fatty acid synthase

Abstract

This invention relates to proteins or polypeptides involved in fatty acid synthesis, such as fatty acid synthases (FAS). The proteins comprise one or more polypeptide chains, wherein said polypeptide chains contain one or more subunits having at least one amino acid substitution as defined herein, thereby resulting in altered fatty acid synthesis activity. The invention also relates to nucleic acid molecules encoding the domains of said proteins or polypeptides, and host cells containing said nucleic acid molecules (e.g., in carrier form). The invention further describes a method for producing fatty acids, comprising culturing the host cells of the invention, and the use of the proteins, nucleic acids, or host cells of the invention for the production of biofuels, fine chemicals, flavoring compounds, and the determination of fungicides and actinomycetes. Furthermore, the invention describes computer simulation methods capable of identifying molecules that can selectively alter the activity of enzyme domains present in FAS or alter a step in type I FAS fatty acid synthesis, said methods comprising identifying and simulating molecules that interact with at least one amino acid in the proteins as defined herein under physiological conditions. Additionally, a method is provided for identifying compounds capable of altering the activity of at least one enzyme in FAS, the method comprising contacting a candidate compound with FAS as defined herein and determining whether said candidate compound exhibits any interaction with FAS. Finally, a protein, a mutant ACP, was provided, which resulted in altered FAS activity.

Description

Technical Field

[0001] This invention relates to proteins or polypeptides involved in fatty acid synthesis, such as fatty acid synthases (FAS). The protein comprises one or more polypeptide chains, wherein the polypeptide chains contain one or more subunits having at least one amino acid substitution as defined, thereby resulting in an alteration of fatty acid synthase activity. The corresponding one or more substitutions are present within the ACP subunit or within at least one subunit of the FAS I complex, and the one or more substitutions are not present within the catalytic site or catalytic center of the corresponding subunit. The invention also relates to nucleic acid molecules encoding the domains of said proteins or polypeptides, and to host cells containing said nucleic acid molecules (e.g., in carrier form). The invention further describes a method for producing fatty acids, comprising culturing host cells according to the invention, and the use of proteins, nucleic acids, or host cells according to the invention in the production of biofuels, fine chemicals, flavoring compounds, and the determination of fungicides and actinomycetes. Furthermore, the invention describes a computer simulation method for identifying molecules capable of selectively altering the activity of enzymatic domains present in FAS, or altering the effect of FAS I on fatty acid synthesis steps, the method comprising identifying and simulating molecules that interact with at least one amino acid in the protein as defined herein under physiological conditions. Furthermore, the present invention provides a method for identifying compounds capable of altering at least one enzymatic activity of FAS, comprising the steps of contacting a candidate compound with FAS as defined herein and determining whether the candidate compound interacts with the FAS in any way. Finally, the present invention provides a protein, namely a mutated or truncated ACP, which leads to alteration of FAS activity. Background Technology

[0002] The biosynthesis of fatty acids (FAs) follows a sequential and repeating enzymatic cycle, utilizing acetyl-CoA, malonyl-CoA, and NAD(P)H ( Figure 1A(Chan, DI, and Vogel, HJ (2010), Biochemistry J.430, 1–19. 10.1042 / BJ20100462. Acyl transporter proteins (ACPs) and their post-translational activation via the phosphate pantothenic acid (Ppant) cofactor (derived from coenzyme A) on serine residues are essential, Chan, DI, and Vogel, HJ (2010), see above. Fatty acid biosynthesis requires active transport of ACP-based biosynthetic acyl intermediates (acetyl and malonyl groups) as covalent adducts bound to the Ppant group. In yeast, transacylases transfer the acetyl group (AT domain) of acetyl-CoA or the malonyl group (MPT domain) of malonyl-CoA to the ACP. The acetyl group is first transferred to the thiol, the active site of ketone synthase (KS domain), where it undergoes a condensation reaction with the malonyl group bound to ACP. The resulting Ppant-bound β-ketoacyl-ACP is reduced to β-hydroxyacyl-ACP by ketone reductase (KR domain) and then transferred to dehydratase (DH domain). The Ppant-β-hydroxyacyl group undergoes dehydration to generate α-enoyl-ACP, which is then reduced by enoyl reductase (ER domain) to give an alkylacyl-ACP with a two-carbon chain extension. This alkylacyl moiety is then transferred to the thiol at the KS active site, where it undergoes another round of condensation with the MPT-activated malonyl-ACP. This cycle repeats until a C16 / C18 alkylacyl-ACP is generated, which is then transferred to a free coenzyme A molecule by the MPT domain.

[0003] Although this biosynthetic cycle is generally conserved, fatty acid biosynthesizing enzymes have evolved two structural solutions. Bacteria, plants, and mitochondria possess independent fatty acid synthase (FAS) enzymes and ACPs (type II systems), White SW et al., 2005, Biochemistry, Vol. 74: 791–831, 10.1146 / annurev.biochem.74.082803.133524. Type II ACPs interact with different enzymes via diffusion-dependent processes. The second is an integrated, large, multi-subunit type I FAS system, Schweizer E and Hofmann J., 2004, Reviews of Microbiology and Molecular Biology, Vol. 68: 501–517, 10.1128 / MMBR.68.3.501-517.2004. Type I systems are either X-shaped dimers (α2), a typical characteristic of metazoan FAS, where each polypeptide chain contains all catalytic domains and is linked to the ACP via a flexible linker; or multi-barrel structures. In Corynebacterium / Mycobacterium / Nocardia (CMN) bacteria, all enzymatic activity is concentrated in a single polypeptide chain (α6); while in fungal FAS, it is concentrated in two polypeptide chains (α6β6). These barrel structures are characterized by a central ring on the equatorial plane, from which two dome-shaped structures extend axially. In this structure, the ACP is located inside the FAS barrel structure and attached via two flexible linkers. The ACPs in CMN bacteria and fungal FAS, in addition to containing the standard four-helix bundle domains found in bacterial and metazoan ACPs, also extend into four helices (domains), Günenc AN et al., 2022; included in *Macroprotein Complexes IV: Structure and Function*, edited by JR Harris and J. Marles-Wright, Springer International Press, pp. 1–33, 10.1007 / 978-3-031-00793-4_1. It is believed that the high efficiency of type I fatty acid synthesis systems is due to the separation of the fatty acid biosynthetic reaction chambers and the spatial proximity of the catalytic sites.

[0004] Using a common carrier protein to transport substrates and intermediates raises two potential operational challenges: 1) Hydrophobic ACP loads need to avoid contact with the polar cellular environment during transfer from one enzyme active site to another. In type II ACPs, this is achieved by isolating the bound intermediate into a hydrophobic slit during transfer. Upon arrival at the next enzyme, the ACP-acyl chain flips out of its isolated state and extends into the active site via a "spring knife" mechanism. 2) The common ACP surface, particularly its Ppant arms, is heterogeneous, but this reduces the number of potential interaction sites that need to be sampled during specific recognition. For example, in type II fatty acid biosynthesizers, positively charged active site slits interact with negatively charged ACP α-helices 2 and 3 (Chan, DI, and Vogel, HJ (2010), see above). However, how these two operational challenges are addressed in type I FASs remains unclear, primarily due to limited structural understanding of ACP transport processes in type I systems.

[0005] The compartmentalization of the ACP and the rapid transfer between various enzyme active sites may eliminate the need for a switchblade-like isolation mechanism in fungal FAS. Although NMR studies of isolated yeast FAS ACP domains suggest the possibility of such an isolation mechanism, the larger ACP in *Saccharomyces cerevisiae* is involved in a wider range of interactions. It utilizes both the canonical and structural ACP domains to bind to the AT and KS enzymatic domains (Singh K. et al., 2020, *Cell*, Vol. 180: 1130-1143.e20, 10.1016 / j.cell.2020.02.034), while the DH domain binds only to the canonical domain. Furthermore, the β subunit of *Saccharomyces cerevisiae* has been reported to exist in two conformational states—rotational and non-rotational—which are associated with different spatial locations of the ACP domain (Singh, K. et al., (2020), see above). The non-rotational conformation corresponds to the binding state of the ACP-KS domain, while the rotational conformation corresponds to the binding state of the ACP-AT domain and can be further stabilized by the newly discovered γ subunit. Fatty acid biosynthesis requires a sequential and ordered cyclic interaction between ACP and a total of six catalytic domains. Figure 1A Two large-scale conformational rearrangements alone may not be sufficient to support the interactions of all these ACPs in fatty acid biosynthesis.

[0006] In view of the above, the present invention aims to identify the structural domains and amino acids involved in FAS enzyme activity, including the catalytic domain, and also to identify the interacting domains. The polypeptides or proteins according to the invention contain at least one amino acid substitution present in one of the subunits of the ACP or FAS I complex, and the one or more substitutions are not located within the catalytic site or catalytic center of the corresponding subunit. In particular, the interactions of ACP-related sites (including interactions with active sites in FAS) enable the identification of suitable molecules, including mutant ACP molecules, thereby achieving the regulation of FAS activity. This, on the one hand, provides improved means and methods for the production of fatty acids in microorganisms, particularly for the production of fatty acids suitable for biofuels, fine chemicals, and other compounds; on the other hand, it also allows for the identification of compounds suitable for use as bactericides or actinomycete bactericides. Summary of the Invention

[0007] According to the present invention, the above-mentioned objective has been achieved by the polypeptides or proteins involved in fatty acid synthesis as described in the claims, which do not involve the catalytic sites of the enzymes in the FAS I complex. Specifically, in a first aspect, the present invention provides a polypeptide or protein comprising one or more polypeptide chains, wherein the polypeptide chain comprises:

[0008] i) One or more subunits containing the following amino acid sequence:

[0009] SEQ ID No. 1 (ER domain);

[0010] SEQ ID No. 2 (AT domain);

[0011] SEQ ID No. 3 (KS domain);

[0012] SEQ ID No. 4 (DH domain);

[0013] SEQ ID No. 5 (KR domain);

[0014] SEQ ID No. 6 (MPT domain); and / or

[0015] SEQ ID No. 7 (ACP domain);

[0016] ii) Having at least one amino acid substitution at positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 of the amino acid sequence corresponding to SEQ ID No. 1 in the ER domain; and / or having at least one amino acid substitution at positions 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 of the amino acid sequence corresponding to SEQ ID No. 2 in the AT domain; and / or having at least one amino acid substitution at positions 68-131 of the amino acid sequence corresponding to SEQ ID No. 3 in the KS domain; and / or having at least one amino acid substitution in the DH domain corresponding to SEQ ID No. The amino acid sequence 4 has an amino acid substitution at positions 189-310; and / or has an amino acid substitution at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 in the KR domain corresponding to the amino acid sequence of SEQ ID No. 5; and / or has at least one amino acid substitution at positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 in the MPT domain corresponding to the amino acid sequence of SEQ ID No. 6; and / or has a substitution in the ACP domain corresponding to SEQ ID No. 7 has at least one amino acid substitution at positions 5-11, 20-25, 34-105, and 135-147;

[0017] The amino acid sequence containing at least one substituted amino acid has at least 70%, or preferably at least 80%, 90%, or 95% sequence identity with the corresponding amino acid sequences of SEQ ID No. 1 and / or SEQ ID No. 2 and / or SEQ ID No. 3 and / or SEQ ID No. 4 and / or SEQ ID No. 5 and / or SEQ ID No. 6 and / or SEQ ID No. 7, thereby resulting in an alteration in fatty acid synthesis activity.

[0018] The positions listed in ii) above indicate the amino acids responsible for the interaction between the ACP and FASI subunits within the corresponding domains of SEQ ID Nos. 1 to 7. The amino acid substitutions in these FAS I proteins or polypeptides are not located within the catalytic center of the corresponding FAS I protein. Unless otherwise stated, the terms "catalytic site" and "catalytic center" are used interchangeably. In another aspect, the present invention relates to nucleic acid molecules encoding the proteins or polypeptides described herein, wherein the nucleic acid molecules preferably further comprise a vector nucleic acid sequence, more preferably an expression vector sequence and / or a promoter nucleic acid sequence and a terminator nucleic acid sequence, and / or other regulatory nucleic acid sequences and / or the nucleic acid molecules preferably comprise dsDNA, ssDNA, cDNA, LNA, PNA, CNA, RNA, or mRNA, or combinations thereof.

[0019] Furthermore, the present invention provides a host cell containing and preferably expressing the nucleic acid molecule of the present invention, wherein the host cell is preferably selected from bacterial cells, fungal cells and algal cells.

[0020] Furthermore, the present invention discloses a method for producing fatty acid cells, the method comprising the step of culturing host cells according to the present invention. Specifically, the method is a method for producing fatty acids with specific chain lengths, such as short-chain fatty acids (C6 to C12) and long-chain fatty acids (C16 to C18).

[0021] Furthermore, the present invention also provides applications of the proteins, nucleic acids, or host cells described herein, including biofuel preparation, fine chemical production, and flavor substance synthesis; the applications also relate to the screening and determination of bactericides and actinomycete bactericides.

