Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
Abstract
:1. Introduction
2. Results
2.1. Ambiguity in How Cyanobacteria Rubisco Mutations Improve RDE Fitness
2.2. An Analytical Pathway for the Directed Evolution of Rubisco Using an RDE Screen
2.3. Determination of a Suitable E. coli Strain for RDE3
2.4. Directed Evolution of RsRubisco in the RDE3 Screen
2.5. The RbcL Substitution Y345F Enhances RsRubisco Carboxylation without Impairing Enzyme Biogenesis
3. Discussion
3.1. Mutating RbcL Amino Acid 345 Has Alternate Effects on Form I Rubisco Biogenesis and Catalysis
3.2. How Do the K83Q andR252L Substitutions Function Cooperativly to Reduce RsRubisco Biogenesis?
3.3. Understanding the Basis of RDE Selection
3.4. Pathways for Evolving RsRubisco Further—Why and How?
4. Materials and Methods
4.1. Comparing the PRK Sensitivity of Different E. coli Strains
4.2. Library Construction and Rubisco Selection Using the RDE Screening
4.3. Rubisco Content and Catalysis
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
Raf1 | Rubisco accumulation factor 1 |
Raf2 | Rubisco accumulation factor 2 |
BSD2 | Bundle Sheath Defective II Rubisco chaperone |
RbcX | Rubisco specific chaperone |
cfu | Colony forming units |
RuBP | Ribulose-1,5-bisphosphate |
SC/O | Specificity for CO2 over O2 |
kcatC | Substrate saturated carboxylation rate |
kcatO | Substrate saturated oxygenation rate |
KC | Apparent Km for CO2 |
KC21%O2 | Apparent Km for CO2 under ambient (20.6% (v/v)) O2 |
KO | Apparent Km for O2 |
PRK | Phosphoribulokinase |
Appendix A
Rubisco Origin | Mutation (s) | Plant RbcL Numbering | KC (μM) | KC21%O2 (μM) | kcatC (s−1) | kcatC/KC (s−1·mM−1) | kcatC/KC21%O2 (s−1· mM−1) | KO (μM) | kcatO (s−1) | kcatO/KO | SC/O | KRuBP (μM) | Rubisco (%CSP) | RDE Strain |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Synchococcus PCC6301 (L8S8) | WT | - | 273 | - | 11.4 | 42 | - | - | - | - | 44.0 | 64 | 1.0 | E. coli K12 Ref [29] |
M259T | 262 | 237 | - | 12.8 | 54 | - | - | - | - | 43.0 | 67 | 6.0 | ||
A8S/M259T | 10/262 | 216 | - | 11.4 | 53 | - | - | - | - | 41.0 | 48 | 5.5 | ||
F342S/M259T | 345/262 | 207 | - | 10.0 | 48 | - | - | - | - | 38.0 | 34 | 3.5 | ||
Synchococcus PCC6301 (L8S8) | WT | - | 200 | 242 | 11.8 | 59 | 48.7 | 1191 | 1.7 | 1.42 | 41.5 | 44 | 0.2 | MM1 Ref [30] |
F345I | 345 | 208 | 266 | 10.1 | 49 | 38.0 | 908 | 1.2 | 1.33 | 36.6 | 31 | 3.4 | ||
I174V | 174 | 233 | 283 | 10.2 | 44 | 36.0 | 1174 | 1.2 | 1.02 | 42.9 | 33 | 1.1 | ||
F345L | 345 | 245 | - | 9.8 | 40 | - | - | - | - | 40.0 | 93 | 1.9 | ||
Q212L | 212 | 370 | 444 | 9.9 | 27 | 22.3 | 1252 | 0.9 | 0.72 | 37.4 | 31 | 1.7 | ||
