| Many Terpenoids natural products are highly valuable in pharmaceutics and other industries. Such as antimalarial artemisinin, anticancer Taxol, ant-oxidative lycopene, sandalwood oil (one of the most highly prized essential oils). Particularly, those Terpenoids most commonly produced in plants, the direct extraction and chemical synthesis of these compounds is often expensive and technically challenging. As the interpretation of these natural products synthesis pathway, recent advances may enable the efficient, cost-effective production of these limited natural resources in microbial metabolic engineering. Via the safe engineering, industrial microorganisms that encode product-specific enzymes, and even entire metabolic pathways, microbial-derived synthetic or semisynthetic Terpenoids may soon replace plant-derived Terpenoids as the primary source of these valuable compounds. The high valuable antioxidants and colorants lycopene and the main component α-santalol o, natural perfume sandalwood oil were selected as the representative compounds for biosynthesis study.1. Biosynthesis of valuable lycopene in Streptomyces avermitilis.(1) Chassis selection. Lycopene are lipid-soluble pigments produced by diverse species, which play a crucial role in the antioxidants with health benefits for humans. Currently, metabolic engineering has the potential to produce these molecules more cost-effectively Large efforts have been focused on using Escherichia coli and yeast as production hosts. Notably, Streptomyces are abundant source of bioactive secondary metabolites and producers, revealing a much higher metabolic potential of bioactive production, and Streptomyces should be an ideal chassis to bioactive agent production with better tolerance and much more precursor titer of target compounds. Here we select S. avermitilis as chassis to synthesis lycopene.(2) The selection of lycopene synthesis gene cluster. A silent lycopene biosynthetic gene cluster in S. avermitilis is used as the lycopene synthesis gene cluster. Lycopene is not only a popular antioxidant, but also perhaps enable convenient colorimetric screening because of its pigment properties. Here, it has facilitated the investigation or optimization of expression elements, including promoter insulator and culture conditions. As expected, these elements circumvent the stringent endogenous regulatory system and activate the gene cluster of lycopene.(3) The selection of promoters from Synthetic Promoters Library. Preliminary research in our laboratory, we developed a quantitative method based on flow cytometry and a super folder green fluorescent protein (sfGFP) at single-cell resolution in Streptomyces. With this sophisticated quantitative method, some 200 native or synthetic promoters and 200 ribosomal binding sites (RBSs) were characterized in a high-throughput format. In this research, seven synthetic promoters with gradient strength were selected to express the lycopene gene cluster, we inserted series of promotors the cassettes upstream of the terpene biosynthetic cluster in S. avermitilis. However, the result showed only promotors of relatively low strength could produce lycopene of modest titer, and using three promotors, which are much stronger than kasOp* as we characterized before, failed to produce detectable lycopene by HPLC.(4) Introduction of the RiboJ insulator. The same as regulatory elements, expression element such as promoter or RBS are responsible for the transcriptional or translational activities of its downstream genes. However, the elements might unpredictably interfere with each other, effect the promoter escaping process, or the RNA second structure formatted. This may be the cause of the above phenomenon. Here, we introduced the RiboJ insulator in the cassettes of lycopene production to eliminate the unanticipated interaction in Streptomyces avermitilis. As we expected, the correlation between lycopene titer and promotor strength was observed, and the highest titer reached 82.02 ±8.69 mg/g (DCW) after five days, increasing six folds compared with that without RiboJ.The extent of overproduction validates the strength of our synthetic promoters, and the correlation between lycopene titer and promoter strength highlights the predictive power endowed by addition of insulator. Indeed, induction of these synthetic promotors and insulator circumvent stringent endogenous regulatory system, not only activate lycopene gene cluster but also lead to lycopene overproduction.2. Biosynthesis of valuable α-santalene in Saccharomyces cerevisiae.(1) Chassis selection. The first biosynthesis step in α-santalol, which the main component of sandalwood oil, is the generation of farnesyl diphosphate (FPP) from Dimethylallyl diphosphate (DMAPP) and Isoprenyl diphosphate(IPP),catalyzed by FPP synthase (FPPS). FPP is cyclized by santalene synthase (SaSSy), which produces a mixture of santalenes (α-,β-and β-epi-santalene) and α-exo-bergamotene. Since SaSSy generated four structurally similar products, it seemed plausible that a single, multi-substrate cytochrome P450 dependent monooxygenase (CYP450) could oxidize α-,β-and β-epi--santalene and a-exo-bergamotene to produce α-,β-and β-epi-santalols and a-exo-bergamotol, respectively. Because CYP450 dependent monooxygenase relies on eukaryotic systems, prokaryotes is typically unsuitable for the expression of eukaryotic P450 enzymes. In this study, yeast is chosen as the chassis strains, such as olefin metabolic advantage yeast Yanxnvia lipolytica. model organisms Saccharomyces cerevisiae. Comprehensive consideration the initial output, proportion of the santalene product, and genetic operations tools, the S. cerevisiae is a better one.(2) Overexpression of tHMGl and ERG20. In order to verify the modification of MVA pathway could increase the precursor pool for isoprenoid synthesis enabling efficient conversion to the target compound a-santalene, two of the main regulatory steps of the MVA pathway catalyzed by 3-hydroxyl-3-methyl-glutaryl-CoAreductase (HMGR) and FPP synthase were overexpressed by GAL1-10p induced promoter system. To circumvent post-transcriptional regulation of HMGR, a truncated HMGR(tHMGR) was introduced. The genetic operation led to a sevenfold increase in a-santalene production.(3) Metabolic engineering of the chassis cells. After the operation of tHMGl and ERG20 effectively, the genes of MVA pathway were all overexpressed, including tHMGl do triple overexpressed. In order to minimize the flow to the biosynthetically related sterols that need the same precursor as a-santalene, FPP, the native promoter (ERG9p) of squalene synthase (SQS) was replaced with a methionine repressed sensing MET3p. Then, to minimize the flux towards farnesol two genes, LPP1 and DPP1, encoding enzymes with FPP dephosphorylation activity have been knockout. At the beginning of the study, using GAL1-10p galactose induced system, to enhance the induction effect, GAL1, GAL10 and GAL7 related galactose metabolism were also been knockout. Firstly, a low copy plasmid pRS416 was used to express exogenous gene, followed the high copy plasmid pRS426 was used. Through the above operation, the production of a-santalene increased about 15-fold. Through the quantitative, the yeast produced 182 mg/L a-santalene in synthetic medium after 3 days by shake flask fermentation. After use codon optimized SanSyn gene replacement the SaSSy, a-santalene yield was 404.74±12 mg/L by BY4742-MVA-426-SanSyn strain. It provides a solid foundation for subsequent a-santalol production.In this research, the chassis selection of Streptomyces could offer a new idea chassis for many Terpenoids biosynthesis in microorganisms. The S. cerevisiae chassis constructed by this work is also suitable for other kinds of sesquiterpenes biosynthesis. |
