Novel proline 4-hydroxylase and use thereof
阅读说明：本技术 新型脯氨酸4-羟化酶及其应用 (Novel proline 4-hydroxylase and use thereof ) 是由 孙际宾 赵晶 刘超 周文娟 郑平 马延和 于 2018-08-06 设计创作，主要内容包括：本发明公开了一种改造脯氨酸4-羟化酶的方法,该方法通过在待改造脯氨酸4-羟化酶的特定位点之间的区段进行整体替换,和/或在特定位点进行氨基酸突变能显著提高所得脯氨酸4-羟化酶的活性和热稳定性,从而得到稳定性和活性优异的脯氨酸4-羟化酶。本发明的脯氨酸4-羟化酶可以应用于生产反式-4-羟基-L-脯氨酸或以反式-4-羟基-L-脯氨酸为前体的下游产物。(The invention discloses a method for modifying proline 4-hydroxylase, which can remarkably improve the activity and the thermal stability of the obtained proline 4-hydroxylase by carrying out integral replacement on a section between specific sites of the proline 4-hydroxylase to be modified and/or carrying out amino acid mutation on the specific sites, thereby obtaining the proline 4-hydroxylase with excellent stability and activity. The proline 4-hydroxylase of the invention can be applied to the production of trans-4-hydroxy-L-proline or downstream products using trans-4-hydroxy-L-proline as a precursor.)
1. A method of engineering a proline 4-hydroxylase, the method comprising:
1) retrieving the amino acid sequence of the polypeptide to be modified with proline 4-hydroxylase function;
if the amino acid residue at position 145-146 in the amino acid sequence of the polypeptide to be modified is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase to be engineered which corresponds to position 145-146 of SEQ ID NO:2 is N, G, the amino acid residue which corresponds to position 182 of SEQ ID NO:2 is D or R or S;
replacing the amino acid segment from the 16 th position after the amino acid residue G to the 4 th position before the amino acid residue D or R or S with the amino acid sequence shown in SEQ ID NO. 13 to obtain the modified polypeptide with the function of proline 4-hydroxylase;
or
2) Replacing the 90 th amino acid residue in the amino acid sequence of the polypeptide to be modified or the 90 th amino acid residue corresponding to SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to G from R, so as to obtain the modified polypeptide with the function of the proline 4-hydroxylase;
or
3) Replacing the 112 th amino acid residue in the amino acid sequence of the polypeptide to be modified or the 112 th amino acid residue corresponding to SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to be modified from E to P or A, preferably P, so as to obtain the modified polypeptide with the function of the proline 4-hydroxylase;
or
4) Replacing the amino acid residue at the 260 th position in the amino acid sequence of the polypeptide to be modified or the amino acid residue corresponding to the 260 th position of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to be modified from A to P, so as to obtain the modified polypeptide with the function of the proline 4-hydroxylase;
or
5) A combination of 1) and 2) above, or a combination of 1) and 3), or a combination of 1) and 4).
2. The method as claimed in claim 1, wherein the amino acid sequence with proline 4-hydroxylase function to be modified comprises the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11, preferably the amino acid sequence with proline 4-hydroxylase function to be modified is shown in SEQ ID NO. 2 or SEQ ID NO. 11.
3. A proline 4-hydroxylase, said proline 4-hydroxylase:
1) if the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13;
or
2) The amino acid residue at position 90 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 90 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is G;
or
3) The amino acid residue at position 112 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 112 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is P or A, preferably P;
or
4) The amino acid residue at position 260 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 260 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is P;
or
5) If the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13; and is
The amino acid residue at position 90 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 90 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is G;
or
6) If the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13; and is
The amino acid residue at position 112 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 112 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is P or A, preferably P;
or
7) If the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13; and is
The amino acid residue at position 260 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 260 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is P;
or
8) Derived from the proline 4-hydroxylase of any one of the above 1) to 7), is formed by substituting, deleting or adding one or more, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, and most preferably 1 amino acid residue in the amino acid sequence of the proline 4-hydroxylase of any one of the above 1) to 7), and has the function of the proline 4-hydroxylase of any one of the above 1) to 7).