L348M | 348 | 287 | - | 11.3 | 39 | - | - | - | - | 35.1 | 84 | - | ||
N115S | 115 | 191 | - | 9.0 | 47 | - | - | - | - | 43.5 | 44 | - | ||
A272G | 272 | 244 | - | 9.7 | 40 | - | - | - | - | 40.2 | 34 | - | ||
K339R | 339 | 148 | - | 10.1 | 68 | - | - | - | - | 43.0 | 35 | - | ||
M262T | 262 | - | - | - | - | - | - | - | - | - | - | 0.9 | ||
M169L | 169 | - | - | - | - | - | - | - | - | - | - | 0.8 | ||
L161M | 161 | - | - | - | - | - | - | - | - | - | - | 1.1 | ||
Synech.PCC7002 (L8S8) | WT | 132 | - | 6.7 | 51 | - | - | - | - | - | 52 | - | BL21(DE3) Ref [58] | |
E49VS/D82GS | E51VS/D96GS | 200 | - | 14.6 | 74 | - | - | - | - | - | 50 | - | ||
Synchococcus PCC6301 (L8S8) | WT | - | 248 | - | 12.9 | 52 | - | - | - | 0.9 | 56.1 | - | - | MM1 Ref [27] |
F140I | 140 | 129 | - | 19.4 | 150 | - | - | - | 2.9 | 51.3 | - | - | ||
C172G | 172 | 493 | - | 12.8 | 26 | - | - | - | 0.4 | 59.6 | - | - | ||
V189I | 189 | 160 | - | 14.3 | 89 | - | - | - | 1.8 | 49.2 | - | - | ||
V221I | 221 | 181 | - | 2.5 | 14 | - | - | - | 0.3 | 57.9 | - | - | ||
V346L | 346 | 256 | - | 4.3 | 17 | - | - | - | 0.3 | 56.9 | - | - | ||
V346I | 346 | 274 | - | 7.2 | 26 | - | - | - | 0.5 | 54.4 | - | - | ||
S398C | 398 | 322 | - | 18.6 | 58 | - | - | - | 1.0 | 56.2 | - | - | ||
I465V | 465 | 298 | - | 14.3 | 48 | - | - | - | 0.9 | 53.7 | - | - | ||
F345I | 345 | 274 | - | 11.2 | 41 | - | - | - | 0.8 | 52.1 | - | - | ||
F140I/F345I | 140/345 | 218 | - | 15.6 | 72 | - | - | - | 1.2 | 59.3 | - | - | ||
Thermosynech elongatus (L8S8) | WT | - | - | 107 | 6.6 | - | 70 | - | - | - | 53.3 | 36 | 6.6 | RDE2 XL1-B Ref [22] |
F345I | 345 | - | 101 | 6.9 | - | 47 | - | - | - | 48.0 | - | 14.6 | ||
P415A | 415 | - | 111 | 6.9 | - | 62 | - | - | - | 52.6 | - | 10.4 | ||
V300A | 300 | - | 135 | 6.6 | - | 49 | - | - | - | 51.9 | - | 7.1 | ||
F345I/P415A | 345I/415 | - | 102 | 4.8 | - | 47 | - | - | - | 49.2 | - | 17.5 | ||
P415A/V98MS | 415/112S | - | 93 | 9.5 | - | 100 | - | - | - | 56.3 | - | 20.4 | ||
P415A/A48VS | 415/58S | - | 105 | 8.8 | - | 84 | - | - | - | 48.0 | - | 9.7 | ||
P415A/H37LS | 415/39S | - | 100 | 8.4 | - | 84 | - | - | - | 49.7 | - | 11.1 | ||
P415A/Y36NS/G112DS | 415/38S/122S/123S | - | 94 | 9.3 | - | 99 | - | - | - | 47.7 | - | 9.7 | ||
P415A/Y36NS/R51HS | 415/38S/65S | - | 120 | 8.0 | - | 68 | - | - | - | 51.8 | - | 13.6 | ||
P415A/A398T/A48VS | 415/398/58S | - | 105 | 8.1 | - | 75 | - | - | - | 45.5 | - | 13.3 | ||
P415A/L74M/D397N | 415/74/397 | - | 92 | 8.2 | - | 90 | - | - | - | 51.8 | - | 12.5 | ||
P415A/A414T | 415/414 | - | 102 | 8.0 | - | 78 | - | - | - | 55.8 | - | 13.9 | ||
