Model-Guided Systematic Metabolic Engineering for Enhanced Spinosad Biosynthesis in Saccharopolyspora spinosa NHF132.

BackgroundSpinosad, a secondary metabolite produced by Saccharopolyspora spinosa, is a polyketide macrolide insecticide with low toxicity and environmental friendliness. Owing to the high level of DNA methylation and unclear regulatory mechanisms, gene engineering to increase spinosad production is challenging. Limited improvements in yield have been observed with heterologous expression or partial overexpression of the 74-kb spinosyn gene cluster (spn), and research on the overexpression of the complete spinosyn gene cluster is lacking.ResultsThe plasmid pCM265-spn was constructed using CRISPR/Cas9-mediated Transformation-Associated Recombination cloning to enable the overexpression of the complete spn gene cluster in Sa. spinosa. The engineered strain Sa. spinosa-spn achieved a 124% increase in spinosad yield (693 mg/L) compared to the wild type (309 mg/L). The overexpression of the spn gene cluster also delayed spore formation and reduced hyphal compartmentalization by influencing the transcription of related genes (bldD, ssgA, whiA, whiB, and fstZ). Transcriptional analysis revealed significant upregulation of genes in the spn gene cluster, thereby enhancing secondary metabolism. Additionally, optimization of the fermentation medium through response surface methodology further increased spinosad production to 920 mg/L.ConclusionsThis study is the first to successfully overexpress the complete spn gene cluster in Sa. spinosa, significantly enhancing spinosad production. These findings have significance for further optimization of spinosad biosynthesis.

Effects of Glucose and Phosphate on Spinosad Fermentation by Saccharopolyspora spinosa

Spinosad, a highly effective insecticide, has an excellent environmental and mammalian toxicological profile. Global market demand for spinosad is huge and growing. However, after much effort, there has been almost no improvement in the spinosad yield from the original producer, Saccharopolyspora spinosa Here, we report the heterologous expression of spinosad using Saccharopolyspora erythraea as a host. The native erythromycin polyketide synthase (PKS) genes in S. erythraea were replaced by the assembled spinosad gene cluster through iterative recombination. The production of spinosad could be detected in the recombinant strains containing the whole biosynthesis gene cluster. Both metabolic engineering and UV mutagenesis were applied to further improve the yield of spinosad. The final strain, AT-ES04PS-3007, which could produce spinosad with a titer of 830 mg/liter, has significant potential in industrial applications. This work provides an innovative and promising way to improve the industrial production of spinosad. At the same time, it also describes a successful method of heterologous expression for target metabolites of interest by replacing large gene clusters.

Stepwise increase of spinosad production in Saccharopolyspora spinosa by metabolic engineering

Advanced Strategies for Production of Natural Products in Yeast.

dTSR enables efficient improvement of heterologous production of spinosad in Saccharopolyspora erythraea.

Spinosad is a potent insecticide that exhibits an excellent environmental and mammalian profile. However, spinosad production in the original producer, Saccharopolyspora spinosa, is insufficient for the huge global demand. Great efforts have been exerted to improve the production of spinosad. Strategies for spinosad overproduction in actinomycetes are reviewed in this article, including metabolic engineering of the precursor and spinosyn biosynthetic pathway, introduction of regulatory genes, genome-scale metabolic model-guided engineering, mutagenesis, genome shuffling, fermentation process optimization, omics analysis, and the heterologous biosynthesis of spinosad in other actinomycetes. Furthermore, highly productive industrial strains should be used as heterologous hosts for enhancing spinosad biosynthesis in the future. To accelerate the engineering process, the CRISPR/Cas9 system should be established in Sa. spinosa for large-scale genome editing. Notably, the regulatory mechanism of spinosad biosynthesis remains unclear. Thus, the combining multi-omics analysis with high-throughput screening of chemical elicitors would be a promising approach in characterizing the regulatory and signal transduction mechanisms and improving spinosad production in Sa. Spinosa.

Combinatorial metabolic engineering strategy of precursor pools for the yield improvement of spinosad in Saccharopolyspora spinosa

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Metabolic modeling to understand and redesign microbial systems

Due to the increasing demand for microbially manufactured products in various industries, it has become important to find optimal designs for microbial cell factories by changing the direction of metabolic flow and its flux size by means of metabolic engineering such as knocking out competing pathways and introducing exogenous pathways to increase the yield of desired products. Recently, with the gradual cross-fertilization between computer science and bioinformatics fields, machine learning and intelligent optimization-based approaches have received much attention in Genome-scale metabolic network models (GSMMs) based on constrained optimization methods, and many high-quality related works have been published. Therefore, this paper focuses on the advances and applications of machine learning and intelligent optimization algorithms in metabolic engineering, with special emphasis on GSMMs. Specifically, the development history of GSMMs is first reviewed. Then, the analysis methods of GSMMs based on constraint optimization are presented. Next, this paper mainly reviews the development and application of machine learning and intelligent optimization algorithms in genome-scale metabolic models. In addition, the research gaps and future research potential in machine learning and intelligent optimization methods applied in GSMMs are discussed.

