Abstract
BackgroundMetabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. Glucosinolates represent a good example of such compounds as they are thought to be the cancer-preventive agents in cruciferous plants. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. In the same study, we showed that co-expression of a sixth Arabidopsis gene, γ-glutamyl peptidase 1 (GGP1), resolved a metabolic bottleneck, thereby increasing BGLS accumulation. However, the accumulation did not reach the expected levels, leaving room for further optimization.ResultsTo optimize heterologous glucosinolate production, we have in this study performed a comparative metabolite analysis of BGLS-producing N. benthamiana leaves in the presence or absence of GGP1. The analysis revealed that the increased BGLS levels in the presence of GGP1 were accompanied by a high accumulation of the last intermediate, desulfoBGLS, and a derivative thereof. This evidenced a bottleneck in the last step of the pathway, the transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to desulfoBGLS by the sulfotransferase AtSOT16. While substitution of AtSOT16 with alternative sulfotransferases did not alleviate the bottleneck, experiments with the three genes involved in the formation and recycling of PAPS showed that co-expression of adenosine 5'-phosphosulfate kinase 2 (APK2) alone reduced the accumulation of desulfoBGLS and its derivative by more than 98% and increased BGLS accumulation 16-fold.ConclusionAdjusting sulfur metabolism by directing sulfur from primary to secondary metabolism leads to a remarkable improvement in BGLS accumulation and thereby represents an important step towards a clean and efficient production of glucosinolates in heterologous hosts. Our study emphasizes the importance of considering co-substrates and their biological nature in metabolic engineering projects.
Highlights
Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products
The accumulation of BGLS was, concomitant with a disproportionately higher accumulation of a putative intermediate, the glutathione conjugate GS-B (~100-fold higher at 6 dpi). This problem was solved by the discovery of g-glutamylpeptidase 1 (GGP1), which was able to cleave GS-B in vitro, and whose presence in BGLS-producing N. benthamiana leaves led to a strong reduction in GS-B accumulation (> 99% at 6 dpi) and a substantial increase in BGLS levels (~4-fold at 6 dpi) [4]
The coding sequences (CDSs) of the remaining genes were amplified from existing cDNA clones: adenosine 5’-phosphosulfate kinase 2 (APK2) (At4g39940) from ABRC clone u21470; ATP sulfurylase 1 (ATPS1) (At3g22890) from ABRC clone u10843; UGT74B1SUR1 (At1g24100-At2g20610) from ORF2nat [6]; and AtSOT16 (At1g74100), AtSOT17 (At1g18590), AtSOT18 (Ag1g74090), and PAPS synthetase (PAPS-S) from published cDNA clones [7,8]
Summary
Metabolic engineering in heterologous organisms is an attractive approach to achieve efficient production of valuable natural products. We have recently demonstrated that it is feasible to engineer benzylglucosinolate (BGLS) in the non-cruciferous plant Nicotiana benthamiana by transient expression of five genes from Arabidopsis thaliana. We demonstrated the production of benzylglucosinolate (BGLS) in leaves of N. benthamiana by transient co-expression of five known biosynthetic genes from Arabidopsis thaliana [4]. The accumulation of BGLS was, concomitant with a disproportionately higher accumulation of a putative intermediate, the glutathione conjugate GS-B (~100-fold higher at 6 dpi) This problem was solved by the discovery of g-glutamylpeptidase 1 (GGP1), which was able to cleave GS-B in vitro, and whose presence in BGLS-producing N. benthamiana leaves led to a strong reduction in GS-B accumulation (> 99% at 6 dpi) and a substantial increase in BGLS levels (~4-fold at 6 dpi) [4]. The discovery of GGP1 showed that the transient system was useful for assessing the feasibility of engineering a given pathway, and for gene discovery
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