P415A/I393M | 415/393 | - | 90 | 7.8 | - | 87 | - | - | - | 48.1 | - | 16.6 | ||
P415A/A398T | 415/398 | - | 110 | 6.4 | - | 59 | - | - | - | 50.5 | - | 16.6 | ||
Synchococcus PCC6301 (L8S8) | WT | - | - | 251 | 12.6 | - | 50 | - | - | - | 46.0 | 44 | 1.3 | |
F345I | 345 | - | 285 | 10.3 | - | 36 | - | - | - | 42.0 | - | 14.4 | ||
F140I | 140 | - | 211 | 11.1 | - | 53 | - | - | - | 46.0 | - | 6.3 | ||
V189I | 189 | - | 186 | 10.6 | - | 57 | - | - | - | 39.0 | NA | 6.2 | ||
RhodospirIllum Rubrum (L2) | WT | - | 149 | - | 12.3 | 83 | - | 159 | 1.4 | 9.0 | 9.0 | 63 | 0.2 | MM1 Ref [26] |
D117V | 129 | 196 | - | 7.3 | 38 | - | 199 | 1.5 | 8.0 | 5.3 | 55 | 0.6 | ||
D117H | 129 | 192 | - | 7.5 | 39 | - | 153 | 1.1 | 7.0 | 5.3 | 48 | 0.5 | ||
H44Q | 56 | 301 | - | 9.3 | 31 | - | 185 | 1.2 | 6.0 | 5.3 | 56 | 0.9 | ||
H44N | 56 | 204 | - | 9.8 | 48 | - | 116 | 1.0 | 9.0 | 5.5 | 59 | 0.7 | ||
Methanococcoides burtonii (L2–L10) | WT | - | 57 | 1337 | 0.6 | 11 | 0.5 | 11 | 0.1 | 8.0 | 1.3 | 4 | 7.0 | MM1 Ref [40] |
E138V | 146 | 66 | 543 | 1.0 | 15 | 1.8 | 35 | 0.4 | 11.0 | 1.5 | 2 | 8.0 | ||
K332E | 336 | 78 | 892 | 1.2 | 15 | 1.4 | 24 | 0.3 | 13.0 | 1.3 | 5 | 6.0 | ||
T421A | 400 | - | - | 1.3 | - | - | - | - | - | 1.7 | - | 9.0 | ||
M423V | 402 | - | - | 1.1 | - | - | - | - | - | - | - | 7.0 | ||
H379R | 360 | - | - | 0.6 | - | - | - | - | - | - | - | 6.0 | ||
Y471N | 471 | - | - | 0.9 | - | - | - | - | - | - | - | 4.0 | ||
G327S | 331/insert\332 | - | - | 0.7 | - | - | - | - | - | - | - | 6.0 | ||
Synechococcus PCC6301 (L8S8) | WT | - | 186 | - | 3.5 | 22 | - | - | - | - | - | 38 | - | R.capsulatus SBI/II-mutant Ref [31] |
F342V | 345 | 256 | - | 2.4 | 12 | - | - | - | - | - | 21 | - | ||
D103V | 106 | 459 | - | 2.4 | 7 | - | - | - | - | - | 34 | - | ||
D103N | 106 | 325 | - | 1.8 | 6 | - | - | - | - | - | 36 | - | ||
D103E | 106 | 301 | - | 1.3 | 3 | - | - | - | - | - | 33 | - | ||
Synechococcus PCC6301 (L8S8) | WT | - | 186 | - | 4.9 | 26 | - | 850 | - | - | - | 27 | - | R.capsulatus SBI/II-mutant Ref [43] |
D103V | 106 | 388 | - | 4.9 | 13 | - | 866 | - | - | - | 25 | - | ||
A375V | 378 | 143 | - | 0.6 | 4 | - | 1088 | - | - | - | 34 | - | ||
D103V/A375V | 106/378 | 294 | - | 3.1 | 10 | - | 1153 | - | - | - | 269 | - | ||
Synechococcus PCC6301 (L8S8) | WT | - | 190 | 247 | 4.3 | 21 | - | 841 | - | - | - | 29 | - | R.capsulatus SBI/II-mutant Ref [28] |
A375T | 378 | 146 | - | 0.5 | 3 | - | 1076 | - | - | - | 37 | - | ||
M259T | 262 | 147 | 209 | 4.4 | 27 | - | 595 | - | - | - | 30 | - | ||
A375V/M259T/M57IS | 378/262/81S | 93 | 126 | 2.2 | 22 | - | 716 | - | - | - | 64 | - | ||
R214H | 217 | 682 | - | 3.6 | 6 | - | 2152 | - | - | - | 30 | - | ||