[0022] Another aspect of the present invention relates to a computer simulation method for identifying molecules. Therefore, this computer simulation method can identify molecules capable of selectively altering a stage in fatty acid synthesis of FAS I. The method includes identifying and simulating molecules that interact with at least one amino acid as defined herein under physiological conditions. Specifically, the at least one amino acid is: at least one amino acid corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, or 500-505 of the amino acid sequence of SEQ ID No. 1 in the ER domain; and / or at least one amino acid corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, or 542-554 of the amino acid sequence of SEQ ID No. 2 in the AT domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 3 in the KS domain; and / or corresponding to SEQ ID No. 3 in the DH domain. 4. Amino acids at positions 189-310 of the amino acid sequence; and / or amino acids in the KR domain corresponding to positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 of the amino acid sequence in SEQ ID No. 5; and / or at least one amino acid in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 of the amino acid sequence in SEQ ID No. 6; and / or at least one amino acid in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 of the amino acid sequence in SEQ ID No. 7.

[0023] Furthermore, this invention provides a computer simulation method for identifying molecules capable of selectively altering the activity of enzyme domains contained in type I fatty acid synthase (FAS I). Molecules identified and modeled by this method exhibit an interaction with at least one of the following amino acids under physiological conditions, resulting in an alteration of at least one enzyme activity in FAS: at least one amino acid corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, or 500-505 of the amino acid sequence in the ER domain; and / or at least one amino acid corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, or 542-554 of the amino acid sequence in the AT domain; and / or corresponding to positions 3 in the KS domain. At least one amino acid at positions 68-131 of the amino acid sequence; and / or an amino acid at positions 189-310 of the amino acid sequence of SEQ ID No. 4 in the DH domain; and / or an amino acid at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, or 602-643 of the amino acid sequence of SEQ ID No. 5 in the KR domain; and / or at least one amino acid at positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, or 396-415 of the amino acid sequence of SEQ ID No. 6 in the MPT domain; and / or at least one amino acid at positions 5-11, 20-25, 34-105, or 135-147 of the amino acid sequence of SEQ ID No. 7 in the ACP domain.

[0024] Furthermore, the present invention provides a method for identifying compounds capable of altering the activity of at least one enzyme of FAS I, the method comprising: contacting a candidate compound with FAS I and determining whether the candidate compound satisfies the following conditions: (i) interacting with at least one of the following amino acids: at least one amino acid corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, 500-505 of the amino acid sequence of SEQ ID No. 1 in the ER domain; and / or at least one amino acid corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, 542-554 of the amino acid sequence of SEQ ID No. 2 in the AT domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 3 in the KS domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 3 in the DH domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 1 in the DH domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 1 in the DH domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 1 in the ER ... The amino acid at positions 189-310 of the amino acid sequence No. 4; and / or the amino acid at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, 602-643 of the amino acid sequence of SEQ ID No. 5 in the KR domain; and / or at least one amino acid at positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, 396-415 of the amino acid sequence of SEQ ID No. 6 in the MPT domain; and / or at least one amino acid at positions 5-11, 20-25, 34-105, 135-147 of the amino acid sequence of SEQ ID No. 7 in the ACP domain; and / or (ii) is a competitor of ACP when interacting with at least one of the following amino acids: corresponding to SEQ ID No. 4 in the ER domain. At least one amino acid corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 of the amino acid sequence of SEQ ID No. 1; and / or at least one amino acid corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 of the amino acid sequence of SEQ ID No. 2 in the AT domain; and / or at least one amino acid corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 3 in the KS domain; and / or an amino acid corresponding to positions 189-310 of the amino acid sequence of SEQ ID No. 4 in the DH domain; and / or a amino acid corresponding to position SEQ ID No. 4 in the KR domain.The amino acids at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 of the amino acid sequence of SEQ ID No. 5; and / or at least one amino acid in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 of the amino acid sequence of SEQ ID No. 6; and / or at least one amino acid in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 of the amino acid sequence of SEQ ID No. 7.

[0025] In another aspect, the present invention provides a protein having the mutation in SEQ ID No. 7, wherein the position of the mutation corresponds to at least one amino acid at positions 5-11, 20-25, 34-105, and 135-147 of the amino acid sequence of SEQ ID No. 7.

[0026] In one aspect, the present invention provides a fungicide or actinomycete biocide whose mechanism of action is FAS I acting on fungi and actinomycetes.

[0027] Detailed Description of the Invention

[0028] In a first aspect, the present invention relates to a protein (peptide) involved in fatty acid synthesis, said protein comprising one or more polypeptide chains, wherein said polypeptide chain comprises:

[0029] i) One or more subunits containing the following amino acid sequence:

[0030] SEQ ID No. 1 (ER domain);

[0031] SEQ ID No. 2 (AT domain);

[0032] SEQ ID No. 3 (KS domain);

[0033] SEQ ID No. 4 (DH domain);

[0034] SEQ ID No. 5 (KR domain);

[0035] SEQ ID No. 6 (MPT domain); and / or

[0036] SEQ ID No. 7 (ACP domain);

[0037] ii) The ER domain contains at least one amino acid substitution at positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 of the amino acid sequence corresponding to SEQ ID No. 1; and / or the AT domain contains at least one amino acid substitution at positions 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 of the amino acid sequence corresponding to SEQ ID No. 2; and / or the KS domain contains at least one amino acid substitution at positions 68-131 of the amino acid sequence corresponding to SEQ ID No. 3; and / or the DH domain contains at positions 189-310 of the amino acid sequence corresponding to SEQ ID No. 4; and / or the KR domain contains at least one amino acid substitution corresponding to SEQ ID No. 1. The amino acid sequence of 5 has amino acid substitutions at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643; and / or has at least one amino acid substitution at positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 in the ACP domain.

[0038] Wherein, the amino acid sequence containing at least one amino acid substitution has at least 70%, or preferably at least 80%, 90%, or 95% sequence identity with the corresponding amino acid sequences of SEQ ID No. 1, and / or SEQ ID No. 2, and / or SEQ ID No. 3, and / or SEQ ID No. 4, and / or SEQ ID No. 5, and / or SEQ ID No. 6, and / or SEQ ID No. 7; thereby causing an alteration in the activity of fatty acid synthesis.

[0039] The positions listed in ii) above identify the amino acids responsible for the interaction between ACP and the FAS I subunits in each of the domains of SEQ ID Nos. 1 to 7. The amino acid substitutions in the FAS I protein or polypeptide are not located within the catalytic center of the corresponding FASI protein.

[0040] Unless otherwise stated herein, the term “FAS” refers to the FAS I complex of yeast or actinomycetes.

[0041] By obtaining structural snapshots of FAS I and ACP, the inventors identified relevant intermediate steps in fatty acid (FA) biosynthesis and revealed the enzymes constituting the FAS I complex and related amino acid residues present in the ACP molecule. This allows for the atomic-level resolution of the fatty acid biosynthesis mechanism in fungal FAS, which can be applied to biotechnology development. Specifically, the inventors identified amino acids involved in the interactions between ACP and the various subunits of FAS I, as well as among different subunits of FAS I. These amino acids play crucial roles in both the individual enzyme activity and the overall enzyme activity of the FAS I complex.

[0042] The amino acid regions identified above have been confirmed as relevant regions / sites for the interaction between ACP and FAS I. Changing at least one amino acid at these relevant sites will alter the interaction between ACP and the corresponding enzyme. It is worth noting in this regard that the inventors have also identified regions in adjacent enzymes that promote ACP binding and interact with the active site of another enzyme. Changing at least one amino acid present in the enzyme or ACP will have a direct or indirect effect on enzyme activity, ultimately leading to changes in the yield of the FAS I complex.

[0043] The sites listed in section ii) above indicate the amino acids of each domain in SEQ ID Nos. 1 to 7, which are responsible for the interaction between ACP and the subunits of FAS I. The amino acid substitutions in these FAS I proteins or polypeptides do not occur within the catalytic center of the corresponding FAS I protein. For example, as... Figure 3 As shown and described below, when ACP is present at the MPT active site, the amino acids that interact with it in the ER and AT domains are also involved. If this interaction is altered (e.g., interrupted), the activity of the MPT active site will also change, although the MPT active site itself and the associated ACP sites present in the MPT active site remain unchanged.

[0044] Depending on the enzyme affected, the result will be alterations in the produced fatty acids, such as changes in the chain length of the fatty acid molecules. In some cases, the identified amino acids and regions can be used to identify compounds or molecules that can inhibit the activity of at least one enzyme, thereby exhibiting toxicity to fungi or actinomycetes.

[0045] In other words, by identifying the relevant sites on FAS I and ACP, suitable inhibitor molecules can be developed to target them.

[0046] As used herein, the term "involved in fatty acid synthesis" refers to proteins or polypeptides involved in the synthesis of fatty acid molecules within cells. Specifically, the proteins involved in fatty acid synthesis are members of the FAS I complex in yeast or actinomycetes, or ACP proteins.

[0047] In other words, on the one hand, the proteins involved in fatty acid synthesis can be ACP proteins or polypeptides.

[0048] The term "alteration of fatty acid synthesis activity" refers to changes in the synthesized products (e.g., changes in fatty acid chain length) or changes in the rate of enzymatic reactions of at least one enzyme in the FAS I complex. Furthermore, this alteration may be due to changes in the assembly and / or structure of the FAS I complex. The alteration can be an inhibition, enhancement, or reduction of enzyme activity or enzymatic reactions. This alteration may be due to changes in the activity of one or more enzymes in FAS I. Ultimately, this alteration may lead to an increase or decrease in the selective yield of specific fatty acids and fatty acid derivatives.

[0049] For example, this activity can be affected by increasing or decreasing the affinity and affinity between the interacting enzyme structural regions in the ACP-FASI complex.

[0050] Furthermore, in this document, the term "interaction" refers to a change in affinity or cohesion between different regions of FAS I, or between at least one region of FAS I and ACP, occurring at at least one amino acid residue or region of amino acid residues in the FAS I complex or ACP as defined herein. Specifically, the interaction refers to an enhancement of affinity or cohesion between the involved binding partners. For example, an enhancement of affinity or cohesion leads to a decrease in the rate of the enzymatic reaction of the enzyme, which is a member of the FAS I complex. Interactions can occur in ways that include at least one of the following attractive forces: covalent interaction (i.e., formation of a chemical bond), formation of a salt bridge via electrostatic interaction, hydrogen bonding involving hydrogen bond donors and acceptors, or hydrophobic association mediated by van der Waals forces.

[0051] As used herein, the term “physiological conditions” refers to conditions present within a cell (e.g., a host cell, or a fungal or actinomycete cell) that enable an interaction between a molecule or compound and an amino acid residue as defined herein.

[0052] As used herein, the term "percentage (%) identity" refers to the sequence similarity between two amino acid sequences. Identity can be determined by comparing positions or sequences; alignment is for comparison purposes. When equivalent positions in the compared sequences are occupied by the same amino acid, the molecules at that position are considered identical.

[0053] As used herein, the term "functional equivalent" refers to an amino acid sequence that is not 100% identical to the amino acid sequence described herein, but contains amino acid additions and / or insertions and / or deletions and / or substitutions, which do not adjust or alter the activity or function of a protein or peptide as defined herein. For example, functional equivalents include amino acid sequences with conserved amino acid substitutions or minor deletions and / or insertions, provided that these modifications do not materially affect their in vitro and / or in vivo fatty acid synthase activity.

[0054] Generally, those skilled in the art will recognize that certain amino acid substitutions in the amino acid sequence of a protein or polypeptide do not affect the protein's (secondary or tertiary) structure, function, and / or activity. Amino acid sequences having such "natural" amino acid substitutions are also within the scope of this invention compared to the amino acid sequences disclosed herein.

[0055] Furthermore, the sequences can be optimized using protein design software, including Rosetta, RosettaFold, AlphaFold2, Gromacs, Swiss-Modeller, Modeller, Quark, Prime, Phyre, Homelette, AMBER, Abalone, ADF, Avogadro, CHARMM, BOSS, CP2k, Desmond, DiscoveryStudio, GROMOS, LAMMPS, MacroModel, MAPS, Materials Studio, MBN Explorer, MDynaMix, MOE, OpenMM, Orac, NAMD + VMD, NWChem, Protein Local Optimization Program, Q, Quantum ATK, SAMSON, Scigress, TINKER, TeraChem, and Yasara, to adapt the specific protein or peptide to the host cell. Additionally, suitable substitutions or at least one amino acid as defined herein can also be determined using the corresponding protein design software.

[0056] As used herein, the term "competitor" refers to a molecule that can prevent a substrate molecule from binding to the enzyme's active site or interact with the substrate molecule to compete for the same binding site; that is, it competes with the substrate for binding to the same binding site. Such competitors (e.g., modified ACP molecules with modified or mutated amino acid sequences) compete with native ACP for the same binding site, thereby interacting with and / or interrupting the enzymatic reaction. The protein or polypeptide types described in this invention, particularly type I FAS variants, possess fatty acid synthase I activity in vitro and / or in vivo.