4. The proline 4-hydroxylase of claim 3, wherein the amino acid sequence of the proline 4-hydroxylase is represented by SEQ ID NO 3, 4, 5, 6, 7, 8, 9, 12.
5. Use of a polypeptide for the production of trans-4-hydroxy-proline or a downstream product that is precursor to trans-4-hydroxy-L-proline, said polypeptide being:
1) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
2) The proline 4-hydroxylase of claim 3 or 4.
6. The use according to claim 5, wherein the polypeptide has an amino acid sequence as shown in SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9, 11, 12 or the amino acid sequence of the polypeptide is as shown in SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9, 11, 12.
7. A host cell comprising genes encoding:
1) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
2) The proline 4-hydroxylase of claim 3 or 4.
8. The host cell of claim 7, wherein the polypeptide has an amino acid sequence as set forth in SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9, 11, 12 or an amino acid sequence as set forth in SEQ ID NO 2, 3, 4, 5, 6, 7, 8, 9, 11, 12.
9. A process for producing trans-4-hydroxy-L-proline or a downstream product that is precursor to trans-4-hydroxy-L-proline, the process comprising:
1) culturing the host cell of claim 7 or 8, and
2) optionally isolating the trans-4-hydroxy-L-proline produced or the downstream product of which trans-4-hydroxy-L-proline is a precursor from the culture system of 1);
alternatively, the method comprises:
1) the following polypeptides are used to catalyze L-proline to produce trans-4-hydroxy-L-proline:
a. a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
b. The proline 4-hydroxylase of claim 3 or 4; and
2) optionally isolating the obtained trans-4-hydroxy-L-proline from the catalytic system of 1).
10. A method of constructing a strain producing trans-4-hydroxy-L-proline or a downstream product from which trans-4-hydroxy-L-proline is a precursor, the method comprising causing the strain to comprise a polypeptide which is:
a. a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
b. The proline 4-hydroxylase of claim 3 or 4.
Technical Field
The invention relates to the field of biotechnology. In particular, the invention relates to novel proline 4-hydroxylase enzymes, genes encoding them and host cells comprising the enzymes or genes encoding them and their use in the production of trans-4-hydroxy-L-proline.
Background
trans-4-hydroxy-L-proline (hydroxyproline for short) is an amino acid with unique physiological activity, is easy to dissolve in water, can be extensively used in the fields of medicine, chemical industry, animal feed and beauty treatment industry, etc., and its market prospect is extensive, and because the expensive hydroxyproline is mainly used for synthesizing side chain of carbapenem antibiotic (meropenem, etc.), carbapenem is third-generation antibiotic, and is one of atypical β -lactam antibiotics with extensive antimicrobial spectrum, and its antimicrobial spectrum is extensive and its antimicrobial activity is strong, and its global general market is over 30 billion dollars.
trans-4-hydroxy-L-proline can be produced by hydroxylation of L-proline by proline 4-hydroxylase, and currently, the engineering bacteria for producing amino acids such as L-proline are mainly corynebacterium glutamicum (corynebacterium glutamicum), escherichia coli (e.coli), and the like, but none of these strains has a gene encoding proline 4-hydroxylase. Therefore, the discovery of proline-4-hydroxylase which can be expressed in L-proline-producing bacteria and has high catalytic activity is the key to the industrial production of trans-4-hydroxy-L-proline.
However, the only proline 4-hydroxylase that has been reported to be industrially used is proline 4-hydroxylase derived from
Therefore, the proline 4-hydroxylase with better expression level and catalytic performance is urgently needed in the field, so that the level of trans-4-hydroxy-L-proline produced by a biological method can be improved.
Disclosure of Invention
The invention aims to provide a novel proline 4-hydroxylase, a method for modifying the proline 4-hydroxylase, the proline 4-hydroxylase with improved stability and activity and the application thereof in producing trans-4-hydroxy-L-proline.
