Overexpression of Sedoheptulose-1,7-Bisphosphatase Enhances Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes.

Alexander Hammel,Frederik Sommer,Timo Mühlhaus,Michael Schroda,David Zimmer,Mark Stitt

doi:10.3389/fpls.2020.00868

Abstract

The productivity of plants and microalgae needs to be increased to feed the growing world population and to promote the development of a low-carbon economy. This goal can be achieved by improving photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7-bisphosphatase (SBP1) up to threefold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the nuclear transgene represented ∼0.3% of total cell protein. Photosynthetic rate and growth were significantly increased in SBP1-overexpressing lines under high-light and elevated CO2 conditions. Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This analysis also revealed that the 11 CBC enzymes represent 11.9% of total cell protein in Chlamydomonas. Here, the range of concentrations of CBC enzymes turned out to be much larger than estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates are often but not always higher than the binding site concentrations of the enzymes for which they act as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates for overexpression in subsequent engineering steps.

Highlights

An increased productivity of plants and microalgae is required to feed the growing world population and to promote the development of a low-carbon economy
The Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7-bisphosphatase (SBPase) has been shown to exert strong metabolic control over RuBP regeneration at light saturation, as the irreversible reaction that it catalyses is positioned at the branch point between regenerative (RuBP regeneration) and assimilatory portions of the CBC
We chose to use the genomic version of the gene including all seven exons and six introns to adapt it to the Modular Cloning (MoClo) syntax (Weber et al, 2011; Patron et al, 2015)

Summary

Introduction

An increased productivity of plants and microalgae is required to feed the growing world population and to promote the development of a low-carbon economy. Engineering efforts that have resulted in increased biomass are the rewiring of photorespiration (Kebeish et al, 2007; Nolke et al, 2014), the improvement of linear electron transport between the photosystems (Chida et al, 2007; Simkin et al, 2017b), or the overexpression of distinct Calvin-Benson cycle (CBC) enzymes [for recent reviews see Kubis and Bar-Even (2019) and Simkin et al (2019)] The rationale behind the latter approach is that the rising concentration of atmospheric CO2 caused by the burning of fossil fuels increases the velocity of the carboxylation reaction of Rubisco and inhibits the competing oxygenation reaction. The overexpression of SBPase alone (Lefebvre et al, 2005; Tamoi et al, 2006; Feng et al, 2007; Rosenthal et al, 2011; Fang et al, 2012; Ding et al, 2016; Driever et al, 2017; Simkin et al, 2017a) or of the cyanobacterial bifunctional SBPase/FBPase (BiBPase) (Miyagawa et al, 2001; Yabuta et al, 2008; Ichikawa et al, 2010; Gong et al, 2015; Ogawa et al, 2015; Kohler et al, 2017; De Porcellinis et al, 2018) resulted in marked increases in photosynthesis and biomass yields in tobacco, lettuce, Arabidopsis thaliana, wheat, tomato, rice, soybean, in the cyanobacterium Synechococcus, and in the microalgae Euglena gracilis and Dunaliella bardawil

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