R214H/A375S | 217/378 | 264 | - | 0.6 | 2 | - | 1731 | - | - | - | 25 | - | ||
T327A | 330 | 273 | - | 2.8 | 11 | - | 1303 | - | - | - | 13 | - | ||
V186I | 189 | 106 | 145 | 2.8 | 28 | - | 686 | - | - | - | 25 | - | ||
V186I/T327A | 189/330 | 110 | 133 | 2.1 | 18 | - | 1189 | - | - | - | 25 | - | ||
S325L | 328 | 111 | - | 4.0 | 36 | - | 931 | - | - | - | 251 | - | ||
S325L/T327A | 328/330 | 142 | - | 2.6 | 21 | - | 577 | - | - | - | 77 | - | ||
Ralstonia eutropha (Cupriavidus necator; L8S8) | WT | - | 37 | - | 3.8 | 102 | - | 1149 | - | - | - | 34 | - | R.eutropha H16ΔLS mutant Ref [44] |
Y347V | 345 | 35 | - | 4.1 | 117 | - | 1139 | - | - | - | - | - | ||
A380V | 378 | 34 | - | 0.3 | 9 | - | 1435 | - | - | - | 939 | - | ||
T330A | 328 | 44 | - | 2.8 | 64 | - | 1087 | - | - | - | 29 | - | ||
Y348C | 346 | 24 | - | 1.6 | 25 | - | 674 | - | - | - | 36 | - | ||
T330A/A380V | 328/378 | 28 | - | 0.7 | 25 | - | 1069 | - | - | - | 769 | - | ||
Y348C/A380V | 346/378 | 20 | - | 0.7 | 35 | - | 652 | - | - | - | 581 | - |
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Amino Acid Mutation | aTrait Selected | Rubisco (Solubility) | kcatC | KC | KCair | kcatC/KC | kcatC/KCair | SC/O | b Selection Host | Reference | |
---|---|---|---|---|---|---|---|---|---|---|---|
SeRubisco (Synechococcus sp. PCC 6301)—Expressed in E. coli at ~1% (w/w) of the Cell Soluble Protein. | |||||||||||
F140I | solubility | c WT | c ↑ 46% | ↓ 48% | - | ↑ 288% | - | ↓ 9% | MM1 | [27] | |
↑ 4.9-fold | ↓ 12% | - | ↓ 16% | - | ↑ 6% | WT | RDE2 | [22] | |||
V189I | d solubility | WT | ↑ 8% | ↓ 35% | - | ↑ 71% | - | ↓ 12% | MM1 | [27] | |
↑ 4.8-fold | ↓ 16% | - | ↓ 26% | - | ↑ 14% | ↓ 15% | RDE2 | [22] | |||
- | ↓ 25% | ↓ 44% | ↓ 41% | ↑ 32% | ↑ 8% | - | R. capsulatus | [28] | |||
M262T | d solubility | ↑ 6-fold | ↑ 13% | ↓ 13% | - | ↑ 29% | - | WT | RDE(K12) | [29] | |
↑ 4-fold | - | - | - | - | - | - | MM1 | [22] | |||
- | WT | ↓ 23% | ↓ 15% | ↑ 23% | ↑ 18% | - | R. capsulatus | [28] | |||
F345I | solubility | ↑ 8-fold | ↓ 14% | ↑ 4% | ↑ 10% | ↓ 17% | ↓ 22% | ↓ 13% | MM1 | [30] | |
↑ 6-fold | ↓ 15% | ↑ 10% | - | ↓ 21% | - | ↓7% | MM1 | [27] | |||
↑ 11.1-fold | ↓ 16% | - | ↑ 13% | - | ↓ 28% | ↓ 8% | RDE2 | [22] | |||
F345L | solubility | ↑ 7-fold | ↓ 17% | ↑ 22% | - | ↓ 32% | - | ↓4% | MM1 | [30] | |
F345V | solubility | - | ↓ 25% | ↑ 38% | - | ↓ 45% | - | ↓ 45% | R. capsulatus | [31] | |
Te-Rubisco (Thermosynechococcus elongatus BP-1)—Expressed in E. coli at ~6% (w/w) of the Soluble Protein. | |||||||||||
F345I | solubility | ↑ 2.2-fold | ↓ 27% | - | WT | - | ↓ 24% | ↓ 10% | RDE2 | [22] | |
