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bioRxiv · 10.1101/862599

A synthetic Calvin cycle enables autotrophic growth in yeast

Gassler, T.·Sauer, M.·Gasser, B.·Mattanovich, D.·Steiger, M. G.
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Abstract

The methylotrophic yeast Pichia pastoris is frequently used for heterologous protein production and it assimilates methanol efficiently via the xylulose-5-phosphate pathway. This pathway is entirely localized in the peroxisomes and has striking similarities to the Calvin-Benson-Bassham (CBB) cycle, which is used by a plethora of organisms like plants to assimilate CO2 and is likewise compartmentalized in chloroplasts. By metabolic engineering the methanol assimilation pathway of P. pastoris was re-wired to a CO2 fixation pathway resembling the CBB cycle. This new yeast strain efficiently assimilates CO2 into biomass and utilizes it as its sole carbon source, which changes the lifestyle from heterotrophic to autotrophic. In total eight genes, including genes encoding for RuBisCO and phosphoribulokinase, were integrated into the genome of P. pastoris, while three endogenous genes were deleted to block methanol assimilation. The enzymes necessary for the synthetic CBB cycle were targeted to the peroxisome. Methanol oxidation, which yields NADH, is employed for energy generation defining the lifestyle as chemoorganoautotrophic. This work demonstrates that the lifestyle of an organism can be changed from chemoorganoheterotrophic to chemoorganoautotrophic by metabolic engineering. The resulting strain can grow exponentially and perform multiple cell doublings on CO2 as sole carbon source with a {micro}max of 0.008 h-1. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/862599v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@c13591org.highwire.dtl.DTLVardef@a3e4d4org.highwire.dtl.DTLVardef@427f5org.highwire.dtl.DTLVardef@db8675_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Gassler, T., Sauer, M., Gasser, B., Mattanovich, D., Steiger, M. G.. 2019-12-03. A synthetic Calvin cycle enables autotrophic growth in yeast. https://doi.org/10.1101/862599

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