Phosphorylation is an important protein post-translational modification and a valuable tool in medicinal chemistry for improving the pharmacological properties of small molecules, but rare in natural product biosynthesis. Here we report phosphorylation in cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides. We identify an unusual kinase domain embedded in the C-terminal macrocyclase encoded in cyanobactin biosynthetic gene clusters and link it to the production of phosphorylated dolichospermamides and aphanizomenamides. Heterologous expression, domain deletion and site directed mutagenesis confirm the role of the kinase domain, while transplantation of this domain into the sphaerocyclamide pathway leads to the production of phosphorylated sphaerocyclamides. The kinase domain acts on both linear and cyclic substrates with preference for Tyr residues. Collectively, these findings expand the diversity of post-translational modifications in cyanobactins and establish a versatile strategy for the engineering and combinatorial biosynthesis of phosphorylated peptides.

Microcystins (MCs) are a family of chemically diverse toxins produced by numerous distantly related cyanobacteria. They are potent inhibitors of eukaryotic protein phosphatases 1 and 2A and are responsible for the toxicosis and death of wild and domestic animals around the world. Microcystins are synthesized on large enzyme complexes comprised of peptide synthetases, polyketide synthases, and additional modifying enzymes. Bioinformatic analysis identified the presence of an additional uncharacterized enzyme in the microcystin ( mcy ) biosynthetic gene cluster in Fischerella sp. PCC 9339, which we named McyK, that lacked a clearly defined role in the biosynthesis of microcystin. Further bioinformatic analysis suggested that McyK belongs to the inosaminephosphate amidinotransferase family and could be involved in synthesizing homo amino acids. Quadrupole time -of -flight tandem mass spectrometry (Q-TOFMS/MS) analysis confirmed that Fischerella sp. PCC 9339 produces MC-Leucine 2 -Homoarginine 4 (MC-LHar) and [Aspartic acid 3 ]MC-Leucine 2 -Homoarginine 4 ([Asp 3 ]MC- LHar) as the dominant chemical variants. We hypothesized that the McyK enzyme might be involved in the production of microcystin variants containing homoarginine (Har) in the strain. Heterologous expression of a codon-optimized mcyK gene in Escherichia coli confirmed that McyK is responsible for the synthesis of L-Har. These results confirm the production of MC-LHar, a novel microcystin chemical variant [Asp 3 ]MC-LHar, and a new microcystin biosynthetic enzyme involved in supply of the rare homo-amino acid Har to the microcystin biosynthetic pathway in Fischerella sp. PCC 9339. This study provides new insights into the logic underpinning the biosynthesis of microcystin chemical variants and broadens our knowledge of structural diversity of the microcystin family of toxins.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 μM to >7.2 μM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Cyanobactins are linear and cyclic post-translationally modified peptides. Here we show that the prenyl-d-Arg-containing autumnalamide A is a member of the cyanobactin family. Biochemical assays demonstrate that the AutF prenyltransferase targets the guanidinium moiety in arginine and homoarginine and is a useful tool for biotechnological applications.

Laxaphycins are a family of cyclic lipopeptides with synergistic antifungal and antiproliferative activities. They are produced by multiple cyanobacterial genera and comprise two sets of structurally unrelated 11- and 12-residue macrocyclic lipopeptides. Here, we report the discovery of new antifungal laxaphycins from Nostoc sp. UHCC 0702, which we name heinamides, through antimicrobial bioactivity screening. We characterized the chemical structures of eight heinamide structural variants A1-A3 and B1-B5. These variants contain the rare non-proteinogenic amino acids 3-hydroxy-4-methylproline, 4-hydroxyproline, 3-hydroxy-d-leucine, dehydrobutyrine, 5-hydroxyl β-amino octanoic acid, and O-carbamoyl-homoserine. We obtained an 8.6-Mb complete genome sequence from Nostoc sp. UHCC 0702 and identified the 93 kb heinamide biosynthetic gene cluster. The structurally distinct heinamides A1-A3 and B1-B5 variants are synthesized using an unusual branching biosynthetic pathway. The heinamide biosynthetic pathway also encodes several enzymes that supply non-proteinogenic amino acids to the heinamide synthetase. Through heterologous expression, we showed that (2S,4R)-4-hydroxy-l-proline is supplied through the action of a novel enzyme LxaN, which hydroxylates l-proline. 11- and 12-residue heinamides have the characteristic synergistic activity of laxaphycins against Aspergillus flavus FBCC 2467. Structural and genetic information of heinamides may prove useful in future discovery of natural products and drug development.

(+)-Valencene and (+)-nootkatone are high value-added sesquiterpenoids found in grapefruit. The synthesis of (+)-nootkatone by chemical oxidation from (+)-valencene cannot meet the increasing demand in natural aromatics markets. Development of a viable bioprocess using microorganisms is attractive. According to the yields of β-nootkatol and (+)-nootkatone by strains harboring different expression cassettes in the resting cell assay, premnaspirodiene oxygenase from Hyoscyamus muticus (HPO), cytochrome P450 reductase from Arabidopsis thaliana (AtCPR) and alcohol dehydrogenase (ADH1) from Saccharomyces cerevisiae were finally selected and overexpressed in CEN·PK2-1Ca, yielding β-nootkatol and (+)-nootkatone with 170.5 and 45.6 mg L-1 ethyl acetate, respectively. A combinational engineering strategy including promoter change, regulator ROX1 knockout, squalene pathway inhibition, and tHMGR overexpression was performed to achieve de novo (+)-valencene production. Subsequent culture investigations found that galactose as the induced carbon source and a lower temperature (25 °C) were beneficial to target accumulation. Also, replacing the inducible promoters (GAL1) of HPO and AtCPR with constitutive promoters (HXT7 and CYC1) dramatically increased the β-nootkatol accumulation from 108.2 to 327.8 mg L-1 ethyl acetate in resting-cell experiments using (+)-valencene as a substrate. Finally, the total terpenoid titer of the engineered strain of PK2-25 using glucose as a carbon source was improved to 157.8 mg L-1 cell culture, which was 56 times the initial value. We present a new candidate for production of (+)-valencene and its related sesquiterpenoids with attraction for industry.

以酿酒酵母为宿主菌株,重构其体内代谢途径,生物合成瓦伦西亚烯及其衍生物.该研究在酿酒酵母菌株中引入来源于纯天仙子的细胞色素P450单加氧酶(cytochrome P450 monooxygenase,HPO)、来源于黄扁柏的瓦伦西亚烯氧化酶(valencene oxidase,CnVO),并构建了它们对应的4个突变体HPOM、CnVO-3、CnVO-4、CnVO-34,与来源于拟南芥的细胞色素还原酶(cytochrome P450 reductase,AtCPR)组合表达,通过静息细胞试验,筛选得到表达HPO和HPOM的菌株催化瓦伦西亚烯的效果最优,选取HPOM作为后续试验的基础.进一步引入来自黄扁柏的瓦伦西亚烯合成酶(valencene synthase,CnVS),过表达醇脱氢酶(alcohol dehydrogenase,ADH1),截短的3-羟基-3-甲基戊二酰辅酶A还原酶(truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase,tHMG1).最终获得1株原位生产瓦伦西亚烯及其衍生物的重组酵母菌株PK2-24,进行3L发酵罐的发酵试验,158 h后总萜产量达310.94 mg/L,较原始菌株提高了111倍.该研究为利用酵母规模化生产瓦伦西亚烯及其衍生物奠定了重要基础.