P415A | solubility | ↑ 1.6-fold | ↑ 5% | - | WT | - | WT | WT | RDE2 | [22] | |
P415A | F345I | solubility/kinetic | ↑ 2.7-fold | ↓ 27% | - | WT | - | ↓ 24% | ↓ 8% | RDE2 | [22] |
P415A | V98MS | solubility/kinetic | ↑ 3.1-fold | ↑ 44% | - | ↓ 13% | - | ↑ 44% | ↑ 6% | RDE2 | [22] |
P415A | A48VS | kinetic | ↑ 1.5-fold | ↑ 33% | - | WT | - | ↑ 35% | ↓ 10% | RDE2 | [22] |
P415A | H37LS | kinetic | ↑ 1.7-fold | ↑ 27% | - | WT | - | ↑ 28% | ↓ 7% | RDE2 | [22] |
P415A | Y36NS/G112DS | kinetic | ↑ 1.5-fold | ↑ 41% | - | ↓ 12% | - | ↑ 60% | ↓ 10% | RDE2 | [22] |
P415A | L74M/D397N | solubility/kinetic | ↑ 1.9-fold | ↑ 24% | - | ↓ 14% | - | ↑ 45% | = WT | RDE2 | [22] |
RsRubisco Isoform | RbcL Residue Number | Rubisco | KC | kcatC | KcatC/KC | SC/O | KO | kcatO | kcatO/KO | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|
11 | 83 | 252 | 345 | a (% CSP) | (μM) | (s−1) | (mM.s−1) | mol·mol−1 | (μM) | (s−1) | (mM·s−1) | |
WT | Val | Lys | Arg | Tyr | 4.9 ± 0.4 a | 60 ± 1 a | 3.7 ± 0.2 a | 60 ± 1 | 58 ± 1 a | 1724 ± 280 | 1.8 | 1.0 |
YZ1 | Ile | Gln | Leu | Phe | 2.9 ± 0.1 b | 66 ± 1 a | 4.7 ± 0.1c | 70 ± 1 | 57 ± 1 a | 1296 ± 207 | 1.6 | 1.3 |
YZ1a | Val | Gln | Leu | Phe | 3.3 ± 0.4 b | 66 ± 1 a | 4.5 ± 0.1c | 68 ± 2 | - | - | - | - |
YZ1b | Ile | Gln | Leu | Tyr | 3.0 ± 0.1 b | 61 ± 1 a | 3.8 ± 0.1 a | 63 ± 1 | - | - | - | - |
YZ1c | Val | Gln | Leu | Tyr | 3.6 ± 0.a b | 63 ± 1 a | 3.9 ± 0.1 ab | 63 ± 1 | - | - | - | - |
YZ1d | Ile | Gln | Arg | Phe | 4.6 ± 0.1 a | 62 ± 1 a | 4.6 ± 0.1c | 76 ± 1 | - | - | - | - |
YZ1e | Ile | Lys | Leu | Phe | 4.5 ± 0.3 a | 63 ± 1 a | 4.3 ± 0.1bc | 70 ± 3 | - | - | - | - |
YZ1f | Val | Gln | Arg | Tyr | 5.1 ± 0.4 a | 61 ± 2 a | 3.9 ± 0.2 ab | 65 ± 1 | 57 ± 1 a | 1220 ± 199 | 1.4 | 1.1 |
YZ1g | Val | Lys | Leu | Tyr | 5.3 ± 0.2 a | 62 ± 4 a | 3.5 ± 0.1 a | 57 ± 3 | - | - | - | - |
YZ1h | Ile | Lys | Arg | Phe | 4.6 ± 0.1 a | 61 ± 1 a | 4.4 ± 0.1a | 73 ± 1 | - | - | - | - |
YZ1i | Val | Lys | Arg | Phe | 4.7 ± 0.2 a | 64 ± 1 a | 4.6 ± 0.1a | 71 ± 2 | 59 ± 1 a | 1551 ± 293 | 1.9 | 1.2 |
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Zhou, Y.; Whitney, S. Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction. Int. J. Mol. Sci. 2019, 20, 5019. https://doi.org/10.3390/ijms20205019
Zhou Y, Whitney S. Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction. International Journal of Molecular Sciences. 2019; 20(20):5019. https://doi.org/10.3390/ijms20205019
Chicago/Turabian StyleZhou, Yu, and Spencer Whitney. 2019. "Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction" International Journal of Molecular Sciences 20, no. 20: 5019. https://doi.org/10.3390/ijms20205019