Engineering metabolic pathways in Amycolatopsis japonicum for the optimization of the precursor supply for heterologous brasilicardin congeners production

Abstract

The isoprenoid brasilicardin A is a promising immunosuppressant compound with a unique mode of action, high potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin has been hampered since the producer strain Nocardia terpenica IFM0406 synthesizes brasilicardin in only low amounts and is a biosafety level 2 organism. Previously, we were able to heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum. Four brasilicardin congeners, intermediates of the BraA biosynthesis, were produced. Since chemical synthesis of the brasilicardin core structure has remained elusive we intended to produce high amounts of the brasilicardin backbone for semi synthesis and derivatization. Therefore, we used a metabolic engineering approach to increase heterologous production of brasilicardin in A. japonicum. Simultaneous heterologous expression of genes encoding the MVA pathway and expression of diterpenoid specific prenyltransferases were used to increase the provision of the isoprenoid precursor isopentenyl diphosphate (IPP) and to channel the precursor into the direction of diterpenoid biosynthesis. Both approaches contributed to an elevated heterologous production of the brasilicardin backbone, which can now be used as a starting point for semi synthesis of new brasilicardin congeners with better properties.

Modulating DHA-Producing Schizochytrium sp. toward Astaxanthin Biosynthesis via a Seamless Genome Editing System

“…We got 5–10 colonies per plate when hmgs , hmgr , or crtZ was overexpressed, while less than 5 colonies per plate were obtained when either idi , crtE , or crtIBY was expressed. This suggests that overexpressing endogenous idi , crtE , or crtIBY alone might affect the recovery of the colonies, probably via the accumulation of excess toxic intermediates such as IPP, DMAPP, and GGPP. − HPLC results showed that overexpressing hmgs , hmgr , idi , crtE , crtIBY , or crtZ individually in strain M326 all led to increased astaxanthin levels when compared with those in strain M326 (Figure c,d). Although the biomass was slightly reduced in all transformants, both the astaxanthin titer and yield of each transformant were increased, especially by overexpressing hmgr , idi , or crtE (Figure c).…”

Genome Assembly and Genetic Traits of the Pleuromutilin-Producer Clitopilus passeckerianus DSM1602

“…Transcription analysis will help to identify the most competing pathways. Moreover, pathway genes of the primary metabolism such as the mevalonate pathway are now accessible and allow for a targeted manipulation of metabolic fluxes towards terpenoid biosynthesis [ 71 , 72 ].…”

A novel LysR‐type regulator negatively affects biosynthesis of the immunosuppressant brasilicardin

“…The recent use of heterologous host systems and genetic manipulations of the regulatory genes and precursor pathways turned out to be efficient in the improvement of production yield of brasilicardin congeners [8,56], serving as a starting point for a semi-synthetic approach. The latest advances in the total synthesis of brasilicardins (however, limited by low total yield) [57,58] combined with a biosynthetic approach could allow further improvement of the The average values of three technical replicates obtained for each strain were normalized to A. japonicum::pPS1+pIJ (100%), standard deviations were calculated using Excel spreadsheet and are presented on the graph production yields.…”