In a first aspect, the present invention provides a method of engineering a proline 4-hydroxylase, the method comprising:
1) retrieving the amino acid sequence of the polypeptide to be modified with proline 4-hydroxylase function;
if the amino acid residue at position 145-146 in the amino acid sequence of the polypeptide to be modified is N, G, the amino acid residue at position 182 is D or R or S; or, if the amino acid residue corresponding to position 145-146 of SEQ ID NO 2 in the amino acid sequence of the proline 4-hydroxylase to be engineered is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO 2 is D or R or S;
replacing the amino acid segment from the 16 th position after the amino acid residue G to the 4 th position before the amino acid residue D or R or S with the amino acid sequence shown in SEQ ID NO. 13 to obtain the modified polypeptide with the function of proline 4-hydroxylase;
or
2) Replacing the 90 th amino acid residue in the amino acid sequence of the polypeptide to be modified or the 90 th amino acid residue corresponding to SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to G from R, so as to obtain the modified polypeptide with the function of the proline 4-hydroxylase;
or
3) Replacing the 112 th amino acid residue in the amino acid sequence of the polypeptide to be modified or the 112 th amino acid residue corresponding to SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to be modified from E to P or A, preferably P, so as to obtain the modified polypeptide with the function of the proline 4-hydroxylase;
or
4) Replacing the amino acid residue at the 260 th position in the amino acid sequence of the polypeptide to be modified or the amino acid residue corresponding to the 260 th position of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to be modified from A to P, so as to obtain the modified polypeptide with the function of the proline 4-hydroxylase;
or
3) A combination of 1) and 2) above, or a combination of 1) and 3), or a combination of 1) and 4).
In a specific embodiment, the amino acid sequence with the function of proline 4-hydroxylase to be modified comprises the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; preferably, the amino acid sequence with the function of proline-4-hydroxylase to be modified is shown as SEQ ID NO. 2 or SEQ ID NO. 11.
In a preferred embodiment, the altered polypeptide having proline 4-hydroxylase function has increased thermostability.
In a preferred embodiment, the method further comprises the step of measuring the activity or thermostability of the resulting polypeptide having proline 4-hydroxylase function.
In a preferred embodiment, the amino acid sequence of the modified polypeptide with proline 4-hydroxylase function is shown in SEQ ID NO 3, 4, 5, 6, 7, 8, 9 and 12.
In a second aspect, the present invention provides a proline 4-hydroxylase having:
1) if the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13;
or
2) The amino acid residue at
or
3) The amino acid residue at position 112 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 112 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is P or A, preferably P;
or
4) The amino acid residue at
or
5) If the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13; and is
The amino acid residue at
or
6) If the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13; and is
The amino acid residue at position 112 in the amino acid sequence of the proline 4-hydroxylase, or the amino acid residue corresponding to position 112 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase, is P or A, preferably P;
or
7) If the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase is N, G, the amino acid residue at position 182 is D or R or S; alternatively, if the amino acid residue in the amino acid sequence of the proline 4-hydroxylase corresponding to position 145-146 of SEQ ID NO. 2 is N, G, the amino acid residue corresponding to position 182 of SEQ ID NO. 2 is D or R or S;
the amino acid sequence segment from the 16 th position after the above amino acid residue G to the 4 th position before the above amino acid residue D or R or S in the amino acid sequence of the proline 4-hydroxylase is the amino acid sequence shown in SEQ ID NO. 13; and is
The amino acid residue at
or
8) Derived from the proline 4-hydroxylase of any one of the above 1) to 7), is formed by substituting, deleting or adding one or more, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, and most preferably 1 amino acid residue in the amino acid sequence of the proline 4-hydroxylase of any one of the above 1) to 7), and has the function of the proline 4-hydroxylase of any one of the above 1) to 7).
In a preferred embodiment, the proline 4-hydroxylase is derived from the proline 4-hydroxylase of any one of the above 1) to 7), is formed by adding one or several, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, and most preferably 1 amino acid residues at any one end of the amino acid sequence of the proline 4-hydroxylase of any one of the above 1) to 7), and has the function of the proline 4-hydroxylase of any one of the above 1) to 7).
In a specific embodiment, the amino acid sequence of the proline 4-hydroxylase is shown as SEQ ID NO 3, 4, 5, 6, 7, 8, 9 and 12.
In a third aspect, the invention provides the use of a polypeptide for the production of trans-4-hydroxy-proline or a downstream product from which trans-4-hydroxy-L-proline is a precursor, the polypeptide being:
1) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
2) The proline 4-hydroxylase of the second aspect.