[0057] In one embodiment, the at least one amino acid substitution is present in the amino acid sequence shown in SEQ ID No. 7, which represents the amino acid sequence of ACP.

[0058] The inventors have recognized that the ER domain contains at least one amino acid corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 of the amino acid sequence in SEQ ID No. 1; and / or the AT domain contains at least one amino acid corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 of the amino acid sequence in SEQ ID No. 2; and / or the KS domain contains at least one amino acid corresponding to positions 68-131 of the amino acid sequence in SEQ ID No. 3; and / or the DH domain contains at least one amino acid corresponding to positions 189-310 of the amino acid sequence in SEQ ID No. 4; and / or the KR domain contains at least one amino acid corresponding to positions 68-131 of the amino acid sequence in SEQ ID No. 1. The amino acids at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 of the amino acid sequence No. 5; and / or at least one amino acid in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 of SEQ ID No. 6; and / or at least one amino acid in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 of SEQ ID No. 7 participate in the enzymatic reaction and / or the binding of ACP to the active site of the enzyme in different forms. In other words, this correlation is not only related to the catalytic site of the relevant enzyme, but also to the interaction and binding of adjacent amino acid regions with ACP, thereby enabling the enzymatic reaction to proceed accordingly. For example, as shown herein and discussed below, structural elements distant from the active site often also participate in ACP binding. Furthermore, yeast-specific ACP domains mediate interactions with adjacent regions of other enzymes (as discussed below). The canonical ACP domain is universally essential for interaction with all enzymatic domains of FAS. Ser180, corresponding to Ser44 in SEQ ID No. 7, is particularly important.

[0059] On the one hand, the affinity and cohesion between ACP and structural elements of the FAS I complex, based on various types of interactions, play a crucial role in ACP binding and the binding force between ACP and structural elements. This binding force is particularly critical for the rate of enzymatic reactions and the process of ACP switching from one active site to another. Therefore, the movement of ACP within the FAS I complex can be altered and, in some cases, interrupted. When the enzymatic cascade reaction is substantially blocked, the FAS I complex is correspondingly inhibited.

[0060] In one embodiment of the present invention, the relevant amino acid is at least one of the following: at least one amino acid in the ER domain corresponding to positions 40-45, 65-70, 92-109, 126-140, 160-170, 175-188, 314-324, 290-301, 467-477 in the amino acid sequence of SEQ ID No. 1; and / or at least one amino acid in the AT domain corresponding to positions 163, 274, 364, 401, 403, 405, 406, 428 in the amino acid sequence of SEQ ID No. 2; and / or at least one amino acid in the KS domain corresponding to positions 95-102, 109-130 in the amino acid sequence of SEQ ID No. 3; and / or the amino acid in the DH domain corresponding to position 198 in the amino acid sequence of SEQ ID No. 4; and / or SEQ ID No. The amino acids in the KR domain at positions 39-62, 178-184, 354-364, 372-392, 409-413, 545-557, and 603-642 in the amino acid sequence of SEQ ID No. 5; and / or at least one amino acid in the MPT domain at positions 20-28, 48-64, 81-96, and 102-110 in SEQ ID No. 6; and / or at least one amino acid in the ACP domain at positions 40-49, 51-70, and 72-105 in SEQ ID No. 7.

[0061] That is, the protein or peptide according to the present invention comprises: at least one amino acid substitution in the ER domain corresponding to positions 40-45, 65-70, 92-109, 126-140, 160-170, 175-188, 314-324, 290-301, 467-477 in the amino acid sequence of SEQ ID No. 1; and / or at least one amino acid substitution in the AT domain corresponding to positions 163, 274, 364, 401, 403, 405, 406, 428 in the amino acid sequence of SEQ ID No. 2; and / or at least one amino acid substitution in the KS domain corresponding to positions 95-102, 109-130 in the amino acid sequence of SEQ ID No. 3; and / or an amino acid substitution in the DH domain corresponding to position 198 in the amino acid sequence of SEQ ID No. 4; and / or in SEQ ID No. Amino acid substitutions in the KR domain at positions 39-62, 178-184, 354-364, 372-392, 409-413, 545-557, and 603-642 in the amino acid sequence of SEQ ID No. 5; and / or at least one amino acid substitution in the MPT domain at positions 20-28, 48-64, 81-96, and 102-110 in SEQ ID No. 6; and / or at least one amino acid substitution in the ACP domain at positions 40-49, 51-70, and 72-105 in SEQ ID No. 7.

[0062] It should be noted that, unless otherwise stated, the terms "protein" and "peptide" are used interchangeably in this article.

[0063] In one embodiment, the relevant amino acids identified according to the present invention are shown in Table I:

[0064]

[0065] As described above, the present invention provides a nucleic acid molecule encoding a protein or polypeptide according to the present invention. The present invention also provides a nucleic acid molecule comprising a corresponding nucleotide exchange that results in amino acid substitutions according to the present invention.

[0066] In one embodiment, the nucleic acid molecule of the present invention further comprises:

[0067] i) A vector nucleic acid sequence, preferably an expression vector sequence; and / or

[0068] ii) Promoter and terminator nucleic acid sequences; and / or

[0069] iii) Other regulatory nucleic acid sequences.

[0070] In another embodiment, the nucleic acid molecule of the present invention comprises double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), complementary DNA (cDNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), circular nucleic acid (CNA), RNA or mRNA, or a combination thereof.

[0071] In one embodiment, the nucleic acid molecule of the present invention preferably comprises a nucleic acid sequence identical to that of naturally occurring nucleic acids, or a nucleic acid sequence that has been non-currently optimized for use in host cells, except for the addition of the amino acid substitutions described in the present invention. The nucleic acid molecule used in the present invention is preferably a nucleic acid expression construct. A nucleic acid expression construct according to the present invention is an expression cassette comprising a nucleic acid molecule according to the present invention, or an expression vector comprising a nucleic acid molecule or expression cassette according to the present invention. Typically, the nucleic acid expression construct comprises regulatory sequences, such as promoter and terminator sequences, which are operatively linked to a nucleic acid sequence encoding a polypeptide of the present invention.

[0072] In another aspect, the present invention provides a host cell containing a nucleic acid molecule according to the present invention, and preferably expressing the nucleic acid molecule.

[0073] The host cell can be selected from bacterial cells, fungal cells, actinomycete cells, algal cells, or host cells of eubacteria.

[0074] For example, when using true bacteria, Escherichia coli or Bacillus subtilis can be used.

[0075] In one implementation, the host cell is a non-oil-producing yeast. Suitable non-oil-producing yeasts include members of genera selected from the group consisting of: *Saccharomyces*, *Schizosaccharomyces*, such as *Saccharomyces cerevisiae*, and *Pichia pastoris*, *Kluyveromyces lactis*, and *Ustilago maydis*. For oil-producing yeasts representing suitable host cells, genera can be selected from *Candida*, *Rhodotorula*, *Yarrowia*, *Cryptococcus*, *Rhodotorula*, *Lipomyces*, and *Trichosporium*. A particularly suitable example of a host cell is *Y. lipolytica*. Additionally, the host cell can also be an actinomycete, such as mycobacteria.

[0076] Suitable algal cells can be members selected from the following genera: Chlamydomonas, Chlorella, Haematococcus, Dunaliella, Nanochloroypsis, Thalassiorao, Brown finger algae, Purple phallotoxum, or Scenedesmus.

[0077] For example, in the use of host cells to produce biofuels according to the present invention, Yersinia species, such as Yersinia lipolytica, can be used.

[0078] In another aspect, the present invention relates to a method for producing fatty acids. This method includes the steps of culturing host cells according to the invention and / or expressing proteins involved in fatty acid synthesis according to the invention, thereby achieving fatty acid synthesis accordingly. For example, the yield of short-chain fatty acid derivatives of C6 to C12 fatty acids (such as C6, C8, C10, and C12 fatty acids) can be increased. The fatty acids can exist in fatty acid form or in the form of esters or thioesters containing said fatty acids. Furthermore, the overall yield of long-chain fatty acids (i.e., fatty acids of C16 and / or C18 length) can also be increased. The product distribution of fatty acids produced by the FAS I complex is also affected according to changes in the activity of each subunit enzyme in the FAS I complex.

[0079] This method includes a method for producing fatty acids, which includes the step of expressing a nucleic acid molecule according to the invention, preferably expressed in a host cell according to the invention, or expressing a protein or polypeptide according to the invention.

[0080] In one embodiment of the method of the present invention, the method is used to produce short-chain (C6 to C12) fatty acids, wherein the protein or polypeptide preferably contains at least one of the following substitutions: Met56Val, Met56Ile, Val26Leu, Val26Ile, Lys62Arg, Gln58Glu, Lys64Arg, Lys83Arg, Asn326Asp, Asn326Gln, Asn326Glu, Asn328Asp, Asn328Gln, Asn328Glu, Gln333His, Gln333Glu, Arg398Glu, Arg398His, His413Asp, His413Asn, His413Glu, His413Gln, His413Tyr, His413Arg; or SEQ ID No. 6 Lys37Arg, Lys37Glu, Lys37Gln, Gly42His, Gly42Tyr, Gly42Phe, Asn48Asp, Asn48Gln, Asn48Glu, Asn48His, As in No.7 n48Arg, Asn48Tyr, Thr45Met, Thr45His, Thr45Asn, Thr45Asp, Thr45Gln, Thr45Glu, Leu51Arg, Leu51Tyr, Lys64Glu, Ly The mutations described herein may be s64Arg, Glu67Asn, Glu67Asp, Glu67Gln, Glu67His, Glu67Tyr, Ser98Tyr, Ser98His, Ser98Glu, Ser98Gln, Ser98Arg, Ser98Lys, Ser99Tyr, Ser99His, Ser99Glu, Ser99Gln, Ser99Arg, or Ser99Lys, or express nucleic acid molecules encoding the aforementioned proteins or polypeptides, preferably expressed in the host cells described in this invention. The specific mutations described above affect the MPT / ACP interaction, ultimately leading to increased yields of short-chain fatty acid derivatives of C6 to C12 fatty acids.

[0081] On the other hand, the present invention relates to the use of proteins or polypeptides according to the invention, nucleic acids according to the invention, and / or host cells according to the invention for various purposes. For example, said uses include the production of biofuels. As mentioned above, biofuels containing short-chain fatty acids are ideal. Furthermore, the use of proteins or polypeptides according to the invention, nucleic acid molecules according to the invention, and / or host cells according to the invention for the production of fine chemicals is also envisioned.

[0082] Furthermore, the applications also include the determination of fungicides and / or biocides. Specifically, interfering with, and particularly inhibiting, the activity of the FAS I complex can provide corresponding biocides (i.e., bactericides) and fungicides. That is, in addition to suitable fungal inhibitors, it is envisioned that biocides, including mycobactericidal agents, can be provided. In this regard, the applications also include the determination of reagents capable of reducing the production of mycolic acid in actinomycetes. That is, as described above regarding mycobactericidal agents, reducing the production of mycolic acid in actinomycetes can ultimately weaken the action of mycobacteria or actinomycetes. While not bound by theory, it can be considered that reducing the production of mycolic acid in actinomycetes affected by the FAS I complex weakens or depletes cell wall components, thereby enhancing the killing effect on actinomycetes (e.g., mycobacteria).

[0083] In another aspect, a computer simulation (in silico) method is provided. Specifically, in another aspect, a computer simulation method is provided for identifying molecules capable of selectively altering the fatty acid synthesis steps of FAS I, the method comprising: identifying and simulating molecules that interact under physiological conditions with the following sites: at least one amino acid in the ER domain corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, 500-505 of the amino acid sequence of SEQ ID No. 1; and / or at least one amino acid in the AT domain corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, 542-554 of the amino acid sequence of SEQ ID No. 2; and / or at least one amino acid in the KS domain corresponding to positions 68-131 of the amino acid sequence of SEQ ID No. 3; and / or at least one amino acid in the DH ... The amino acid at positions 189-310 of the amino acid sequence No. 4; and / or the amino acid at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, 602-643 of the amino acid sequence in the KR domain corresponding to positions 36-66, 176-170, 242-276, 293-306, 325-335, 353-358, 396-415 of the amino acid sequence in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, 396-415 of the amino acid sequence in the ACP domain corresponding to positions 5-11, 20-25, 34-105, 135-147 of the amino acid sequence in the ACP domain.

[0084] In another aspect of this disclosure, a computer simulation method is provided for identifying molecules capable of selectively altering the activity of enzymatic domains present in FAS, wherein the identified and simulated molecules, under physiological conditions, correspond to at least one amino acid in the ER domain at positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, 500-505 of the amino acid sequence of SEQ ID No. 1; and / or at least one amino acid in the AT domain at positions 5-141, 162-166, 273-277, 362-368, 291-430, 542-554 of the amino acid sequence of SEQ ID No. 2; and / or at least one amino acid in the KS domain at positions 68-131 of the amino acid sequence of SEQ ID No. 3; and / or at least one amino acid in the DH domain corresponding to the position of SEQ ID No. 1. The amino acids at positions 189-310 of the amino acid sequence No. 4; and / or the amino acids in the KR domain corresponding to positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, 602-643 of the amino acid sequence of SEQ ID No. 5; and / or at least one amino acid in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, 396-415 of SEQ ID No. 6; and / or at least one amino acid in the ACP domain corresponding to positions 5-11, 20-25, 34-105, 135-147 of SEQ ID No. 7 exhibit interaction; thereby causing at least a change in the enzymatic activity of FAS.

