TY  - JOUR
A1  - He, Hai
A1  - Edlich-Muth, Christian
A1  - Lindner, Steffen N.
A1  - Bar-Even, Arren
T1  - Ribulose Monophosphate Shunt Provides Nearly All Biomass and Energy Required for Growth of E. coli
JF  - ACS Synthetic Biology
N2  - The ribulose monophosphate (RuMP) cycle is a highly efficient route for the assimilation of reduced one-carbon compounds. Despite considerable research, the RuMP cycle has not been fully implemented in model biotechnological organisms such as Escherichia coli, mainly since the heterologous establishment of the pathway requires addressing multiple challenges: sufficient formaldehyde production, efficient formaldehyde assimilation, and sufficient regeneration of the formaldehyde acceptor, ribulose 5-phosphate. Here, by efficiently producing formaldehyde from sarcosine oxidation and ribulose 5-phosphate from exogenous xylose, we set aside two of these concerns, allowing us to focus on the particular challenge of establishing efficient formaldehyde assimilation via the RuMP shunt, the linear variant of the RuMP cycle. We have generated deletion strains whose growth depends, to different extents, on the activity of the RuMP shunt, thus incrementally increasing the selection pressure for the activity of the synthetic pathway. Our final strain depends on the activity of the RuMP shunt for providing the cell with almost all biomass and energy needs, presenting an absolute coupling between growth and activity of key RuMP cycle components. This study shows the value of a stepwise problem solving approach when establishing a difficult but promising pathway, and is a strong basis for future engineering, selection, and evolution of model organisms for growth via the RuMP cycle.
KW  - ribulose monophosphate cycle
KW  - methylotrophy
KW  - metabolic engineering
KW  - growth selection
KW  - carbon labeling
KW  - flux modeling
KW  - formaldehyde assimilation
Y1  - 2018
U6  - https://doi.org/10.1021/acssynbio.8b00093
SN  - 2161-5063
VL  - 7
IS  - 6
SP  - 1601
EP  - 1611
PB  - ACS
CY  - Washington, DC
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  -