In a specific embodiment, the polypeptide has an amino acid sequence shown as SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, or the amino acid sequence of the polypeptide is shown as SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12.
In a preferred embodiment, the downstream product from which trans-4-hydroxy-L-proline is a precursor is a carbapenem antibiotic side chain.
In a fourth aspect, the present invention provides a host cell comprising genes encoding:
1) a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
2) The proline 4-hydroxylase of the second aspect.
In a specific embodiment, the polypeptide has an amino acid sequence shown as SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, or the amino acid sequence of the polypeptide is shown as SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12.
In a preferred embodiment, the host cell is a bacterium; including but not limited to: coli (E.coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Serratia marcescens (Serratia marcescens), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and Bacillus subtilis (Bacillus subtilis).
In a fifth aspect, the present invention provides a process for the production of trans-4-hydroxy-L-proline or a downstream product from which trans-4-hydroxy-L-proline is a precursor, the process comprising:
1) the host cell of the fourth aspect, and
2) optionally isolating the trans-4-hydroxy-L-proline produced or the downstream product of which trans-4-hydroxy-L-proline is a precursor from the culture system of 1);
alternatively, the method comprises:
1) the following polypeptides are used to catalyze L-proline to produce trans-4-hydroxy-L-proline:
a. a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
b. A proline 4-hydroxylase of the second aspect; and
2) optionally isolating the obtained trans-4-hydroxy-L-proline from the catalytic system of 1).
In a sixth aspect, the present invention provides a method of constructing a strain producing trans-4-hydroxy-L-proline or a downstream product from which trans-4-hydroxy-L-proline is a precursor, the method comprising causing the strain to comprise a polypeptide which is:
a. a polypeptide having an amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 11; or
b. The proline 4-hydroxylase of the second aspect.
In a preferred embodiment, the method comprises allowing the strain to express the polypeptide.
In a further preferred embodiment, the method comprises allowing the strain to comprise an expression vector expressing the polypeptide or integrating a gene encoding the polypeptide into the genome of the strain.
In a preferred embodiment, the host cell is a bacterium; including but not limited to: coli (E.coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Serratia marcescens (Serratia marcescens), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and Bacillus subtilis (Bacillus subtilis).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a schematic representation of L-proline hydroxylation catalyzed by proline-4-hydroxylase to produce trans-4-hydroxy-L-proline;
FIG. 2 shows the alignment of the amino acid sequence shown in SEQ ID NO. 2 with the amino acid sequence annotated as proline 4-hydroxylase.
Detailed Description
The inventors have conducted extensive and intensive studies and unexpectedly found that the activity of the obtained proline 4-hydroxylase mutant can be significantly improved by replacing the whole region between the specific sites of the original proline 4-hydroxylase; the inventor also unexpectedly finds that the mutation of the amino acid of the segment overall replacement combined with the specific site can improve the thermostability of the proline 4-hydroxylase while obviously improving the activity of the proline 4-hydroxylase, thereby obtaining the proline 4-hydroxylase with excellent stability and activity. The present invention has been completed based on this finding.
Definition of terms
The terms "polypeptide" or "polypeptide of the invention" or "proline 4-hydroxylase of the invention" or "proline 4-hydroxylase" or "L-proline 4-hydroxylase" as used herein have the same meaning and are used interchangeably herein to refer to a protein having the activity of catalysing the production of trans-4-hydroxy-L-proline from L-proline. This polypeptide is naturally absent in E.coli and therefore belongs to an exogenous protein.
The proline 4-hydroxylase of the invention is a proline 4-hydroxylase with improved activity, thermostability or both after being modified. The modification is that the whole segment is replaced between the specific sites of the proline 4-hydroxylase to be modified, or the amino acid mutation is carried out at the specific sites of the proline 4-hydroxylase to be modified, or the combination of the two technical means.
