-culturing of di fferent marine fungi; 79 compounds were isolated from marine fungi and bacteria; and only 18 compounds were disclosed from co-culturing di fferent marine bacteria. The metabolites by co-culture of marine fungi and bacteria accounted for the largest proportion (51% of all metabolites of marine microbial co-culture). Alkaloids were the largest group with ≥51.9%, whereas macrolides were the lowest group with <0.65%. Just only one macrolide was identified from

the co-cultures of di fferent marine bacteria. Furthermore, co-cultures of di fferent marine bacteria did not produce cyclopeptides, phenylpropanoids and steroids, and co-cultures of di fferent marine fungi did not induce the biosynthesis of terpenoids.

Several studies sugges<sup>t</sup> that *Aspergillus* spp. are the most common fungi that co-fermented with other microbes and produce numerous novel skeletons. The majority of these NPs have antimicrobial or/and antitumor activities. However, some significant restrictions obstruct the development of the co-culture technology; e.g., cryptic and undefined biosynthesis routes and the producers of NPs from the co-cultivation of two or more microorganisms, the particularities of strains and environmental and nutritional requirements, the instability of the ecological relationship, the uncertainty of the interaction relationship and the high contamination probability. Therefore, new technology and equipment need to be created, such as metabolomics analysis and molecular network technology. The new mechanisms of chemical communication of microbes (through direct/mediate contact) also need to be further investigated. In conclusion, co-culture is still shrouded in mystery as a prospective experimental tool for novel bioactive NPs. This article embodies the value and diversity of NPs from the co-cultivation of marine-derived microorganisms and it is considered as a guided reference for studying NPs.

**Author Contributions:** J.C. and P.Z. conceived and wrote the review; X.Y. and B.W. o ffered important advice to improve the review; M.E., H.Z. and H.W. revised the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research work was supported by National Natural Science Foundation of China (number 81773628 and 41776139), and the 111-National Overseas Expertise Introduction Center for Green Pharmaceutical Discipline Innovation (number D17012).

**Conflicts of Interest:** The authors declare no conflict of interest.