[0085] In one embodiment, the method involves: at least one amino acid in the ER domain corresponding to positions 40-45, 65-70, 92-109, 126-140, 160-170, 175-188, 314-324, 290-301, 467-477 of the amino acid sequence in SEQ ID No. 1; and / or at least one amino acid substitution in the AT domain corresponding to positions 163, 274, 364, 401, 403, 405, 406, 428 of the amino acid sequence in SEQ ID No. 2; and / or at least one amino acid substitution in the KS domain corresponding to positions 95-102, 109-130 of the amino acid sequence in SEQ ID No. 3; and / or at least one amino acid substitution in the DH domain corresponding to position 198 of the amino acid sequence in SEQ ID No. 4; and / or at least one amino acid substitution in the KR domain corresponding to position SEQ ID No. 1; and / or at least one amino acid substitution in the AT domain corresponding to positions 163, 274, 364, 401, 403, 405, 406, 428 of the amino acid sequence in SEQ ID No. 2; and / or at least one amino acid substitution in the KS domain corresponding to positions 95-102, 109-130 of the amino acid sequence in SEQ ID No. 3; and / or at least one amino acid substitution in the KR domain corresponding to position 198 of the amino acid sequence in SEQ ID No. 4; and / or at least one amino acid substitution in the KR domain corresponding to position 198 of the amino acid sequence in SEQ ID No. 4; and / or at least one amino acid substitution in the AT domain corresponding to positions 163, 274, 364, 401, The amino acid substitutions at positions 39-62, 178-184, 354-364, 372-392, 409-413, 545-557, and 603-642 of the amino acid sequence No. 5; and / or at least one amino acid substitution in the MPT domain corresponding to positions 20-28, 48-64, 81-96, and 102-110 of SEQ ID No. 6; and / or at least one amino acid substitution in the ACP domain corresponding to positions 40-49, 51-70, and 72-105 of SEQ ID No. 7.

[0086] Preferably, the amino acid is one of the amino acids shown in Table 1 above.

[0087] In other words, the computer simulation method according to the first and other embodiments involves identifying molecules capable of selectively altering the synthesis process of the FAS I complex. This method includes applying suitable software based on data obtained from the identified FAS I complex. The corresponding datasets are available under their respective EMDB IDs or PDB IDs, as shown in Table 2.

[0088] Table 2. Cryo-electron microscopy data collection and optimization, in conjunction with Figure 1. Figure 2 and Figure 3 Related

[0089]

[0090]

[0091] The corresponding molecules that interact under physiological conditions are identified and simulated using appropriate software tools for design purposes. As mentioned above, interaction with the identified amino acid refers to interaction through attraction, including covalent or non-covalent bonding, such as chemical bonding, salt bridge formation through electrostatic interactions, hydrogen bonding through hydrogen bond donors and acceptors, or hydrophobic association through small or large van der Waals forces.

[0092] These molecules, identified and simulated, are capable of interacting with at least one identified amino acid. These molecules should be able to alter the activity of the FAS I complex, thereby affecting at least the rate of the enzymatic reaction of one of the enzymes within the FAS I complex. For example, these molecules could serve as candidate inhibitors of enzyme activity, or they could increase the yield or selectivity of fatty acid production from the FAS I complex.

[0093] Those skilled in the art are familiar with suitable tools that can achieve this purpose. These tools include software such as Autodock, AutoDock Vina, Dock, FlexAID, LeDock, Glide, Molecular Manipulation Environment (MOE), rDock, SEED, SwissDock, CSD Gold, Chimera, DockThor, etc.

[0094] In another aspect of the computer simulation method, a computer simulation approach is provided for identifying molecules capable of selectively altering the activity of enzyme domains present in FAS, wherein the identified and simulated molecules exhibit interactions with at least one defined amino acid under physiological conditions. As described above, suitable existing tools can be used to perform this operation. The identified and simulated molecules may interact with amino acids present at the corresponding enzyme catalytic site, or, as a substitute or complement, with adjacent regions of the same or different enzymes in the FAS I complex. These molecules may allow for the selective increase of the yield of specific fatty acid molecules, such as short-chain or long-chain fatty acid molecules as defined herein.

[0095] On the other hand, a method is provided for identifying compounds capable of altering the activity of at least one enzyme in the FAS I complex. The method comprises: contacting a candidate compound with a protein or peptide according to the invention or with the entire FAS I complex, and determining whether the candidate compound is capable of binding with at least one amino acid in the ER domain corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, 500-505 of the amino acid sequence in SEQ ID No. 1; and / or at least one amino acid in the AT domain corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, 542-554 of the amino acid sequence in SEQ ID No. 2; and / or at least one amino acid in the KS domain corresponding to positions 68-131 of the amino acid sequence in SEQ ID No. 3; and / or at least one amino acid in the DH domain corresponding to positions 189-310 of the amino acid sequence in SEQ ID No. 4; and / or at least one amino acid in the KR domain corresponding to positions 68-131 of the amino acid sequence in SEQ ID No. 3. The amino acids at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 of the amino acid sequence No. 5; and / or at least one amino acid in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 of SEQ ID No. 6; and / or at least one amino acid in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 of SEQ ID No. 7 interact with each other.In addition, or alternatively, determine whether the candidate compound is an ACP competitor that interacts with at least one of the following amino acids: at least one amino acid in the ER domain corresponding to positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, 500-505 of the amino acid sequence in SEQ ID No. 1; and / or at least one amino acid in the AT domain corresponding to positions 5-141, 162-166, 273-277, 362-368, 291-430, 542-554 of the amino acid sequence in SEQ ID No. 2; and / or at least one amino acid in the KS domain corresponding to positions 68-131 of the amino acid sequence in SEQ ID No. 3; and / or at least one amino acid in the DH domain corresponding to positions 189-310 of the amino acid sequence in SEQ ID No. 4; and / or at least one amino acid in the KR domain corresponding to positions 68-131 ... The amino acids at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 of the amino acid sequence No. 5; and / or at least one amino acid in the MPT domain corresponding to positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 of SEQ ID No. 6; and / or at least one amino acid in the ACP domain corresponding to positions 5-11, 20-25, 34-105, and 135-147 of SEQ ID No. 7.

[0096] This method may include determining changes in activity using LC / MS methods known to those skilled in the art and described in the examples. This method is particularly suitable for high-throughput screening of compound libraries. Candidate compounds may serve as a starting point for development or as a starting compound for a suitable active agent. The active agent may be a fungicide or an actinomycete. Typically, the step of contacting the candidate compound with the FAS I complex is performed in cell culture or computer simulation. Suitable cells for this method include host cells expressing the FAS I complex as described herein, wherein the protein or polypeptide according to the invention may or may not be present. That is, the FAS I complex present in the host cells may be mutant or natural.

[0097] In another aspect, the present invention relates to a protein having a mutation of at least one amino acid at positions corresponding to positions 5-11, 20-25, 34-105, and 135-147 of SEQ ID No. 7, or not containing the amino acid sequence of positions 1 to 79 of SEQ ID No. 7.

[0098] That is, in another aspect, the present invention relates to modified ACP polypeptides or proteins wherein at least one amino acid at the position described herein is mutated. The mutation can be any type of mutation described herein. In a preferred embodiment, the mutation is a substitution of at least one amino acid in the ACP domain at positions 40-49, 51-70, and 72-105 of SEQ ID No. 7, or occurs in the ACP domain consisting of amino acids 80-169 of SEQ ID No. 7 or a functionalized fragment thereof. The corresponding proteins having the above-described mutations are particularly suitable as competitors for ASP binding in the FAS I complex. In particular, this application proposes that ACP polypeptides or proteins comprising at least one of the above-described substitutions or at least one amino acid substitution of SEQ ID No. 7 shown in Table 1 selectively alter the activity of the FAS I complex by correspondingly changing the enzymatic reaction rate.

[0099] In another aspect of the invention, proteins having the mutation shown in SEQ ID No. 7 are particularly suitable for use as fungicides or actinomycete biocides.

[0100] For example, when used as a fungicide or actinomycete biocide, the protein may be provided in a form suitable for pharmaceutical or agricultural compositions. Those skilled in the art are fully aware of how to provide suitable compositions accordingly. Attached Figure Description

[0101] Figure 1. Structure of Saccharomyces cerevisiae fatty acid synthase (FAS) at a resolution of 1.9 Å.

[0102] (A) Illustration of the yeast fatty acid biosynthesis cycle; ACP: acyl carrier protein, AT: acetyltransferase, MPT: malonyl-palmitoyltransferase, KS: ketone synthase, KR: ketone reductase, DH: dehydratase, ER: enoyl reductase, FMN: flavin mononucleotide.

[0103] (B) Visualization of chemical details in the 1.9 Å resolution structure. One of the six asymmetric units of FAS (top left) is extracted and displayed in the center. The individual enzyme domains are displayed in the same manner as in (A). The location of the magnified region (iv) in the asymmetric unit is marked. (i) FMN bound to the ER active site. Electron density shows a 22° kink in the isochloropyrazine ring system, consistent with its reducing role in fatty acid biosynthesis. (ii) Recognition of FMN by amino acids and their associated water network at the ER active site. (iii) NADP+ bound in the ER active site cleft, with its nicotinamide group stacked between the FMN isochloropyrazine and His740 (corresponding to amino acid position 184 in SEQ ID No. 1); meanwhile, the solvent network facilitates the recognition of the ADP moiety. (iv) Recognition of NADP+ by amino acids and solvent in the cavity of the KR active site. (v) Malonyl-CoA in a post-hydrolyzed state was found in the MPT active site. Coenzyme A interacts with multiple residues at the MPT active site; simultaneously, malonyl is covalently linked to Ser1808 (corresponding to amino acid 244 in SEQ ID No. 6) and interacts with Arg1834 (corresponding to amino acid 270 in SEQ ID No. 6).

[0104] Figure 2 The ACP domain is shown at a representative position in fatty acid synthesis intermediates.

[0105] (A) (i) In biochemically synchronized FAS, only three unique ACP densities were observed within each dome structure. (ii) In unsynchronized FAS, two scenarios can be envisioned: the ACP domains remain unresolved (right panel), or more than three densities are observed (left panel). Both scenarios imply that the binding of the three ACP domains to different enzyme domains within the FAS dome structure is asynchronous.

[0106] (B) Distribution of the ACP domain in non-circulating FAS (FASx, left panel), FAS with acetyl-CoA, malonyl-CoA and NADP+ (FASamn, middle panel), and FAS with acetyl-CoA and malonyl-CoA (FASam, right panel).

[0107] (C) Image processing scheme for classifying different binding states of ACP.

[0108] (D) Structural diagram of the FAS asymmetric unit. As shown in the figure, ACP is visualized at the position representing the five intermediates in fatty acid (FA) biosynthesis.

[0109] Figure 3 Interaction between ACP and the FAS enzyme domain.

[0110] Interactions of ACP with MPT (i, ii), ER (iii, iv), and KR domains (v, vi). The α2 and α3 helices of ACP contact residues outside the cleft of the MPT active site (i), while the α4 helix and the loop between the α4 and α5 helices contact structural fragments of the ER domain (positions 874-880, corresponding to amino acids 318-324 of SEQ ID No. 1) and the AT domain (positions 402-406, corresponding to amino acids 402-406 of SEQ ID No. 2) (ii). At the ER domain, the α3 helix of ACP contacts the ER loop (positions 653-658, corresponding to amino acids 97-102 of SEQ ID No. 1), while the α4 helix is ​​adjacent to a structural fragment of the KS domain (positions 1066-1071, corresponding to amino acids 96-101 of SEQ ID No. 3) (iii). Furthermore, the α2 helix of ACP contacts the adjacent AT fragment (positions 402-407, corresponding to amino acids 402-407 of SEQ ID No. 2), while the ACP residues (positions 198-201, corresponding to amino acids 62-65 of SEQ ID No. 7) are close to the ER ring (positions 847-851, corresponding to amino acids 291-295 of SEQ ID No. 1) (iv). At the KR domain, both canonical ACP and structural ACP participate in binding. The α2 and α3 helices of ACP contact residues near the KR active site (v), while the canonical domain, based on the DM1 helix, contacts residues 365-375, corresponding to amino acids 38-48 of SEQ ID No. 5 (vi). The ACP ring between the α4 / 5 helices also contacts the adjacent KS residues (positions 1092-1095, corresponding to amino acids 122-125 of SEQ ID No. 3) (vi).

[0111] Figure 4. Structural rearrangement when substrate and ACP are combined.

[0112] (A) Comparison of overlap between empty MPT domain, malonyl-CoA-bound MPT domain, and ACP-bound MPT domain.

