CIESC Journal ›› 2019, Vol. 70 ›› Issue (1): 189-198.doi: 10.11949/j.issn.0438-1157.20180813

• Biochemical engineering and technology • Previous Articles     Next Articles

Exploring the key structural properties affecting the function of multi-step phytoene dehydrogenase CrtI

Chen CHEN1,2(),Ying WANG1,2,Hong LIU1,2,Yan CHEN1,2,Mingdong YAO1,2(),Wenhai XIAO1,2   

  1. 1. Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin 300072, China
    2. SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China
  • Received:2018-07-18 Revised:2018-10-26 Online:2019-01-05 Published:2018-10-29
  • Contact: Mingdong YAO E-mail:993203668@qq.com;mingdong.yao@tju.edu.cn

Abstract:

A kind of universal enzyme that catalyzes a multi-step continuous reaction in an organism plays an important role in the biological metabolic process. As a typical representative, phytoene dehydrogenase (CrtI) can catalyze multi-step continuous dehydrogenation to produce products of great value such as lycopene. Herein, the catalytic function of CrtI was studied in Saccharomyces cerevisiae. Firstly, by combining design and screening of three heterologous enzymes CrtE, CrtB and CrtI in the lycopene synthesis pathway, CrtI was confirmed as the main limiting factor, and CrtI from Blakeslea trispora exerted excellent catalytic function. Through bioinformatics and protein structural analysis the key residue S311 of BtCrtI was explored, which linked and maintained the key secondary structure of active center domain. Subsequently, the results of saturation mutation showed that the type of amino acid residue mutated at S311 had a significant effect on the structure and function of the active center domain. This provided a novel structural point for the design and modification of enzymes. Meanwhile, another interesting finding is that the various activity of CrtI mutants did not disturbing the carotenoid metabolic flow in our biosynthesis pathway. Therefore, CrtI is crucial to improve the yield and purity of lycopene.

Key words: enzyme, synthetic biology, biocatalysis, phytoene dehydrogenase(CrtI), multi-step dehydrogenation, enzyme active center structure

CLC Number: 

  • Q 554+.3

Fig.1

Multi-step sequential dehydrogenations catalyzed by CrtI"

Table 1

Plasmid and strains involved in this study"

Plasmids/strainsDescriptionSource

plasmids

pJET1.2/blunt

blunt-end PCR fragments cloning vector

purchased

PCY01pJET1.2/blunt possessing TRP1 homologous arm,TCYC1-AaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1our lab
PCY02pJET1.2/blunt possessing TRP1 homologous arm,TCYC1-PaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1our lab
PCY03pJET1.2/blunt possessing TRP1 homologous arm,TCYC1-XdCrtI-PGAL10-PGAL1-PaCrtB-TPGK1our lab
PCY04pJET1.2/blunt possessing TRP1 homologous arm,TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1our lab
PCY05pJET1.2/blunt possessing LEU2 homologous arm with LEU2 marker, TACT1-tHMG1-PGAL10-PGAL1-PaCrtE-TGPM1our lab
PCY06pJET1.2/blunt possessing LEU2 homologous arm with LEU2 marker, TACT1-tHMG1-PGAL10-PGAL1-SaCrtE-TGPM1our lab
PCY07pJET1.2/blunt possessing LEU2 homologous arm with LEU2 marker, TACT1-tHMG1-PGAL10-PGAL1-AfCrtE-TGPM1our lab
PCY08pJET1.2/blunt possessing LEU2 homologous arm with LEU2 marker, TACT1-tHMG1-PGAL10-PGAL1-BtCrtE-TGPM1our lab
PCY09pJET1.2/blunt possessing LEU2 homologous arm with LEU2 marker, TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1our lab
PCY36-54pJET1.2/blunt possessing TRP1 homologous arm,TCYC1-BtCrtI(S311mutants)-PGAL10-PGAL1-PaCrtB-TPGK1our lab
S. cerevisiaestrains
SyBE_Sc14C10

CEN.PK2-1C(MATα,his3Δ1,leu2-3_112,trp1-289,ura3-52,MAL2-8C,SUC2),Δgal1 Δgal7 Δgal10::HIS3,