Specifically, the proline 4-hydroxylase of the present invention is obtained as follows:
a) the amino acid residue at position 145-146 in the amino acid sequence of the proline 4-hydroxylase to be modified is N, G, and the amino acid residue at position 182 is D or R or S; or, in the amino acid sequence of the proline 4-hydroxylase to be modified, the amino acid residue corresponding to the 145-146 position of SEQ ID NO. 2 is N, G, and the amino acid residue corresponding to the 182 position of SEQ ID NO. 2 is D, R or S, the amino acid segment from the 16 th position after the amino acid residue G to the 4 th position before the amino acid residue D, R or S is replaced by the amino acid sequence shown in SEQ ID NO. 13, so as to obtain the modified proline 4-hydroxylase;
b) or the amino acid residue at
c) or the amino acid residue at position 112 in the amino acid sequence of the proline 4-hydroxylase to be modified, or the amino acid residue corresponding to position 112 of SEQ ID NO. 2 in the amino acid sequence of the proline 4-hydroxylase to be modified is replaced by P or A, preferably P;
d) or the amino acid residue at
e) or combining the above a) and b), a) and c), a) and d).
The modification method can obviously improve the activity of the modified proline 4-hydroxylase. For example, the pure enzyme activity of the engineered proline 4-hydroxylase is more than 2 times that of the wild type.
In view of the teachings of the present invention, it will be appreciated by those skilled in the art that the engineering process of the present invention is not limited to any particular proline 4-hydroxylase, as long as the amino acid sequence of the proline 4-hydroxylase to be engineered meets the requirements set forth above. For example, but not limited to, the amino acid sequence of the proline 4-hydroxylase to be engineered is shown in SEQ ID NO 2 or SEQ ID NO 11; the amino acid sequence of the modified proline 4-hydroxylase is shown in SEQ ID NO 3, 4, 5, 6, 7, 8, 9 and 12.
Based on The knowledge of The prior art, it is well known to those skilled in The art that The alteration of a few amino acid residues in certain regions, e.g., non-critical regions, of a polypeptide does not substantially alter The biological activity, e.g., The sequence resulting from appropriate substitution of certain amino acids does not affect its activity (see Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings pub. Co. P224). Thus, one of ordinary skill in the art would be able to effect such a substitution and ensure that the resulting molecule still possesses the desired biological activity.
Accordingly, the present invention includes not only the proline 4-hydroxylase obtained by the above-described proline 4-hydroxylase modification method, but also further mutants obtained by obvious changes based on the proline 4-hydroxylase obtained by the above-described proline 4-hydroxylase modification method. For example, starting from the modified proline 4-hydroxylase of the present invention, one or more, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 10, more preferably 1 to 3, and most preferably 1, amino acid residue substitutions, deletions or additions are made in the amino acid sequence thereof to form a mutant still having the function of proline 4-hydroxylase. In particular, it will be apparent to those skilled in the art that a mutant formed by the addition of one or more, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue at either end of the amino acid sequence of a protein or polypeptide can function as the original protein or polypeptide. For example, it is apparent that a protein or polypeptide bearing a 6His tag at either end of the amino acid sequence has similar activity to the original protein or polypeptide.
Meanwhile, those skilled in the art know that mutations in which one or more amino acid residues are substituted, deleted, inserted, and added while maintaining the original function of the polypeptide are referred to as conservative mutations, and typical ones among the conservative mutations represent conservative substitutions, which are mutations in which, if the substitution site is an aromatic amino acid, Phe, Trp, and Tyr are substituted for each other; if hydrophobic amino acids, mutual substitution is made in Leu, Ile and Val; if it is a polar amino acid, substitution between Gln and Asn; if it is a basic amino acid, it is substituted with one another in Lys, Arg and His; if it is an acidic amino acid, a mutual substitution between Asp and Glu; if it is an amino acid having a hydroxyl group, Ser and Thr are substituted for each other. Specifically, substitutions between amino acid residues considered conservative substitutions are shown in the following table. These conservative variant mutants can be generated by amino acid substitution, for example, as shown in the following table.
Thus, the present invention includes mutants in which at most 20, preferably at most 10, more preferably at most 3, even more preferably at most 2, and most preferably at most 1 amino acid has been replaced by an amino acid of similar or analogous nature as compared to the amino acid sequence of the proline 4-hydroxylase of the present invention.
The present invention also provides polynucleotides encoding the polypeptides of the invention. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.
Thus, as used herein, "comprising," "having," or "including" includes "comprising," "consisting essentially of … …," "consisting essentially of … …," and "consisting of … …"; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".
