Engineering a Biosynthetic Pathway to Produce (+)-Brevianamides A and B

Abstract

The privileged fused-ring structure comprising the bicyclo[2.2.2]diazaoctane (BDO) core is prevalent in diketopiperazine (DKP) natural products that exhibit potent and diverse biological activities. Typically, only low yields of these compounds can be extracted from native fungal producing strains and accessing them diastereoselectively remains challenging using available synthetic routes. BDO-containing DKPs including (+)-brevianamides A 5 and B 6 are assembled via multi-enzyme biosynthetic pathways incorporating non-ribosomal peptide synthetases, prenyltransferases, flavin monooxygenases, cytochromes P450 and isomerases. To simplify access to this class of alkaloids, we designed an engineered biosynthetic pathway in Escherichia coli, composed of six enzymes sourced from different kingdoms of life. The pathway includes a cyclodipeptide synthase from Streptomyces sp. CMB-MQ030 (NascA), cyclodipeptide oxidase from Streptomyces sp. F5123 (DmtD2/DmtE2), prenyltransferase from Aspergillus sp. MF297-2 (NotF), flavin-dependent monooxygenase from Penicillium brevicompactum (BvnB), and kinases from Shigella flexneri and Thermoplasma acidophilum (PhoN and IPK). Cultivated in glycerol media supplemented with prenol, the engineered E. coli strain produces 5.3 mg/L of (-)-dehydrobrevianamide E 4, which undergoes a previously reported lithium hydroxide rearrangement cascade to yield 5 and 6, with a combined 70% yield and a 94:6 diastereomeric ratio. Additionally, titers of 4 were increased to 20.6 mg/L by enhancing NADPH pools in the engineered strain. Overall, our study combines de novo biosynthetic pathway engineering and chemical synthesis approaches to generate complex indole alkaloids.

Keywords: Biosynthesis; CRISPR; alkaloid; biocatalysis; indole; synthetic biology.

Figure 1.

De novo biosynthesis of diketopiperazines. (a) Origins of genes used in this study. Arrows point to surrogate genes. NRPS = non-ribosomal peptide synthetase, FMO = flavin-dependent monooxygenase, PT = prenyltransferase, CDPS = cyclodipeptide synthase, CDO = cyclodipeptide oxidase. (b) Plasmids designed in this study to assemble the engineered biosynthetic pathway. KanR = kanamycin resistance, CmR = chloramphenicol resistance, AmpR = ampicillin resistance. Additional plasmid, pRARE, harbors spectinomycin resistance. Black rectangles represent T7 promoters. (c) Engineered pathway for the total synthesis of (+)-brevianamide A 5 and (+)-brevianamide B 6. (d) Shunt pathway resulting from a desaturation on the proline ring.

Figure 2.

In vitro reactions of NotF and BvnB assessed via HPLC (absorbance at 254 nm).

Figure 3.

Production of all diketopiperazines in target and shunt pathways. (a) HPLC traces of engineered biosynthetic pathway in E. coli measured at 254 nm absorbance using a Phenomenex Lux^® cellulose column (see SI for details). All HPLC trace alignments with authentic standards shown in Figure S9. Compounds at approximately 6.5, 9.5, and 11.5 minutes were confirmed by LC-MS and NMR to be chloramphenicol and acetylated chloramphenicol derivatives. (b) Quantification of unsaturated and saturated NotF and BvnB product titers from the engineered biosynthetic pathway. Strain descriptions listed in Table S3. Open squares signify the absence of the noted gene.

Figure 4.

Assessment of BvnB substrate preference. (a) HPLC traces of individual and in tandem biocatalytic reactions of compounds 3 and 7 with BvnB (absorbance measured at 254 nm using a Phenomenex Luna^® C18(2) column – see SI for details). Percent conversion of each reaction listed in table. (b) Kinetic parameters for BvnB reactions.

Figure 5.

Increasing NADPH via CRISPR-Cas9 KO of phosphofructokinase (PFK) gene, pfkA. (a) Simplified glycolysis and pentose phosphate pathway, highlighting NADPH production through the oxidative branch of the PPP after KO of pfkA. Dotted line refers to subsequent pyruvate metabolism. Glucose-6P = Glucose-6-phosphate, Fructose-6P = F6P = Fructose-6-phosphate, Fructose-1,6-biP = Fructose-1,6-bisphosphate, DHAP = dihydroxyacetone phosphate, G3P = glyceraldehyde-3-phosphate, G6PDH = Glucose-6-phosphate dehydrogenase, 6-PGL = 6-phosphogluconolactone, 6-PG = 6-phosphogluconate, 6PGD = 6-phosphogluconate dehydrogenase, RU5P = ribulose-5-phosphate, R5P = ribose-5- phosphate, X5P = xylulose-5-phosphate, S7P = sedoheptulose-7-phosphate, E4P = erythrose-4-phosphate. (b) Titers in mg/L of 4 quantified by HPLC analysis after four days of incubation (absorbance measured at 254 nm). Strain descriptions listed in Table S3. Δ refers to the KO of pfkA. P value calculated using Graphpad Prism one-way ANOVA.

Scheme 1.

Accessing (+)-brevianamides 5 and 6. (a) Brevianamide biosynthetic pathway. (b) Lawrence group synthesis of 5 and 6.