Cultivation of Mycolicibacterium spp. Mutants in Miniaturized and High-Throughput Format to Characterize Their Growth, Phytosterol Conversion Ability, and Resistance to the Steroid Products.

This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire-MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC-MS analysis.

Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium.

The chapter describes the bioconversion of phytosterols into androstenedione (AD) by Mycolicibacterium spp. in shake flasks and fermenters, as well as LC-MS-based methods for analysis of phytosterols and steroid products. Phytosterols are derived as by-products of vegetable oil refining and manufacture of wood pulp. They contain the same four-ring nucleus as steroids and may be converted to high-value steroids by removing the sidechain at C17 and minor changes at other sites in the ring structure. Many bacteria, including Mycolicibacterium spp., can degrade phytosterols. Mutants of Mycolicibacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and androstadienedione (ADD). The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic for the cells. Two main strategies have been employed to overcome this challenge: the use of two-phase systems and the addition of chemically modified cyclodextrins. The latter method is used here. Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as hints how to minimize the practical problems due to the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Optimization of FK-506 production in Streptomyces tsukubaensis by modulation of Crp-mediated regulation.

FK-506 is a potent immunosuppressive macrocyclic polyketide with growing pharmaceutical interest, produced by Streptomyces tsukubaensis. However, due to low levels synthesized by the wild-type strain, biotechnological production of FK-506 is rather limited. Optimization strategies to enhance the productivity of S. tsukubaensis by means of genetic engineering have been established. In this work primarily global regulatory aspects with respect to the FK-506 biosynthesis have been investigated with the focus on the global Crp (cAMP receptor protein) regulator. In expression analyses and protein-DNA interaction studies, the role of Crp during FK-506 biosynthesis was elucidated. Overexpression of Crp resulted in two-fold enhancement of FK-506 production in S. tsukubaensis under laboratory conditions. Further optimizations using fermentors proved that the strategy described in this study can be transferred to industrial scale, presenting a new approach for biotechnological FK-506 production. KEY POINTS: • The role of the global Crp (cAMP receptor protein) regulator for FK-506 biosynthesis in S. tsukubaensis was demonstrated • Crp overexpression in S. tsukubaensis was applied as an optimization strategy to enhance FK-506 and FK-520 production resulting in two-fold yield increase.