"corresponds to"
The term "corresponding to" as used herein has the meaning commonly understood by a person of ordinary skill in the art. Specifically, "corresponding to" means the position of one sequence corresponding to a specified position in the other sequence after alignment of the two sequences by homology or sequence identity. Thus, for example, the amino acid sequence shown in SEQ ID NO. 11 corresponds to the amino acid sequence shown in SEQ ID NO. 2 at position 145-146 which is at position 139-140.
Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: computer Molecular Biology (computerized Molecular Biology), Lesk, a.m. ed, oxford university press, new york, 1988; biological calculation: the Informatics and genomic items (Bi ℃ omputing: information and genoprojects), Smith, D.W., eds, academic Press, New York, 1993; computer analysis of Sequence Data (computer analysis of Sequence Data), first part, Griffin, a.m. and Griffin, h.g. eds, humana press, new jersey, 1994; sequence Analysis in molecular biology (Sequence Analysis in molecular biology), von Heinje, g., academic Press, 1987 and Sequence Analysis primers (Sequence Analysis primer), Gribskov, m. and Devereux, j. eds M.C. kton Press, New York, 1991 and Carllo, h. and Lipman, d.s., SIAM J.applied Math.48: 1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG program package (Devereux, J. et al, 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al, 1990). BLASTX programs are publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al, NCBI NLM NIH Bethesda, Md.20894; Altschul, S. et al, 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
Use of polypeptides
Based on the teachings of the present invention, one skilled in the art will appreciate that the polypeptide or proline 4-hydroxylase of the present invention can be used to produce trans-4-hydroxy-proline or downstream products that are precursors to trans-4-hydroxy-L-proline. Particularly, the trans-4-hydroxy-L-proline is produced by using the proline 4-hydroxylase modified by the invention and taking glucose as a substrate, and the yield can reach 2.39 g/L;
in a specific embodiment, the polypeptide of the invention used for the production of trans-4-hydroxy-proline or a downstream product from which trans-4-hydroxy-L-proline is precursor has the amino acid sequence shown in SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12 or the amino acid sequence thereof is shown in SEQ ID No. 2, 3, 4, 5, 6, 7, 8, 9, 11, 12.
Strain for producing trans-4-hydroxy-L-proline or downstream product using trans-4-hydroxy-L-proline as precursor and construction method thereof
The present invention provides a strain producing trans-4-hydroxy-L-proline or a downstream product containing trans-4-hydroxy-L-proline as a precursor, based on the proline 4-hydroxylase of the present invention. The production strain comprises the proline 4-hydroxylase of the invention, for example comprises a gene coding for the proline 4-hydroxylase of the invention, or comprises an expression vector expressing the proline 4-hydroxylase of the invention, or integrates a gene coding for the proline 4-hydroxylase of the invention into the genome of the strain.
The above-mentioned production strain may be a bacterium, including but not limited to: coli (E.coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Brevibacterium flavum (Brevibacterium flavum), Serratia marcescens (Serratia marcescens), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and Bacillus subtilis (Bacillus subtilis).
Process for the production of trans-4-hydroxy-L-proline or downstream products from which trans-4-hydroxy-L-proline is a precursor
On the basis of the proline 4-hydroxylase or the production strain of the invention, the person skilled in the art knows that trans-4-hydroxy-L-proline or downstream products which are precursors to trans-4-hydroxy-L-proline can be produced using the production strain of the invention; an extracellular enzyme catalytic system may also be used to produce trans-4-hydroxy-L-proline or a downstream product using trans-4-hydroxy-L-proline as a precursor by directly using the proline 4-hydroxylase of the present invention.
The invention has the advantages that:
1. the modification method can obviously improve the activity of proline 4-hydroxylase;
2. the point mutation technology can obviously improve the thermal stability of proline 4-hydroxylase;
3. the amino acid sequence substitution method provided by the invention is combined with a point mutation technology, so that the thermal stability and the activity of proline 4-hydroxylase can be obviously improved, and the yield of trans-4-hydroxy-L-proline is improved.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
The reagents and starting materials used in the present invention are commercially available.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种新型的脯氨酸3-羟化酶及其应用