Spinosad (spinosyns A and D) is a mixture of secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. In this study, we applied genome shuffling of S. spinosa to achieve a rapid improvement of spinosad production. Ten strains with subtle improvements in spinosad production were obtained from the populations generated by the mutation with nitrosoguanidine and ultraviolet irradiation, and then they were subjected for recursive protoplast fusion. After four rounds of genome shuffling, a high yielding strain, designated as S. spinosa 4-7, was successfully isolated. Its production reached 547 mg/L, which was increased by 200.55% and 436.27% in comparison with that of the highest parent strain and the original strain, respectively. The subculture experiments indicated that the high producer of S. spinosa 4-7 was stable. Spinosad fermentation experiments by S. spinosa 4-7 were carried out in a 5-L fermentor, and its production of spinosad reached 428 mg/L after 168 h of fermentation.

Spinosad, a member of polyketide-derived macrolides produced in the actinomycete Saccharopolyspora spinosa, has been developed as a broad-spectrum and effective insecticide. The β-oxidation pathway could be an important source of building blocks for the biosynthesis of spinosad, thus the effect of vegetable oils on the production of spinosad in a high-yield strain was investigated. The spinosad production increased significantly with the addition of strawberry seed oil (511.64 mg/L) and camellia oil (520.07 mg/L) compared to the control group without oil (285.76 mg/L) and soybean oil group (398.11 mg/L). It also revealed that the addition of oils would affect the expression of genes involved in fatty acid metabolism, precursor supply, and oxidative stress. The genetically engineered strain, in which fadD1 and fadE genes of Streptomyces coelicolor were inserted, produced spinosad up to 784.72 mg/L in the medium containing camellia oil, while a higher spinosad production level (843.40 mg/L) was detected with the addition of 0.01 mM of thiourea.

Saccharopolyspora spinosa is a well-known actinomycete for producing the secondary metabolites, spinosad, which is a potent insecticides possessing both efficiency and safety. In the previous researches, great efforts, including physical mutagenesis, fermentation optimization, genetic manipulation and other methods, have been employed to increase the yield of spinosad to hundreds of folds from the low-yield strain. However, the metabolic network in S. spinosa still remained un-revealed. In this study, two S. spinosa strains with different spinosad production capability were fermented and sampled at three fermentation periods. Then the total RNA of these samples was isolated and sequenced to construct the transcriptome libraries. Through transcriptomic analysis, large numbers of differentially expressed genes were identified and classified according to their different functions. According to the results, spnI and spnP were suggested as the bottleneck during spinosad biosynthesis. Primary metabolic pathways such as carbon metabolic pathways exhibited close relationship with spinosad formation, as pyruvate and phosphoenolpyruvic acid were suggested to accumulate in spinosad high-yield strain during fermentation. The addition of soybean oil in the fermentation medium activated the lipid metabolism pathway, enhancing spinosad production. Glutamic acid and aspartic acid were suggested to be the most important amino acids and might participate in spinosad biosynthesis.

BackgroundPolyketides, such as spinosad, are mainly synthesized in the stationary phase of the fermentation. The synthesis of these compounds requires many primary metabolites, such as acetyl-CoA, propinyl-CoA, NADPH, and succinyl-CoA. Their synthesis is also significantly influenced by NADH/NAD+. Rex is the sensor of NADH/NAD+ redox state, whose structure is under the control of NADH/NAD+ ratio. The structure of rex controls the expression of many NADH dehydrogenases genes and cytochrome bd genes. Intracellular redox state can be influenced by adding extracellular electron acceptor H2O2. The effect of extracellular oxidoreduction potential on spinosad production has not been studied. Although extracellular oxidoreduction potential is an important environment effect in polyketides production, it has always been overlooked. Thus, it is important to study the effect of extracellular oxidoreduction potential on Saccharopolyspora spinosa growth and spinosad production.ResultsDuring stationary phase, S. spinosa was cultured under oxidative (H2O2) and reductive (dithiothreitol) conditions. The results show that the yield of spinosad and pseudoaglycone increased 3.11 fold under oxidative condition. As H2O2 can be served as extracellular electron acceptor, the ratios of NADH/NAD+ were measured. We found that the ratio of NADH/NAD+ under oxidative condition was much lower than that in the control group. The expression of cytA and cytB in the rex mutant indicated that the expression of these two genes was controlled by rex, and it was not activated under oxidative condition. Enzyme activities of PFK, ICDH, and G6PDH and metabolites results indicated that more metabolic flux flow through spinosad synthesis.ConclusionThe regulation function of rex was inhibited by adding extracellular electron acceptor-H2O2 in the stationary phase. Under this condition, many NADH dehydrogenases which were used to balance NADH/NAD+ by converting useful metabolites to useless metabolites and unefficient terminal oxidases (cytochrome bd) were not expressed. So lots of metabolites were not waste to balance. As a result, un-wasted metabolites related to spinosad and PSA synthesis resulted in a high production of spinosad and PSA under oxidative condition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0098-z) contains supplementary material, which is available to authorized users.