TY  - JOUR
A1  - Rieck, Christoph Paul Kurt
A1  - Geiger, Daniel
A1  - Munkert, Jennifer
A1  - Messerschmidt, Katrin
A1  - Petersen, Jan
A1  - Strasser, Juliane
A1  - Meitinger, Nadine
A1  - Kreis, Wolfgang
T1  - Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae
JF  - Microbiologyopen
N2  - A yeast expression plasmid was constructed containing a cardenolide biosynthetic module, referred to as CARD II, using the AssemblX toolkit, which enables the assembly of large DNA constructs. The genes cloned into the vector were (a) a Δ5‐3β‐hydroxysteroid dehydrogenase gene from Digitalis lanata, (b) a steroid Δ5‐isomerase gene from Comamonas testosteronii, (c) a mutated steroid‐5β‐reductase gene from Arabidopsis thaliana, and (d) a steroid 21‐hydroxylase gene from Mus musculus. A second plasmid bearing an ADR/ADX fusion gene from Bos taurus was also constructed. A Saccharomyces cerevisiae strain bearing these two plasmids was generated. This strain, termed “CARD II yeast”, was capable of producing 5β‐pregnane‐3β,21‐diol‐20‐one, a central intermediate in 5β‐cardenolide biosynthesis, starting from pregnenolone which was added to the culture medium. Using this approach, five consecutive steps in cardenolide biosynthesis were realized in baker's yeast.
Y1  - 2019
U6  - https://doi.org/10.1002/mbo3.925
SN  - 2045-8827
VL  - 8
IS  - 12
PB  - Wiley
CY  - Hoboken
ER  - 
TY  - JOUR
A1  - de la Cruz, Jorge Gonzalez
A1  - Machens, Fabian
A1  - Messerschmidt, Katrin
A1  - Bar-Even, Arren
T1  - Core Catalysis of the Reductive Glycine Pathway Demonstrated in Yeast
JF  - ACS synthetic biology
N2  - One-carbon (C1) compounds are attractive microbial feedstocks as they can be efficiently produced from widely available resources. Formate, in particular, represents a promising growth substrate, as it can be generated from electrochemical reduction of CO2 and fed to microorganisms in a soluble form. We previously identified the synthetic reductive glycine pathway as the most efficient route for aerobic growth on formate. We further demonstrated pathway activity in Escherichia coli after expression of both native and foreign genes. Here, we explore whether the reductive glycine pathway could be established in a model microorganism using only native enzymes. We used the yeast Saccharomyces cerevisiae as host and show that overexpression of only endogenous enzymes enables glycine biosynthesis from formate and CO2 in a strain that is otherwise auxotrophic for glycine. We find the pathway to be highly active in this host, where 0.125 mM formate is sufficient to support growth. Notably, the formate-dependent growth rate of the engineered S. cerevisiae strain remained roughly constant over a very wide range of formate concentrations, 1-500 mM, indicating both high affinity for formate use and high tolerance toward elevated concentration of this C1 feedstock. Our results, as well the availability of endogenous NAD-dependent formate dehydrogenase, indicate that yeast might be an especially suitable host for engineering growth on formate.
KW  - metabolic engineering
KW  - synthetic biology
KW  - one-carbon metabolism
KW  - carbon labeling
KW  - tetrahydrofolate
KW  - glycine cleavage system
Y1  - 2019
U6  - https://doi.org/10.1021/acssynbio.8b00464
SN  - 2161-5063
VL  - 8
IS  - 5
SP  - 911
EP  - 917
PB  - American Chemical Society
CY  - Washington
ER  -