[0113] (B) Comparison of MPT active sites in the presence of malonyl-CoA (left figure) and in the ACP-bound state (right figure).

[0114] (C) Comparison of the overlap of KR active sites in the presence and absence of NADP+.

[0115] (D) The state of the KR active site when there is no NADP+ (left figure), when NADP+ is bound (middle figure), and when NADP+ and ACP are bound simultaneously (right figure).

[0116] (E) Comparison of the overlap between the Ppant arm and NADP+ at ER shows that the two entities repel each other due to steric hindrance.

[0117] (F) Comparison of the overlap of ER active sites when binding NADP+ and ACP. When ACP binds, the loop at positions 847-852 corresponding to amino acids 291-296 of SEQ ID No. 1 shifts inward, resulting in a narrowing of the ER active site cleft.

[0118] Figure 5. Comparison of ACP binding and domains in the FAS systems of bacterial type II and Saccharomyces cerevisiae type I, and... Figure 3 and Figure 4 Related.

[0119] The structures of ACP-bound malonyl-CoA-acyltransferase (MCAT) (Escherichia coli, PDB ID: 6U0J) – which is homologous to the AT and MPT domains of Saccharomyces cerevisiae type I FAS – as well as ACP-bound Escherichia coli dehydratase (FabA, PDB ID: 4KEH6) and ketone synthase (FabB, PDB ID: 6OKC) were compared with the corresponding domains in fungi.

[0120] (A) Acetyltransferase domain.

[0121] (i, left figure) Comparison of overlap between E. coli MCAT enzyme and scFAS AT domain. In addition to the conserved folding of the catalytic domain, the scFAS AT domain also contains structural fragments. (i, right figure) Comparison of overlap between the ACP domain (AcpP) bound to the E. coli MCAT enzyme and the ACP (ACP) bound to the scFAS AT domain.

[0122] (ii and iii) Comparison of ACP (ii) bound to *E. coli* MCAT enzyme and ACP (iii) bound to the scFAS AT domain. Although the Ppant-serine residue (Ser36 in *E. coli* AcpS and Ser180 corresponding to amino acid 44 in scFAS ACP) is located at the entrance of the catalytic crack in both ACP-binding structures, the interaction interface between the ACP and the catalytic domain differs. The scFAS ACP cannot bind in the AcpP-MCAT posture because it is sterically hindered by the AT ring (positions 543-553, corresponding to amino acids 543-553 in SEQ ID No. 2) and the adjacent ER domain fragment (ii, iii). Conversely, the scFAS ACP interacts with the AT catalytic domain via α-helices 2 and 3, with the ER domain fragment via the ring connecting α-helices 3 and 4, and with the AT domain via helix 5 (iii). The buried surface area of ​​the scFAS AT-ACP interaction interface is approximately 1033 Å. 2 It is almost an MCAT-AcpP interaction interface (buried surface area 459 Å) 2 Twice as much as 1.

[0123] (B) Malonyl / palmitoyltransferase.

[0124] (i) Comparison of overlap between E. coli MCAT enzyme and scFAS MPT domain. In addition to the conserved folding of the catalytic domain, the scFAS MPT domain also contains structural fragments.

[0125] (ii and iii) Comparison of AcpP (ii) bound to the *E. coli* MCAT enzyme and ACP (iii) bound to the scFAS MPT domain. The positions of Ppant-serine residues are marked in the figure. AcpP contacts MCAT via its α-helix 2 and the loop connecting α-helices 2 and 3. In contrast, the scFAS ACP contacts the MPT domain via its α-helix 3 and adjacent structural fragments of the ER and AT domains via α-helices 4 and 5, respectively. These additional interactions stabilize ACP.

[0126] (C) Ketone synthase.

[0127] (i) Comparison of overlap between E. coli KS enzyme and scFAS KS domain. Similar to bacterial KS, scFAS KS domain forms a dimer, but in addition to the conserved catalytic domain, scFAS KS also contains structural fragments.

[0128] (ii and iii) Comparison of ACP (AcpP) bound to the *E. coli* KS enzyme (ii) and ACP (ACP) bound to the scFAS KS domain (iii). The position of Ppant-serine is marked in the figure. AcpP contacts KS via α-helices 2 and 3, while in scFAS, the ACP α-helix 3 of the canonical domain contacts the KS domain in a different manner than the AcpP-KS interaction. The interaction between scFAS ACP and KS is further stabilized by the interaction of ACP α-helices 5 and 6 of the domain with the KS structural fragment (positions 1120-1180, corresponding to amino acids 150-210 of SEQ ID No. 3).

[0129] (D) Dehydrating enzymes.

[0130] (i) Comparison of the overlap of domain 12 between Escherichia coli dehydratase and scFAS dehydratase. Escherichia coli dehydratase (FabA) forms a dimer, while in scFAS it exhibits a pseudodimer structure.

[0131] (ii and iii) Comparison of ACP (AcpP) bound to the *E. coli* DH enzyme (ii) and ACP (scFAS ACP) bound to the scFAS DH domain (iii). The position of Ppant-serine is marked in the figure. *E. coli* AcpP interacts with FabA primarily through its α-helix 2. The interaction between ACP and scFAS DH is primarily mediated by α-helix 3, and the postures of bacterial ACP and scFAS ACP relative to their respective DHs differ. In the case of scFAS ACP, the adjacent DH structural elements (positions 1420-1520, corresponding to amino acids 179-279 of SEQ ID No. 4) and the ER domain prevent it from adopting a binding posture similar to AcpP (iii).

[0132] Figure 6 A comparison of the structural characteristics of bacterial type II enoyl reductases and ketone reductases with their fungal type I counterparts. Figure 3 and Figure 4 Related.

[0133] Since there are currently no publicly available structures showing that ACP binds to the KR domain in fungal-like ER (FabK) and type II FAS systems, in such cases, only a broad comparison of the complementarity of ACP recognition between type I and type II systems can be made.

[0134] (A) Enyl reductase.

[0135] (i) Comparison of overlap between Porphyromonas gingivalis FabK enzyme (PDB ID: 4IQL) and scFAS ER domain.

[0136] (ii) Surface schematic of FabK and scFAS ER domains. Both the scFAS ER domain and bacterial FabK have an active site cleft with two entrances and a binding FMN cofactor. The scFAS ER also has additional structural fragments.

[0137] (iii and iv) NADPH / NADP+ enters (iii) through inlet B, while the Ppant group bound to ACP enters (iv) through inlet A.

[0138] (v and vi) In the FabK-NADP+ structure, only the ADP part was resolved (v); an overlap comparison between the scFAS ER that binds NADP+ and FabK (vi) shows that FabK binds NADPH / NADP+ in a similar manner.

[0139] (vii and viii) Comparison of the overlap between the scFAS ER bound to the Ppant group and FabK (vii) shows that FabK's entrance A can also accommodate the Ppant group into the active site in a similar manner. Notably, the FabK inhibitor binds in the Ppant binding pocket and functions as a competitive inhibitor of NADPH / NADP+ (viii).

[0140] (B) Ketoreductase.

[0141] (i) Bacterial KR (FabG, PDB ID: 1Q7B) is a tetramer, while scFAS KR is a dimer.

[0142] (ii, iii, iv) The catalytic folding of KR in both types of FAS systems is highly conserved. The active site cleft of the NADP+-bound E. coli KR (iii) is more open than that of the NADP+-bound scFAS KR (v, left figure); in the scFAS KR, the adjacent loop is tightly attached to the bound co-substrate and forms a pocket for the Ppant arm to enter (v, right figure).

[0143] (vi) Comparison of the overlap between the NADP+-binding FabG and the ACP-binding scFAS KR domains. Since the bound NADP+ and the catalytic tyrosine residues are located in similar positions in both type II and type I KRs, it can be inferred that the Ppant arm in the bacterial KR will be located in a similar position to that observed in the scFAS KR.

[0144] Figure 7 A structural snapshot of the ACP domain in the fatty acid biosynthesis cycle.

[0145] (A) Structural framework of the yeast fatty acid biosynthesis cycle. The figure shows the asymmetric unit of yFAS and the ACP domains occupying different positions, representing intermediates in the fatty acid biosynthesis cycle. At these different positions, the ACP-binding domains are indicated by different colors: AT, MPT, KS, KR, DH, and ER. All other states except the previously reported DH-binding state are resolved in this paper.

[0146] (B) Comparison of ACP domain binding to enzyme domains in the absence of (i) and presence of (ii) yeast-specific ACP domains. When binding to AT (iii, iv), MPT (iii, v), ER (iv, v, vi), and DH (vi) domains, the ACP domains sterically hinder the binding of ACP to neighboring enzyme domains. In the absence of the ACP domain, this binding is unimpeded. This creates a "path of least resistance," defining a "functional compartment" in which the active sites are arranged as shown in (ii).

[0147] The present invention will be further described by way of embodiments, but these embodiments do not constitute a limitation of the present invention. Detailed Implementation

[0148] Materials and methods

[0149] Data and code availability

[0150] The coordinate data of the resolved structures have been stored in the Protein Database (PDB), with accession numbers as follows: PDB: 8PRV, PDB: 8PRW, PDB: 8PS1, PDB: 8PS2, PDB: 8PS8, PDB: 8PS9, PDB: 8PSA, PDB: 8PSF, PDB: 8PSG, PDB: 8PSJ, PDB: 8PSK, PDB: 8PSL, PDB: 8PSM. The electron microscope images have been stored in the Electron Microscopy Database (EMDB) with accession numbers: EMDB: 17839, EMDB: 17840, EMDB: 17842, EMDB: 17843, EMDB: 17846, EMDB: 17847, EMDB: 17848, EMDB: 17851, EMDB: 17852, EMDB: 17853, EMDB: 17854, EMDB: 17855, EMDB: 17856, EMDB: 17859.

[0151] Experimental model and details of research participants

[0152] Yeast culture

[0153] This study used the *Saccharomyces cerevisiae* strain tma17Δ BJ2168 (genotype: MATa prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2 tma17::kanMX), see Singh, K. et al., 2020, as described above. Cells were cultured in YPD medium in an Inforrs 250 L fermenter and collected at the late logarithmic growth stage when OD600 reached 9–10. Cells were then washed with cold double-distilled water and resuspended in a 2x purification buffer (0.05 M BisTris pH 6.5, 0.05 M potassium acetate, 0.01 M magnesium acetate) containing 20% ​​(w / v) sucrose, with 2 mL of buffer added per gram of cells. Cells were then flash-frozen into beads in liquid nitrogen and stored at -80°C until use.

[0154] Bacterial culture

[0155] In order to express -1xUnaG、 -2xUnaG、 -Tes or Tes, transform the corresponding plasmid into chemically competent BL21-AI (ThermoFisher Scientific; for use in...) -Tes or Tes) or BL21 Star (ThermoFisherScientific; for use in -1xUnaG or -2xUnaG) cells. The initial culture was cultured overnight at 37°C in LB medium containing 1% (w / v) glucose. The pre-culture was then inoculated into TB medium, 500 mL of which was prepared to achieve an OD600 of 0.05, and 1% glucose (w / v) and antibiotic were added. The cultures were cultured at 37°C and 180 rpm until an OD600 of 0.6–0.8 was reached. The temperature was then lowered to 18°C, and cultured for another hour. The cultures were then induced by adding 1 mM IPTG (BL21 Star culture) or 1 mM IPTG and 2% (w / v) L-arabinose (BL21-AI culture). Overexpression was then performed at 18°C ​​for 16 hours. Cells were collected by centrifugation (5,000 g, 5 min, 4°C), resuspended in ice-cold 1× PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4), washed, and centrifuged again (5,000 g, 5 min, 4°C). The final precipitate was rapidly frozen in liquid nitrogen and stored at -80°C.

