Natural Products Heterologous Biosynthesis Based On Systems-synthetic Biology

Natural products are a large group of bioactive compounds produced by living organisms. Many of them have pharmacological use in drug discovery and development, and even industrial or agricultural use. Biosynthesis of natural products in heterologous hosts has become increasingly attractive due to new culture techniques that allow for large scale production. Various natural products or their key intermediates have been successfully obtained in heterologous hosts such as E. coli and S. cerevisiae.Heterologous biosynthesis of natural products becomes a key component of synthetic biology. The research of synthetic biology, however, is highly related to systems biology. Both disciplines are newly developed; the models and methods developed for systems biology are also suitable for the research of synthetic biology. Strategies based on their integration (termed systems-synthetic biology) were performed to construct the in silico platform for natural products heterologous biosynthesis research. The key intermediate in erythromycin and other polyketides biosynthesis, 6-deoxyerythronolide B (6dEB), and the key intermediate in taxol biosynthesis, taxadiene, were selected as the representative compounds for study. The main results were achieved and shown as follows:(1) To solve the problem of low yield of 6dEB (as well as other natural products), systems biology methods were used to analyze the theoretical 6dEB yields in heterologous hosts, and in silico efforts were invested onpredicting some potential key gene targets for improving 6dEB biosynthesis. The theoretical capability of different hosts on producing 6dEB was first evaluated by analyzing the maximum theoretical molar yield of 6dEB (MTMY of 6dEB or MTMY6dEB) under the three feasible carbon sources, glucose, propionate, and glycerol. Results support that glucose is the best substrate for 6dEB production from an economic and long-term standpoint, although presently propionate is more favorable in experiment. By comparing with S. cerevisiae and B. subtilis, E. coliis found to be the most efficient heterologous host for 6dEB biosynthesis due to the highest MTMY values under the same conditions. Besides, two strategies including the flux distribution comparison analysis (FDCA) method and the linear minimization of metabolic adjustment-based (LMOMA-Based) method were developed and specifically employed for in silico strain improvement for 6dEB production, and finally we obtained some potential gene targets for future experimental improvement. Further analysis also indicated that the specific growth rate (SGR), the non-growth associated maintenance (NGAM), the specific oxygen uptake rate (SOUR), and the specific carbon source uptake rate (SCUR) are the key factors directly affecting the MTMY of 6dEB production. For instance, increasing the SGR or NGAM decrease the MTMY, while increasing the SOUR or SCUR increase the MTMY. These findings may guide for further experimental improvement of 6dEB yields in practice.(2) The selection of precursor provision pathways and the improvement of precursor provision in heterologous hosts are key problems in the research of natural products heterologous biosynthesis. Here, we tried to solve these problems from an in silico standpoint of view. The central precursor of terpenoids, isopentenyl diphosphate (IPP), proceeds via two separate pathways, the 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway, in different organisms. Previous works far support the utilization of the MVA pathway for IPP over the DXP pathway for terpenoid biosynthesis. In this study, however, the MTMY of IPP (MTMYIPP) and the thermodynamic properties of these two pathways were analyzed and compared in silico, and results indicated that DXP is superior to MVA for IPP provision. Despite the organisms, for either glucose or glycerol as sole carbon source, the MTMYIPP produced by DXP pathway is obviously higher than MVA pathway. If both pathways were adopted, the MTMYIPP would be a bit higher than DXP pathway alone, but not better enough to support introducing a foreign pathway into the host due to a higher metabolic burden. Besides, the MTMYIPP values in either S. cerevisiae or B. subtilis are inferior to E. coli's, regardless of the kinds of carbon sources. Furthermore, FDCA and LMOMA-Based methods were also applied to predict the key gene targets for improving IPP provision in E. coli, which may play a key role in constructing a high-performance heterologous host for terpenoids biosynthesis. (3) Based on above results, E. coli was selected as the host, and DXP pathway was determined to be the IPP provision pathway for biosynthesis of taxadiene– the key intermediate during taxol biosynthesis. The pathway engineering strategy was performed to improve taxadiene production in E. coli. Firstly, dxs, idi, and certain isp genes involved in DXP pathway were over-expressed with T7 promoter to generate more IPP; secondly, pathway engineering methods including promoter replacement and codon usage optimization were also performed on downstream taxadiene synthesis pathway (TXDP) for improving taxadiene biosynthesis. Finally, a high specific taxadiene production strain was successfully obtained, and 876±60 mg L-1 taxadiene was achieved in bioreactor. It confirms that the endogenic DXP pathway in E. coli has powerful potential in supplying IPP and thus an excellent choice for taxol as well as other terpenoids heterologous biosynthesis.(4) P450 enzymes widely exist in natural products biosynthetic pathways. Difficulties in functional expression of these enzymes in prokaryotic hosts such as E. coli are the bottleneck of research on taxol or other natural products biosynthesis. In this paper, bioinformatic strategies were employed to analyze the hydrophobic profile and the transmembrane helices of CYP725A4 sequence, and a molecular phylogenetic tree of CYP725A4 was constructed. Then, based on the results, the tertiary structures of native and N-terminal truncated CYP725A4 were reconstructed via the homology modeling, and similar operations were performed for native and truncated versions of cytochrome P450 reductase used for the functional expression of CYP725A4. Besides, some strategies for improving the functional expression in heterologous hosts were also outlined. These results may give guidances for ulterior works on molecular engineering and effective functional expression of CYP725A4 or other P450s in heterologous hosts.Because of the well representative research targets and the versatility of the research methods, the platform constructed by this work are also suitable for other kinds of natural products biosynthesis research. In future, the platform will get well developed step-by-step with the development of systems biology and synthetic biology.