Producing fast and active Rubisco in tobacco to enhance photosynthesis

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild type in air supplemented with 1% CO2. This study represents a step towards the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Engineering α-carboxysomes into plant chloroplasts to support autotrophic photosynthesis

“…At 1% CO 2 , the transgenic plants showed a slower growth rate than WT plants (Fig. 4 c, d ) and the Hn Rubisco-expressing tobacco plants 44 . We note that although the production of CB Tob did not cause marked effects on the chloroplast ultrastructure (Fig.…”

“…Quantification of the Rubisco content, by examining the regression of Rubisco activity against the concentration of the Rubisco inhibitor carboxyarabinitol-1,5-bisphosphate (CABP) 48 , further revealed that the Rubisco content in the transgenic chloroplasts was ~30% the Nt Rubisco content in WT tobacco chloroplasts (Table 1 and Supplementary Fig. 1 ) and slightly less than that in the Hn Rubisco-expressing tobacco chloroplasts; 44 however, this value is greater than the Rubisco content in transgenic tobacco chloroplasts that produce simplified cyanobacterial α-carboxysomes (~10% the Nt Rubisco level in WT chloroplasts) 38 .…”

“…A total of 20 μg of plasmid DNA was coated with 3.5 mg gold particle (0.5 mg per shot) according to the standard protocol 44 , 73 . Wild-type (WT) tobacco ( Nicotiana tabacum cv .…”

“…Recent studies have provided advanced knowledge about its protein composition, self-assembly, shell-cargo encapsulation, threedimensional structure, and internal Rubisco organization 11,31,[39][40][41][42] . The extensive information available for H. neapolitanus αcarboxysome assembly and functionality makes it an ideal candidate for engineering carbon-assimilating modules in diverse nonnative organisms [29][30][31]39 , for example, with the intent of enhancing H 2 production 43 and expressing a more active Rubisco in tobacco chloroplasts 44 .…”

Posttranscriptional tuning of gene expression over a large dynamic range in synthetic tobacco chloroplast operons

“…Indeed, several metabolites have been produced by transferring entire biosynthetic pathways into plastids (Fuentes et al, 2018; Jensen and Scharff, 2019). Additionally, transferring the carbon-concentrating mechanism (CCM) from cyanobacterium into plastids to improve photosynthesis illustrates ways in which agronomic traits can be modified by plastid engineering (Lin et al, 2014; Long et al, 2018; Chen et al, 2023).…”