[0156] Purification of yeast FAS

[0157] FAS was purified according to the method described by Singh, K. et al. (2020) (see above). Starting with 700 g of frozen cell beads (equivalent to 233 g of yeast wet weight), the cell beads were ground into a fine powder in liquid nitrogen using a Retsch ZM200 grinder. The ground powder was thawed in a 37°C water bath, and purification buffer (0.05 M BisTris pH 6.5, 0.05 M potassium acetate, 0.01 M magnesium acetate) was added to a final concentration of 0.33-fold (from a 10-fold dilution of stock solution). Sucrose powder was then added to 20% (w / v), benzylamidine chloride to 10 mM, and PMSF to 1 mM (from 100 mM propanol stock solution). The extract was incubated on a magnetic stirrer at 25°C for 30 min, and then centrifuged at 30,000 rcf for 30 min at 4°C. After centrifugation, the supernatant was filtered sequentially through three layers of coarse cotton cloth and three layers of Miracloth to obtain the yeast S30 cell extract. Octylglucose neopentyl glycol (OGNG, from a 10% (w / v) stock solution) was added to a final concentration of 0.2% (v / v), and the extract was incubated at 30°C for 30 minutes, followed by centrifugation at 100,000 rcf for 1 hour at 4°C. The supernatant was again filtered through three layers of coarse cotton cloth and three layers of Miracloth. The thus clarified S100 extract was differentially precipitated using polyethylene glycol 400 (PEG; the numbers indicate the average molecular weight of the PEG polymer). PEG400 was added to the yeast S100 extract with stirring at 18°C ​​to a concentration of 20% (v / v), and incubated for 30 minutes. The precipitated protein was removed by centrifugation (4°C, 30,000 rcf, 30 minutes). Subsequently, the PEG400 concentration in the supernatant was increased to 30% (v / v) as described above for precipitation. The FAS-containing precipitate from this step was recovered by centrifugation (4°C, 30,000 rcf, 30 min) and resuspended in purification buffer (containing 2% (w / v) sucrose, 10 mM DTT, and 0.01% (w / v) lauryl maltose neopentyl glycol (LMNG)) on an 18°C ​​track shaker. The resuspended sample was loaded into a 10–45% (w / v) linear sucrose gradient prepared with 10 mM DTT purification buffer and centrifuged at 100,000 rcf for 16 h at 4°C. The gradient fractions were collected in 1 mL portions. FAS-containing fractions were identified by SDS-PAGE. The selected fractions were combined and precipitated with 40% (v / v) PEG400. After centrifugation at 30,000 rcf for 30 minutes, the supernatant was discarded, and the precipitate was resuspended in purification buffer (containing 2% (w / v) sucrose, 10 mM DTT and 0.01% (w / v) LMNG).The resuspension was then loaded into a 10–45% (w / v) linear sucrose gradient prepared with 10 mM DTT purification buffer and centrifuged at 79,000 rcf for 16 h at 4°C. FAS-containing fractions were identified by SDS-PAGE, pooled, and cycled at 18°C ​​for 30 min in the presence of 50 µM malonyl-CoA and 100 µM NADPH (i.e., “cycled” FAS). For FASx samples, the cycling treatment of FAS with the addition of malonyl-CoA and NADPH was omitted (“uncycled” FAS). Proteins were precipitated and concentrated by adding 40% (v / v) PEG400 and then resuspended in purification buffer containing 2% (w / v) sucrose, 10 mM DTT, and 0.01% (w / v) LMNG. A second step is required: the resuspension is loaded into a 10-45% (w / v) linear sucrose gradient prepared with 10 mM DTT purification buffer and centrifuged at 60,000 rcf for 16 hours at 4°C. Subsequently, the FAS-containing fraction is precipitated again with 40% (v / v) PEG400 to obtain the final purified protein formulation at a concentration of approximately 15 mg / mL, dissolved in purification buffer containing 10% (w / v) sucrose, 10 mM DTT, and 0.01% (w / v) LMNG. Protein concentration is determined using the Bradford protein quantification method (BioRad, Munich, Germany) with BSA as the standard. The protein concentration is maintained at approximately 15 mg / mL.

[0158] Purification of recombinant proteins

[0159] for -1xUnaG、 -2xUnaG、 Purification of Tes or Tes: The frozen cell pellet from overexpressing cells was resuspended in resuspension buffer at a ratio of 5 mL / g cells. 2 U / mL DNase (NEB) and 0.33 mg / mL lysozyme were added, and the suspension was incubated at 4°C for 30 min. Subsequently, the cell suspension was homogenized twice using an Emulsiflex C3 homogenizer (Avestin) at 15,000 PSI pressure to lyse the cells. The lysis buffer was centrifuged at 50,000 g for 30 min at 4°C, filtered through a cellulose acetate syringe filter (Sartorius Stedim), and loaded onto a 10 mL Ni-NTA column pre-equilibrated with 20 column volumes of resuspension buffer (0.05 M BisTris pH 6.5, 0.05 M potassium acetate, 0.01 M magnesium acetate, 0.01 M imidazole). After the target protein bound, the column was washed with 10 column volumes of wash buffer (0.05 M BisTris pH 6.5, 0.5 M potassium acetate, 0.01 M magnesium acetate, 10 mM imidazole). During elution, 5 column volumes of elution buffer (0.05 M BisTris pH 6.5, 0.05 M potassium acetate, 0.01 M magnesium acetate, 0.5 M imidazole) were added, and 1 mL of elution fraction was collected per tube. The elution fraction was analyzed by SDS-PAGE, and the corresponding fractions were combined. Then, TEV protease (enzyme to protein ratio 1:50) was added to digest the combined protein solution. Enzymatic digestion was performed in dialysis tubes (6,000–8,000 MWCO) at 4°C with gentle stirring overnight (16 hours) in 5 L of dialysis buffer (0.05 M BisTris pH 6.5, 0.05 M potassium acetate, 0.01 M magnesium acetate, 5% (w / v) sucrose). To remove affinity tags, TEV protease, and undigested protein, the dialyzed protein solution was loaded onto a 10 mL Ni-NTA column equilibrated with 10 column volumes of dialysis buffer. The eluent was collected and analyzed by SDS-PAGE. To concentrate the final protein, a centrifugal filter with an appropriate pore size was used, and the protein concentration was determined by spectroscopic methods using a specific extinction coefficient and molecular weight calculated from the sequence. The final protein solution was aliquoted, frozen in liquid nitrogen, and stored at -80°C.

[0160] Preparation of samples for cryo-electron microscopy (Cryo-EM)

[0161] For FASx samples, “uncirculated” FAS was diluted to 0.5 mg / mL using freezing buffer (0.05 M BisTris pH 6.5, 0.05 M potassium acetate, 0.01 M magnesium acetate, 10 mM DTT). For 1.9 Å resolution FAS structures (FASmn), the cycled FAS was diluted to 2 mg / mL using freezing buffer, and 1 mM malonyl-CoA and 2 mM NADP+ were added. The mixture was incubated at 30°C for 30 min, then transferred to 4°C. For FASamn samples, the cycled FAS was diluted to 2 mg / mL using freezing buffer, and 20 μM acetyl-CoA, 1 mM malonyl-CoA, and 2 mM NADP+ were added. The mixture was incubated at 30°C for 30 min, then transferred to 4°C. For FASam samples, the cycled FAS was diluted to 2 mg / mL using freezing buffer, and 1 mM acetyl-CoA and 1 mM malonyl-CoA were added. The samples were incubated at 30°C for 30 minutes, followed by transfer to 4°C. Samples were kept at 4°C before the preparation of the grid.

[0162] Preparation of cryo-electron microscopy support

[0163] For FAS samples in the presence of malonyl-CoA and NADP+, UltrAuFoil R1.5 / 1.6 300-mesh netting (Quantifoil, Jena) was used. These nettings were pre-floated on a custom-made continuous carbon film, covering approximately 25% of the netting area, for alignment purposes. For FASx and FASam samples, Quantifoil R3.5 / 1 (copper) 200-mesh netting was used; while for FASamn samples, Quantifoil R2 / 2 (copper) 200-mesh netting was used. Shortly before spotting, the nettings were glow-discharged for 30 seconds under low vacuum using a self-built glow discharge instrument. For FASmn and FASamn samples, 4 μL of the corresponding protein solution was dropped onto the glow-discharged nettings, blotted dry with a Vitrobot Mark IV (Thermo Fisher Scientific, Eindhoven) at 4°C and 100% humidity for 6.5 seconds, and then rapidly frozen. For FASx and FASam samples, the particles were adsorbed onto a continuous carbon film attached to a Quantifoil (3.5 / 1) mesh (Quantifoil, Jena, Germany) at 4°C for 2 minutes. The mesh was then transferred to a Vitrobot Mark IV blast freezer, 4 μL of double-distilled water was added to the mesh, and the samples were then blotted dry and vitrified as described above.

[0164] Cryo-electron microscopy data acquisition and image processing

[0165] FAS structure in the presence of NADP+ and malonyl-CoA

[0166] All cryo-electron microscopy (cryo-EM) data were collected in nanoprobe mode on a Titan Krios 300 kV electron microscope equipped with a monochromator (ThermoFisher Scientific, Eindhoven, Netherlands) and an aberration corrector "B-Cor" (CEOS, Heidelberg, Germany). The monochromator potential and excitation parameters were adjusted to 3 kV and 0.8, respectively, to achieve an energy spread of approximately 0.1–0.15 eV for the electron source. The B-Cor corrector was configured to correct off-axis aberrations, coma and higher-order electron optical aberrations caused by beam-image shift, and linear distortion. For each cryo-EM dataset, the B-Cor was fine-tuned using a signal from amorphous carbon to i) correct phase errors introduced by on-axis electron optical aberrations to within 45 degrees at scattering angles ≥ 17 mrad (equivalent to ≤ 1.16 Å resolution); and ii) reduce linear distortion to ≤ 0.2%. This study collected 30,659 movie frames, each containing 40 fractions (see Table 2). A Falcon III direct electron detector (Thermo Fisher Scientific, Eindhoven) was used in electron counting mode with a nominal magnification of 96,000x (approximately 0.615 Å / pixel), underfocus ranging from 0.5 μm to 2.4 μm, a total exposure time exceeding 18.54 seconds, a dose per frame of approximately 1.25 e / Ų, and a total dose of approximately 50 e / Ų. An improved version of EPU software (Thermo Fisher Scientific, Eindhoven) was used for data acquisition via light-image shifting, with data collected at up to 3×3 apertures, acquiring 5 movie frames at each aperture. Unless otherwise specified, all image processing was performed using RELION 3.1 (Zivanov, J., et al., (2020), Journal of the International Union of Crystallography 7, 253–267. 10.1107 / S2052252520000081). Global motion correction and dose weighting were performed using MotionCorr2 integrated into RELION (Zheng, SQ, et al., (2017). Nature Methods 14, 331–332. 10.1038 / nmeth.4193), with a B-factor set to 150. CTF estimation was performed using CTFFIND 4.1.13 (Rohou, A., and Grigorieff, N. (2015). Journal of Structural Biology 192, 216–221. 10.1016 / j.jsb.2015.08.008).Grain selection was performed using Gautomatch (a free software) with a 2D template as a reference, followed by 4x pixel merging and extraction (4.26 Å / pixel, 90-pixel bounding box). Three rounds of referenceless 2D classification were performed to remove defective grains. The remaining grains were re-centered and extracted to a 180-pixel bounding box (2.13 Å / pixel), and then 3D classification was performed under C1 symmetry to distinguish between intact and broken grains. The FAS structure (EMDB-4578) was low-pass filtered to 45 Å and used as a 3D reference. Intact grains were then selected and extracted to a 560-pixel bounding box (0.615 Å / pixel). 3D refinement was then performed with D3 symmetry to obtain a 2.45 Å structure (FSC 0.143 criterion). A CTF refinement was performed on the 3D refined grains to correct defocus and astigmatism in each micrograph. Subsequently, grain motion and the corresponding relative B-factor per frame were estimated using Bayesian polishing. A second round of 3D refinement was performed on the polished particles to obtain a 1.98 Å structure. A third round of CTF refinement was then performed to correct the defocus and astigmatism of each particle. Subsequently, the particles were again Bayesian polished, this time extracted with a 920-pixel bounding box (0.615 Å / pixel), followed by a third round of 3D refinement. Ewald sphere correction was performed on the two resulting half-images (Ewald sphere correction, Zivanov, J., et al., (2018), Elife 7.10.7554 / eLife.42166; Russo, CJ, and Henderson, R. (2018), Ultramicroscopy 187, 26–33. 10.1016 / J.ULTRAMIC.2017.11.001), resulting in a final nominal resolution of 1.87 Å.

[0167] FAS asymmetric units in FASx, FASamn and FASam samples

[0168] Data was acquired on a Titan Krios (Thermo Fisher Scientific) equipped with a Falcon 3 (linear mode) detector, operating at 300 kV. Data collection and processing statistics are summarized in Table 2. The processing of the acquired movie frame images up to the 3D classification step was similar to the steps described above. Subsequently, the selected intact particles were used for another round of 3D classification (C1 symmetry).

[0169] In the FASx dataset, 162,079 particles exhibit a rotational conformation, while 35,927 particles are in a non-rotational conformation. These two groups of particles were processed separately. Selected particles were then used for 3D refinement (C1 symmetry). In particles with a rotational conformation, three ACP densities were found within each dome-shaped structure, located near the AT domain. In particles with a non-rotational conformation, nine ACP densities were found within each dome-shaped structure: three each near the AT, KS, and MPT domains. Since each FAS dome-shaped structure can only contain three ACP domains, particles with a non-rotational conformation were selected for subsequent multi-round 3D classification (C1 symmetry). However, in all obtained FAS 3D categories, each dome-shaped structure contained at least six partial ACP densities, indicating that the ACP domains are randomly distributed within the two dome-shaped structures. To classify different ACP binding states, a strategy involving symmetry expansion and signal subtraction was employed, similar to that described previously (Roh, SH, et al., (2017), Proceedings of the National Academy of Sciences 114, 8259–8264. 10.1073 / pnas.1704725114). For this purpose, particles belonging to the non-rotational conformation were subjected to three-dimensional refinement (D3 symmetry), followed by symmetry expansion using the "relion_particle_symmetry_expand" function in RELION. Masks containing the αβ dimer and the ACP domain at the AT, KS, and MPT domains were fabricated to computationally extract each asymmetric unit from each FAS molecule. These asymmetric units were then subjected to unaligned three-dimensional classification using masks surrounding three possible locations of the ACP domain to classify different ACPs located at different active sites. Four distinct categories were observed, with ACP located in the AT, KS, or MPT domains, or not visible. Particles in each category were then subjected to unaligned 3D classification without any masking to distinguish different conformational states of the αβ dimer. Particles exhibiting rotational conformations were treated in a similar manner to those with non-rotational conformations.

