De Novo Biosynthesis of Apigenin, Luteolin, and Eriodictyol in the Actinomycete Streptomyces albus and Production Improvement by Feeding and Spore Conditioning

Abstract

Nutraceutical compounds as plant flavonoids play an important role in prevention and modulation of diverse heath conditions, as they exert interesting antifungal, antibacterial, antioxidant, and antitumor effects. They also possess anti-inflammatory activities in arthritis, cardiovascular disease or neurological diseases, as well as modulatory effects on the CYP450 activity on diverse drugs. Most flavonoids are bioactive molecules of plant origin, but their industrial production is sometimes hindered due to reasons as low concentration in the plant tissues, presence in only some species or as a complex mixture or inactive glycosides in plant vacuolae. In this work, we describe the de novo biosynthesis of two important flavones, apigenin and luteolin, and one known flavanone, eriodictyol. Their plant biosynthetic pathways have been reconstructed for heterologous expression in Streptomyces albus, an actinomycete bacterium manageable at industrial production level. Also, production levels for apigenin have been improved by feeding with naringenin precursor, and timing for settlement of secondary metabolism has been advanced by spore conditioning. In the cases of eriodictyol and luteolin, their production in this important type of biotechnology-prone bacteria, the actinomycetes, had not been described in the literature yet.

Multifunctional SEVA shuttle vectors for actinomycetes and Gram‐negative bacteria

“…Recently, it has been shown that apigenin can be completely synthesized de novo in S. albus by the heterologous expression of its biosynthetic pathway (Marín et al, 2017). Taking advantage of this, the five genes ( TAL , 4Cl , CHS , CHI , and FNS ) needed for the production of apigenin from L‐tyrosine were selected.…”

“…With this framework, apigenin can be produced by simply feeding cells with a cheap carbon source, without the need for expensive precursors or inducers which generally increases the final production cost. Recently, all of these elements (GenBank accession numbers LT629810.1, LT629809.1, LT629808.1, LT629807.1, LT629806.1, LT629805.1) were assembled to yield the pAPI plasmid, and its functionality was heterologously validated in S. albus (Marín et al, 2017). Given this previous work, it was decided to test the apigenin production yield with two of the newly designed SEVA shuttle vectors with different copy number values.…”

“…Given this previous work, it was decided to test the apigenin production yield with two of the newly designed SEVA shuttle vectors with different copy number values. For that, the constitutive promoter and the complete apigenin biosynthetic pathway were obtained from the pAPI (Marín et al, 2017) plasmid after digestion with HindIII and the blunt end producing enzyme ScaI. The resulting ~ 7,5 kb DNA fragment with all the necessary elements was purified and cloned into two different copy number shuttle vectors developed as part of this work: (a) an integrative (monocopy) vector and (b) a high‐copy number vector.…”

“…To accomplish this, the following ion sets were selected to detect the transitions of the parent ions to the product ions specific to the analytes: naringenin 272 > 119 Da and 272 > 151 Da; apigenin 270 > 117 Da and 270 > 150 Da. Pure flavonoid standards were purchased from Sigma‐Aldrich (apigenin and naringenin) (Marín et al, 2017).…”

Streptomyces as a Source of Antimicrobials: Novel Approaches to Activate Cryptic Secondary Metabolite Pathways

“…Modifying developmental conditions to enhance PCD and MII differentiation leads to the improvement of secondary metabolite production. This approach was recently applied in a study combining the heterologous expression of plant flavonoids in Streptomyces albus with a strategy to enhance PCD and MII differentiation [110]. The same strategy was successful at improving mTGase production from Streptomyces mobaraensis [111].…”