Production of 11α-Hydroxysteroid Derivatives by Corynebacterium glutamicum Expressing the Rhizopus oryzae Hydroxylating System.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industry. Particularly, the 11α-hydroxylation of steroids is a process of biotechnological importance currently carried out at industrial scale for the production of contraceptive drugs and glucocorticoids. This process is performed by several fungal species including Rhizopus nigricans, Aspergillus ochraceus, Aspergillus niger, and Rhizopus oryzae that are used to produce by biotransformation hydroxylated steroids for pharmaceutical purposes (Wang et al., J Steroid Biochem Mol Biol 171:254-261, 2017). However, the development of more efficient biotransformation processes is essential since the steroidal derivatives obtained by the in vivo hydroxylation are often a mixture of hydroxylated compounds in different positions of the steroid molecule. This phenomenon is due to the large number of different CYPs contained in the fungal strains.The genes responsible for the 11α-hydroxylase activity in R. oryzae consisting in the cytochrome CYP509C12 and its redox partner, the reductase RoCPR1, have been chemically synthetized forming a synthetic operon named FUN optimized to be expressed in bacteria. To express this operon, we have selected the strain Corynebacterium glutamicum R31 that is a robust and GRAS bacterial strain widely used for industrial purposes. The synthetic operon has been cloned in the pECXK-99E vector, yielding pXKFUN plasmid, and transformed C. glutamicum R31 to generate C. glutamicum R31 (pXKFUN) strain. This strain is not a steroid degrader and can efficiently transport C19 and C21 steroids across the cytoplasmic membrane (García-Fernandez et al. Catalysts 316:1-12, 2017). C. glutamicum can be used as a clean host for steroid biotransformation, because it does not introduce additional undesired side reactions on the steroids, thus reducing the contamination of the final products (Felpeto-Santero et al., Microbiol Biotechnol 12:856-868, 2019). Here we show a proof of concept that C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes. The protocol below provides detailed information on steroid 11α-hydroxylations by Corynebacterium recombinant strain.

Production of 11α-hydroxysteroids from sterols in a single fermentation step by Mycolicibacterium smegmatis.

11α-hydroxylated steroid synthons are one of the most important commercially pharmaceutical intermediates used for the production of contraceptive drugs and glucocorticoids. These compounds are currently produced by biotransformation using fungal strains in two sequential fermentation steps. In this work, we have developed by a rational design new recombinant bacteria able to produce 11α-hydroxylated synthons in a single fermentation step using cholesterol (CHO) or phytosterols (PHYTO) as feedstock. We have designed a synthetic operon expressing the 11α-hydroxylating enzymes from the fungus Rhizopus oryzae that was cloned into engineered mutant strains of Mycolicibacterium smegmatis that were previously created to produce 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) from sterols. The introduction of the fungal synthetic operon in these modified bacterial chassis has allowed producing for the first time 11αOH-AD and 11αOH-ADD with high yields directly from sterols in a single fermentation step. Remarkably, the enzymes of sterol catabolic pathway from M. smegmatis recognized the 11α-hydroxylated intermediates as alternative substrates and were able to efficiently funnel sterols to the desired hydroxylated end-products.

Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis.

14α-hydroxylated steroids are starting materials for the synthesis of contraceptive and anti-inflammatory compounds in the steroid industry. A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochlioboluslunatus able to hydroxylate steroids has been engineered into a shuttle plasmid named pMVFAN. This plasmid was used to transform two mutants of Mycolicibacterium smegmatis named MS6039-5941 and MS6039 that accumulate 4-androstene-3,17-dione (AD), and 1,4-androstadiene-3,17-dione (ADD), respectively. The recombinant mutants MS6039-5941 (pMVFAN) and MS6039 (pMVFAN) were able to efficiently express the hydroxylating CYP system of C.lunatus and produced in high yields 14αOH-AD and 14αOH-ADD, respectively, directly from cholesterol and phytosterols in a single fermentation step. These results open a new avenue for producing at industrial scale these and other hydroxylated steroidal synthons by transforming with this synthetic operon other Mycolicibacterium strains currently used for the commercial production of steroidal synthons from phytosterols as feedstock.

Identification and expression of the 11ß-steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum.

Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11ß-hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11ß-hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11ß-hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes.

Bioconversion of Phytosterols into Androstadienedione by Mycobacterium smegmatis CECT 8331.

The C19 steroid 1,4-androstadiene-3,17-dione (androstadienedione, ADD) is an added value product used as a synthon in the pharmaceutical industry for the commercial production of corticosteroids, mineralocorticoids, oral contraceptives, and other pharmaceutical steroids. Phytosterol biotransformation catalyzed by microbial whole cells is actually a very well-established research area in white biotechnology. The protocol below provides detailed information on ADD production by the mutant CECT 8331 of Mycobacterium smegmatis mc2155 using phytosterols as raw material in a lab scale. This protocol describes the bioconversion of phytosterols into ADD in a single fermentation step.

Engineering alternative isobutanol production platforms.

A synthetic inducible operon (IbPSO) expressing alsS, ilvC, ilvD and kivD genes encoding a pathway capable to transform pyruvate into 2-isobutyraldehyde has been designed and two recombinant plasmids named pIZIbPSO and p424IbPSO were constructed. The IbPSO containing plasmids can generate in a single transformation event new recombinant isobutanol producer strains and are useful for testing as suitable hosts wild type bacteria in different culture media. In this way we found that Shimwellia blattae (p424IbPSO) was able to produce in flasks up to 6 g l(-1) of isobutanol using glucose as carbon source. Moreover, for the first time, we have demonstrated that isobutanol can be produced from sucrose using Escherichia coli W (ATCC9367) transformed with pIZIbPSO. These robust recombinant strains were also able to produce isobutanol from a raw carbon source like hydrolysed lignocellulosic biomass.

Genome Sequence of the Butanol Hyperproducer Clostridium saccharoperbutylacetonicum N1-4.

Clostridium saccharoperbutylacetonicum is one of the most important acetone-butanol-ethanol (ABE)-generating industrial microorganisms and one of the few bacteria containing choline in its cell wall. Here, we report the draft genome sequence of C. saccharoperbutylacetonicum strain N1-4 (6.6 Mbp; G+C content, 29.4%) and the findings obtained from the annotation of the genome.