[0170] In the FASamn dataset, after 3D classification, all particles exhibited a non-rotational conformation. However, each dome-shaped structure contained six ACP densities, three located in the KR domain and three in the KS domain. Therefore, using the same method as the FASx dataset, particle symmetry expansion and signal subtraction were performed, followed by 3D classification.

[0171] In the FASam dataset, after 3D classification, all particles exhibited a non-rotational conformation. However, each dome-shaped structure contained six ACP densities, three located in the ER domain and three in the KS domain. Therefore, using the same method as the FASx dataset, particle symmetry expansion and signal subtraction were performed, followed by 3D classification.

[0172] Cryo-electron microscopy model construction

[0173] Crystallography Δ The FAS model (PDB-6QL9) was used as the initial model for the 1.87 Å FAS spectrum. It was rigidly coupled to the electron microscopy density using UCSFChimera (Pettersen, EF, et al., (2004), Journal of Computational Chemistry 25, 1605–1612. 10.1002 / jcc.20084). This was then further refined using a rigid model in Refmac5 (Murshudov, et al., (2011), Acta Crystallographica D: Biocrystals 67, 355–367. 10.1107 / S0907444911001314). The model was then subjected to multiple rounds of manual modeling in Coot33 and refinement in Refmac5. Water molecules were incorporated into the spectrum using a combination of Coot and manual modeling methods. After refinement, all water molecules with a B factor higher than 70 Ų were excluded.

[0174] The model obtained from the 1.87 Å FAS map was used as the initial model for the FAS asymmetric unit containing the ACP domain that binds to the enzyme domain. The asymmetric unit containing a single αβ dimer was rigidly attached to the electron microscopy density using UCSF Chimera. These models were then manually modeled multiple times in Coot (Emsley, P., and Cowtan, K. (2004), Acta Crystallographica D: Biocrystals 60, 2126–2132. 10.1107 / S0907444904019158) and refined in Refmac5.

[0175] For model validation, the Fourier shell correlation (FSC) (FSCsum) between the refined model and the spectra was calculated. The model was also refined using the first unfiltered half-map. The model was then compared with the first half-map (FSCwork) and the second half-map (FSCfree) to check for signs of overfitting. In Table 2, RMS bias is from Refmac5, while other values ​​were calculated using the Phenix software package (Adams, PD, et al., (2010), Acta Crystallographica D: Biocrystals 66, 213–221. 10.1107 / S0907444909052925). All images were generated using Pymol36, UCSF Chimera, and ChimeraX (Goddard, TD, et al., (2018), Protein Science 27, 14–25. 10.1002 / pro.3235).

[0176] Enzyme activity assay and semi-quantitative analysis of coenzyme A derivatives

[0177] For the semi-quantitative product analysis of different FAS enzyme activities, 253.8 nM of freshly purified FAS and 500 times molar excess were respectively used. - Subunit or TesA construct mixture. All reactions were incubated at 25°C for 30 min to ensure the added protein bound to FAS and subjected to density gradient centrifugation. The enzymatic reactions were initiated by adding co-substrate to the density gradient peak fractions at final concentrations of 100 µM (acetyl-CoA), 60 µM (malonyl-CoA), and 320 µM (NADPH). After incubation at 30°C for 30 min, the reactions were quenched by liquid nitrogen freezing. For subsequent TesA treatment, 6 µM MTesA was added before freezing. Coenzyme A thioesters were analyzed according to the method adapted from Wolf, J., et al., (2016), Molecular Microbiology 102, 882–908. 10.1111 / mmi.13498. Acetonitrile was used instead of methanol to analyze acyl-CoA up to C20-acyl-CoA and to adjust the gradient to separate more polar derivatives (e.g., malonyl-CoA and free CoA). Metabolites were analyzed by HPLC-MS using an Agilent 6545 QTOF system equipped with an ESI source and coupled to a 1290 series HPLC system. 1 μL of untreated enzyme assay solution was directly injected into a Gemini C18 column (3 µm, 110 Å, 150 × 2 mm). The solvents used were 50 mM ammonium formate (pH 8.1) (A) and acetonitrile (B), with a column temperature of 35°C and the following gradient: 95% A for 1 min, gradient to 30% B over 18 min, gradient to 95% B over 17 min, and gradient to 100% B over 4 min. The QTOF operating parameters were as follows: positive ion mode, gas temperature 200°C, sheath gas temperature 325°C, fragment voltage 180 V, cone voltage 45 V, capillary voltage 4500 V. Data evaluation was performed using MassHunter qualitative navigation software (version B08.00, Agilent Technologies, Santa Clara, USA) by analyzing the mass-to-charge ratio ion chromatograms of the corresponding coenzyme A thioesters [M+H]+ and [M+2H]++. Retention times were confirmed using standards.

[0178] Sequence conservation analysis

[0179] The inventors analyzed the sequence conservation of ACP binding sites at different catalytic sites in *Saccharomyces cerevisiae* and *Y. lipolytica*. To this end, they first used Clustal Omega (Madeira, F., et al., (2022), *Nucleic Acid Research* 50, W276–W279. 10.1093 / nar / gkac240) to generate multiple sequence alignments of the FAS genes in these two species, and then used ChimeraX to map the sequence conservation to different ACP binding states of *Saccharomyces cerevisiae* FAS.

[0180] Quantitative and statistical analysis

[0181] Calculate the distribution of ACP in each asymmetric unit.

[0182] Based on the three-dimensional classification results after signal subtraction, each asymmetric unit was assigned a label. The asymmetric units were then traced back to their corresponding FAS molecules in the RELION particle star file. The collected information was tabulated and analyzed according to the method described by Roh et al. (2017), *Proceedings of the National Academy of Sciences* 114, 8259–8264. 10.1073 / pnas.1704725114. In short, we used normalized mutual information (NMI) for computational analysis to capture the correlation between asymmetric units, an index used to measure conformational correlation between a pair of subunits. To compare the NMI distribution of the data with a random distribution, we generated a simulated dataset of 100 rearranged data points based on the original data. In the simulated data, each particle in the original data was randomly rotated. The distribution of ACP in each asymmetric unit / each FAS molecule was then calculated using PANDAS (Reback, J., et al. (2020), pandas-dev / pandas: Pandas 1.0.3. 10.5281 / ZENODO.3715232).

[0183] Semi-quantitative analysis of coenzyme A derivatives

[0184] All enzymatic reactions were performed in triplicate. For each independent reaction, the total coenzyme A content was expressed as 100%. Mean values ​​and error ranges were calculated based on these values. The relative abundance of a single acyl-CoA group is expressed as its proportion of the total coenzyme A content.

[0185] result

[0186] yeast FAS structure with resolution better than 2 Å

[0187] To reduce potential structural heterogeneity, this study used FAS purified from a *Saccharomyces cerevisiae* strain lacking the γ subunit (Δγ-FAS) (Singh, K., et al., 2020). Prior to cryo-electron microscopy (cryo-EM) mesh preparation, Δγ-FAS was further stabilized by incubation with saturated concentrations of malonyl-CoA and NADP+. Imaging of the mesh using a 300 kV Titan Krios Mono / BCOR microscope yielded an experimental B-factor of approximately 53 Ų, resulting in a nominal resolution of 1.9 Å for the final structure. Local resolution ranged from 1.8–2.2 Å in the central whorl region to 2.2–2.7 Å in the dome region. Such variations in local resolution have been previously reported. Notably, optical improvements in the electron microscope allowed for a proportionally improved resolution, approximately 1 Å better than any previously reported yeast FAS structure.

[0188] In this structure, approximately 7700 water molecules were reliably modeled, averaging 0.33 water molecules per residue (approximately 0.48 per residue in the central wheel-shaped region and approximately 0.18 per residue in the dome region). Higher resolution revealed a 22° kink in the isorhozoline ring system of the FMN at the ER active site. Figure 1B This isotropic kink, i), was not observed in previous structures. This isotropic kink indicates that FMN is in its reduced form, consistent with the reducing function of ER in fatty acid biosynthesis. Figure 1A A hydrogen bond network composed of side chains and water molecules stabilizes FMN within the ER active site. Figure 1B Similarly, the polar contact involving amino acid side chains and water molecules enables the ER and KR active sites to recognize NADP+ ( , ii). Figure 1B (iii and iv). This spectrum is consistent with the malonate of the nucleophile Ser1808-Oγ at the MPT active site and the bound hydrolyzed coenzyme A molecule ( Figure 1B Due to the low local resolution of the MPT region, only a very small number of local water molecules can be located.

[0189] ACP domains at the sites of fatty acid biosynthesis intermediates

[0190] In the biochemically synchronized samples, all six FAS ACP domains were observed to bind to the KS domain. This biochemical synchronization was achieved by incubating FAS with saturated concentrations of malonyl-CoA and NADPH, thereby tracking fatty acid synthesis intermediates. Starting from this "cycled" state, saturated concentrations of Δ... The Δγ-subunit incubates the FAS, stabilizing the ACP domains bound to the AT domains, while the dome region of the FAS is in a rotational conformation (Singh, K., et al., 2020). Both ACP states can be observed in cryo-electron microscopy 3D reconstructions, regardless of whether symmetry is applied. However, in the 1.9 Å FAS structure (Fig. 1), the definition of the ACP domains is poor, essentially unresolved. This is presumably due to biochemical heterogeneity within the FAS dome, manifested as ACP domains occupying non-uniform positions in each functional compartment. If this is indeed the case, then we can expect to observe similar non-fragmented / unfragmented ACP densities as in high-resolution FAS structures, or ACP densities of more than three parts within a single dome. Since the ACP domains are components of the α-subunit, biochemically, a maximum of three can exist within a single FAS dome. Both scenarios suggest that averaging of single-particle images is insufficiently accurate when reconstructing FAS structures in image processing. Overcoming these challenges posed by biochemical heterogeneity, and combining them with solutions to these cryo-electron microscopy technical difficulties, will enable the acquisition of structural information on noteworthy intermediates in the fatty acid biosynthesis cycle, albeit at potentially low resolution. We will describe below how to overcome these image processing challenges.

[0191] The non-uniform binding of ACP to various active sites within a single FAS dome is likely the reason why ACP is difficult to resolve. To test this hypothesis, we first resolved FAS structures without malonyl-CoA and NADPH tracking (unsymmetry applied, i.e., the "uncycled" state, hereinafter referred to as "FASx"). We expected that the distribution of ACP within the FAS dome would be the most random in this case. The results were consistent with expectations; the FASx structures showed 9 low-threshold partial ACP densities per dome (i.e., 18 per FAS), distributed near AT, MPT, and KS. In the asymmetric structure of the cyclic FAS incubated with acetyl-CoA, malonyl-CoA, and NADP+ (hereinafter referred to as "FASamn"), each dome showed 6 partial ACP densities (i.e., 12 per FAS), adjacent to KR and KS (Fig. 2B, middle). In the asymmetric structure of the cyclic FAS containing only acetyl-CoA and malonyl-CoA (hereinafter referred to as "FASam"), six partial ACP densities (i.e., 12 per FAS) were also observed in each dome, located next to ER and KS.

[0192] As mentioned above, more than three ACP domains were observed within the FAS dome, indicating that protein density was averaged during image processing. In the FAS, the large pseudo-symmetric barrel structures appear to be the primary driver of image alignment, rather than the ACP domains, leading to a loss of ACP density during reconstruction. To overcome this obstacle, we employed a strategy: first, a three-dimensional refinement with strict D3 symmetry was performed, followed by symmetry expansion and particle signal subtraction to computationally extract six αβ dimers (asymmetric units) from each FAS particle. Subsequently, the FAS asymmetric units were classified using masks representing individual ACP locations, followed by classification without any masks. This yielded asymmetric unit structures with a resolution of approximately 3 Å, revealing complete, single-site ACP density representing intermediate states in fatty acid biosynthesis.

[0193] The conformation of FAS and the binding mode of ACP to the enzyme domain

[0194] Previous studies have shown that the FAS β-subunits employ two main conformational states: non-rotated and rotated. In the FASx and FASam datasets, researchers also identified two additional hybrid conformational states. In the semi-non-rotated (SNRot) state, the AT and ER domains are non-rotated, while the DH and MPT domains are rotated. The semi-rotated (SRot) state is characterized by an overall rotated conformation, but the AT domain is not fully rotated. The study found that the ACP was located near the AT domain in all four β-subunit conformations, which can be explained by the ACP's ability to pivot around Ser180 (corresponding to amino acid position 44 of SEQ ID No. 7). This rotational capability of the ACP allows it to adapt to the space between AT and ER in different conformations while maintaining interactions through canonical and structural domains. The ACP's location at KS and ER is associated with both non-rotated and semi-non-rotated conformations, consistent with minor changes in the two domains in these conformations. However, the binding of ACP to KR is only associated with the non-rotational conformation. Furthermore, ACP bound to DH was observed to be in a non-rotational state. These findings suggest that the rotated β-subunit conformation specifically participates only in the acetyl loading step, while the condensation and extension phases appear to occur in the non-rotational conformation. Therefore, these novel conformations are likely employed during the ACP transfer from AT to KS or the reverse transfer process.

