posted on 2021-10-19, 17:36authored byTaiwo
S. Akinyemi, Nana Shao, Zhe Lyu, Ian J. Drake, Yuchen Liu, William B. Whitman
Methanococcus
maripaludis is a rapidly
growing, hydrogenotrophic, and genetically tractable methanogen with
unique capabilities to convert formate and CO2 to CH4. The existence of genome-scale metabolic models and an established,
robust system for both large-scale and continuous cultivation make
it amenable for industrial applications. However, the lack of molecular
tools for differential gene expression has hindered its application
as a microbial cell factory to produce biocatalysts and biochemicals.
In this study, a library of differentially regulated promoters was
designed and characterized based on the pst promoter,
which responds to the inorganic phosphate concentration in the growth
medium. Gene expression increases by 4- to 6-fold when the medium
phosphate drops to growth-limiting concentrations. Hence, this regulated
system decouples growth from heterologous gene expression without
the need for adding an inducer. The minimal pst promoter
is identified and contains a conserved AT-rich region, a factor B
recognition element, and a TATA box for phosphate-dependent regulation.
Rational changes to the factor B recognition element and start codon
had no significant impact on expression; however, changes to the transcription
start site and the 5′ untranslated region resulted in the differential
protein production with regulation remaining intact. Compared to a
previous expression system based upon the histone promoter, this regulated
expression system resulted in significant improvements in the expression
of a key methanogenic enzyme complex, methyl-coenzyme M reductase,
and the potentially toxic arginine methyltransferase MmpX.