Engineering improved Rubisco poses a crucial strategy for enhancing photosynthesis but is challenged by the alternate locations of the plastome rbcL gene and nuclear RbcS genes. Here we develop a RNAi-RbcS Nicotiana tabacum (tobacco) master-line, tobRrΔS, amenable to rbcL-rbcS co-engineering by chloroplast transformation. Four tobacco genotypes coding alternative rbcS genes and adjoining 5ˈ-intergenic sequences revealed Rubisco production was highest in the lines incorporating a rbcS gene whose codon use and 5ˈUTR matched rbcL. These lines produced up to 50% the wild-type Rubisco content. Additional tobacco lines coding potato rbcL-rbcS operons examined how the differing mesophyll made small subunits (pS1, pS2, pS3) or the trichome pST-25 subunit influenced Rubisco biogenesis, catalysis, leaf physiology and plant growth. Rubisco levels were ~15% lower in leaves expressing pS3 relative to those producing pS1, pS2, and pST. However, the pS3-subunit increased carboxylation rate (kcatC) by 13% and carboxylation efficiency (CE, kcatC divided by the Km for CO2) by 17% relative to the pS1 and pS2 subunits as a result of the βa-βb loop substitutions Asn-55-His and Lys-57-Ser. By contrast tobacco photosynthesis and growth were most impaired in lines producing the pST-subunit that reduced CE and CO2/O2 specificity of potato Rubisco by 40% and 15% respectively.