Δypl062w::KanMX

our lab
SyBE_Sc14C53

SyBE_Sc14C10,

trp1::TRP1_TCYC1-AaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-PaCrtE-TGPM1

our lab
SyBE_Sc14C22

SyBE_Sc14C10,

trp1::TRP1_TCYC1-PaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-PaCrtE-TGPM1

our lab
SyBE_Sc14C23

SyBE_Sc14C10,

trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-PaCrtE-TGPM1

our lab
SyBE_Sc14C56

SyBE_Sc14C10,

trp1::TRP1_TCYC1-AaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-SaCrtE-TGPM1

our lab
SyBE_Sc14C25

SyBE_Sc14C10,

trp1::TRP1_TCYC1-PaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-SaCrtE-TGPM1

our lab
SyBE_Sc14C26

SyBE_Sc14C10,

trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-SaCrtE-TGPM1

our lab
SyBE_Sc14C59

SyBE_Sc14C10,

trp1::TRP1_TCYC1-AaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-AfCrtE-TGPM1

our lab
SyBE_Sc14C28

SyBE_Sc14C10,

trp1::TRP1_TCYC1-PaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-AfCrtE-TGPM1

our lab
SyBE_Sc14C29

SyBE_Sc14C10,

trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-AfCrtE-TGPM1

our lab
SyBE_Sc14C62

SyBE_Sc14C10,

trp1::TRP1_TCYC1-AaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-BtCrtE-TGPM1

our lab
SyBE_Sc14C31

SyBE_Sc14C10,

trp1::TRP1_TCYC1-PaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-BtCrtE-TGPM1

our lab
SyBE_Sc14C32

SyBE_Sc14C10,

trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-BtCrtE-TGPM1

our lab
SyBE_Sc14C65

SyBE_Sc14C10,

trp1::TRP1_TCYC1-AaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1

our lab
SyBE_Sc14C34

SyBE_Sc14C10,

trp1::TRP1_TCYC1-PaCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1

our lab
SyBE_Sc14C35

SyBE_Sc14C10,

trp1::TRP1_TCYC1-BtCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1

our lab
SyBE_Sc14C130

SyBE_Sc14C10,

trp1::TRP1_TCYC1-XdCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-PaCrtE-TGPM1

our lab
SyBE_Sc14C131

SyBE_Sc14C10,

trp1::TRP1_TCYC1-XdCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-SaCrtE-TGPM1

our lab
SyBE_Sc14C132

SyBE_Sc14C10,

trp1::TRP1_TCYC1-XdCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-AfCrtE-TGPM1

our lab
SyBE_Sc14C133

SyBE_Sc14C10,

trp1::TRP1_TCYC1-XdCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-BtCrtE-TGPM1

our lab
SyBE_Sc14C134

SyBE_Sc14C10,

trp1::TRP1_TCYC1-XdCrtI-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1

our lab
SyBE_Sc14C109-127

SyBE_Sc14C10,

trp1::TRP1_TCYC1-BtCrtI(S311mutants)-PGAL10-PGAL1-PaCrtB-TPGK1,leu2::LEU2_TACT1-tHMG1-PGAL10-PGAL1-TmCrtE-TGPM1

our lab

Fig.2

Comparsion of caroteniods production by CrtE, CrtB and CrtI from diverse species"

Fig.3

Active center structural model of FAD-BtCrtI and substrate (phytoene) complex(a)[cofactor FAD, substrate (phytoene), the key secondary structures β14 and α6 involved in the active center, S311 are labeled respectively; also the key residues and the corresponding interactions are labled by black font]; partial multi-sequence alignment of active center domain of CrtI from different species(b) [the secondary structures β14 and α6 are labled by black squares, and the corresponding site of S311 (BtCrtI) is labled by asterisk]"

Fig.4

Yield difference of dehydrogenation products derived from site-saturation mutagenesis of S311(a); active center structural models of various complex of FAD-mutations of S311 and substrate [(b)-(f)]"

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