[0195] This article and previous studies (see Singh, K., et al., (2020); Leibundgut, M., et al., (2007), Science 316, 288–290. 10.1126 / science.1138249; Lou, JW, and Mazhab-Jafari, MT (2020), Communications Biology 3. 10.1038 / s42003-020-0997-y) capture snapshots that help reveal the determinants on which the FAS enzyme domain recognizes ACP. The ACP canonical domain, containing Ppant-Ser180 (corresponding to amino acid 44 of SEQ ID No. 7) and its flanking α-helices 2 and 3, is located at the opening of the cleft between the active sites of the MPT, KR, and ER domains. Figure 3 The identification of the ACP canonical domains is primarily achieved through electrostatic interactions, consistent with the charge complementarity of the interacting surfaces. These interactions are similar to the associations of the ACP canonical domains with the KS, AT, and DH domains, and therefore appear to be generally conserved in ACP canonical domain-active site interactions. For ACPs binding MPT, KR, and ER, we plotted additional ACP-FAS interaction maps (…). Figure 3 ACP also contacts structural elements near AT and ER at MPT via the ACP α4 helix. Figure 3 (i and ii). At ER, the α2 and α3 helical residues of ACP and the loop region connecting the α3 / 4 helix form extensive contacts, and the structural elements near AT and KS interact with the α2 and α4 helices of ACP, respectively. Figure 3 (iii and iv). At KR, the α2 and α3 helices of ACP contact KR residues outside the active site, while the loop region between the ACP α3 / 4 helices recognizes nearby KR structural elements, and the α4 helix of ACP interacts with KS ( Figure 3 Unlike ER and MPT, ACP bound to KR also utilizes its yeast-specific extended regions (the loop region between the α5 helix and the α5 / 6 helix, and the α7 / 8 helix) to form additional contacts with the coiled helical dimerized fragments of the KR domain.

[0196] therefore, Figure 3The findings, along with previously reported results (see Singh, K., et al., (2020); Leibundgut, M., et al., (2007), Science 316, 288–290. 10.1126 / science.1138249; Lou, JW, and Mazhab-Jafari, MT (2020), Communications Biology 3. 10.1038 / s42003-020-0997-y), indicate that structural elements distant from the active site are universally involved in ACP binding. Furthermore, yeast-specific ACP domains mediate interactions when binding KS, KR, and AT. However, the canonical ACP domain is universally required for interactions with all enzyme domains of FAS.

[0197] Structural changes to accommodate the Ppant arms in the MPT, KR, and ER domains

[0198] Next, the study explored whether local conformational adaptations of the MPT, KR, and ER domains facilitated the recognition of ACP (especially the Ppant arm). The inventors captured two different variants of the Ppant group bound to MPT: the first derived from malonyl-CoA as a separate molecule, and the second in which the Ppant group was covalently linked to Ser180 of ACP (corresponding to amino acid position 44 of SEQ ID No. 7). In both cases, a large conformational change was observed, characterized by the shift of the MPT helical subdomain toward the MPT catalytic site Ser1808 (corresponding to amino acid position 244 of SEQ ID No. 6). Figure 4 A). As a result, Ppant is encapsulated in a clamp-like manner. In the ACP-bound state, the cavity of the MPT active site is enlarged overall, which could explain how malonyl-CoA or malonyl-ACP is distinguished from the longer ACP-bound acyl intermediate (ACP). Figure 4 (B, right). This currently explains the adaptation mechanism of Ppant-acyl adducts with up to 10 carbon atoms (about 16 Å), but how palmitoyl or stearoyl adducts bind remains to be elucidated.

[0199] Similarly, this paper captures two scenarios for NADP+-bound KR domains: in the absence of ACP-bound Ppant arms ( Figure 1B , iv) and in its existence ( Figure 3 In both scenarios, closure of a ring on the nicotinamide-ribose moiety of NADP+ was observed. Figure 4C), which is similar to previously reported results (see Jenni, S., Leibundgut, et al., (2007), Science 316, 254–261. 10.1126 / science.1138248). The closure of the loop forms a pocket that serves as an entry site for the Ppant arm of ACP binding and has a dual purpose: stabilizing NADPH / NADP+ binding and stabilizing the Ppant arm of ACP binding (C). Figure 4 C). The binding of NADPH / NADP+ also leads to the expansion of the constricted channel formed by Asn829, Phe883, Leu930, and Leu936 (corresponding to amino acids 502, 327, 374, and 380 of SEQ ID No. 5), thereby enabling the KR active site to accommodate fatty acyl intermediates longer than 4-6 carbon atoms. Figure 4 D).

[0200] Within the ER domain, the Ppant arm enters the active site through a slit within the FAS dome. Notably, the Ppant arm undergoes an approximately 96° kink within the ER active site. Figure 4 E), allowing the enoyl bond to be positioned between catalytic histidine His740 (corresponding to amino acid position 184 of SEQ ID No. 1) and FMN. Current structural resolution cannot distinguish between cis and trans enoyl bonds, but both configurations can be accommodated at this position. The ER domain, which binds the ACP ( Figure 3 The superposition of nicotinamide with the ER domain bound to NADP+ (Fig. 1D, iii) revealed that the adduct of nicotinamide and the enoyl-ACP Ppant arm occupies the same position near the FMN isorhodin. Figure 4 E). Accompanying this change, when ACP binds, the ER ring segment shrinks the ADPR binding gap by approximately 3 Å, making the binding of NADPH / NADP+ to enoyl-acyl-ACP mutually repulsive ( Figure 4 F).

[0201] In summary, the ACP-Ppant-mediated movement in MPT and the NADPH / NADP+-mediated ring closure in KR collectively generate a clamping motion, thereby enabling the recognition of the Ppant arm and accommodating the continuously increasing chain length of covalent acyl intermediates in fatty acid biosynthesis. In contrast, ER utilizes a pre-formed cleft to position the kinked enoyl-Ppant arm above the isorhodiazine ring of FMN. This cleft shrinks the active site, making the binding of enoyl-acyl-ACP and NADPH / NADP+ mutually exclusive.

[0202] Comparison of ACP-FAS enzyme interactions in type I and type II systems

[0203] This paper elucidates the structure of yeast ACP (yACP) bound to multiple yeast FAS (yFAS) enzyme domains, enabling a comparison of ACP recognition by FAS enzymes in type I and type II systems. To this end, the structure of ACP bound to malonyl-CoA-acyltransferase (MCAT) (PDB ID: 6U0J) was compared with the structures of yFAS-AT (Fig. 5A) and yFAS-MPT (Fig. 5B) bound to yACP, as MCAT performs two transacylation functions in bacteria. Furthermore, the structure of FabB ketoacyl synthase bound to ACP (PDB ID: 6OKC) was compared with yFAS-KS (PDB ID: 6QL9, Fig. 5C) bound to yACP, and the structure of FabB dehydratase bound to ACP (PDB ID: 4KEH) was compared with yFAS-DH (PDB ID: 6WC7, Fig. 5D) bound to yACP.

[0204] Bacterial ACP (AcpP) interacts with MCAT via α-helix 2 (corresponding to α-helix 3 in yACP) and α2 / 3 loops, with a buried surface area (BSA) of 459 Å at the interaction interface. 2 (Figs. 5A and 5B). In yFAS-AT, in addition to the conserved catalytic domain, there are additional structural fragments (Fig. 5A, I left). Although Ppant-serine is located at the opening of the active site cleft in both bacteria and yFAS structures (Fig. 5A, i right), a similar binding posture for AcpP cannot be achieved in yFAS-AT due to spatial conflicts with the structural ring and adjacent ER domain fragments (Fig. 5A, iii). Instead, in yFAS-AT, the α-helices 2 and 3 of yACP (BSA 530 Å) 2 The yACP interacts with the AT catalytic domain, and the α3 / 4 ring of the yACP contacts the adjacent ER fragment (BSA 240 Å). 2 ), while the α-helix 5 of yACP combines with the AT structure domain (BSA 263 Å). 2 Therefore, the interaction interface between yACP and yFAS-AT is almost twice as large as that between MCAT-AcpP.

[0205] yFAS-MPT also possesses structural segments beyond the catalytic domain (Fig. 5B, i). yACP interacts with yFAS-MPT via its α-helix 3 (BSA 238 Ų) and with adjacent structural segments of the ER and AT domains via its α-helices 4 and 5 (BSA 216 Ų) (Fig. 5B, iii). Thus, the interaction surfaces of yACP are redistributed relative to AcpP, integrating the domains of yACP and utilizing adjacent segments in the yFAS architecture while maintaining similar interaction interface sizes.

[0206] Both bacteria and yFAS KS are homodimers, the latter containing additional structural fragments (Fig. 5C, i). AcpP interacts with FabB via its α-helices 2 and 3 (BSA 803 Å). 2 yACP, on the other hand, adopts a different posture from AcpP α-helix 2, utilizing its α-helix 3 to interact with the KS catalytic domain (BSA 377 Å). 2 It interacts with the KS structural segment via α-helices 5 and 6 (BSA 477 Å2). Although yACP utilizes its own structural domains and the KS structural segment, the size of its interaction interface is still comparable to that of AcpP.

[0207] FabA forms a dimer, while yFAS-DH exhibits a pseudo-dimer structure (Fig. 5D, i). The α-helix 2 of AcpP primarily participates in binding with FabA (Fig. 5D, ii), and similarly, the α-helix 3 of yACP primarily participates in the interaction with DH (Fig. 5D, iii). However, considering the structural differences between yFAS-DH and FabA, the binding attitude of yACP on yFAS-DH differs significantly from that of AcpP on FabA. This difference in binding attitude is likely due to the structural elements of DH and the adjacent ER domains.

[0208] Currently, there is no structure for AcpP binding to bacterial KR and ER enzymes. Therefore, we only compared the overall structure and co-substrate binding mode with the fungal ER enzyme FabK (PDB ID: 4IQL, Fig. 6A) and KR enzyme FabG (PDB ID: 1Q7B, Fig. 6B).

[0209] yACP is primarily mediated by its canonical structural domain (BSA 701 Å). 2 It interacts with yFAS-ER, while its structural domains also have a small amount of contact (BSA 69 Å). 2 Both FabK and yFAS-ER have an elongated active site slit with two inlets, A and B. Figure 6(A, i, and ii). In the yFAS-ER, NADPH / NADP+ enters the active site cleft via entrance B (Fig. 6A, iii), while the Ppant arm of yACP enters via entrance A (Fig. 6A, iv). Considering that the ADP portion of NADPH / NADP+ occupies a similar position at entrance B in FabK as it does in the yFAS-ER (Fig. 6A, v, and vi), and that competitive inhibitors of FabK occupy the Ppant arm binding site at entrance A (Fig. 6A, vii, and viii), the ACP recognition mechanism is likely conserved in both systems.

[0210] FabG exists as a tetramer, while yFAS-KR exists as a dimer, but the catalytic folding between the type I and type II systems is highly conserved. Figure 6 B, i, and ii). FabG combined with NADP+ ( Figure 6 B, iii) and yFAS-KR without the prosthetic group ( Figure 6 Similar to B, iv), but the latter is more similar to yFAS-KR bound to NADP+ ( Figure 4 C and Figure 6 B, v) are much more open. Since NADP+ and catalytic tyrosine residues are well superimposed in both FabG and yFAS-KR, the Ppant arm of AcpP in FabG may occupy a similar position as in yFAS-KR. Figure 4 C and Figure 6 B, vi). In addition, the active site cleavage of FabG is more open, which can easily accommodate long fatty acyl chains.

[0211] Redesigning FAS using ACP structural snapshots from fatty acid biosynthesis cycles

[0212] The structure in which yACP binds to multiple yFAS enzyme domains provides a potential pathway for redesigning the fatty acid biosynthetic output of yFAS. Importantly, mapping the FAS sequence of *Yarrowia lipolytica* (an oil-producing and biotechnologically relevant yeast) to the structural snapshot obtained in this paper shows that all interacting ACP and enzyme domain amino acids are conserved. Considering all ACP positions and yFAS conformations, the free volume inside the FAS barrel structure is estimated to be approximately 3.9 × 10⁻⁶. 5 Å 3 However, after incorporating six γ subunits, the volume is reduced to approximately 3.6 × 10⁻⁶. 5 Å 3 This indicates that each yFAS dome can accommodate up to approximately 150 kDa of additional exogenous globular proteins (approximately 300 kDa for the entire FAS). Due to... The subunit is located in the FAS dome and is combined in a specific way, so it can be used as a carrier.

[0213] The inventors conducted a proof-of-concept experiment to assess the feasibility of this idea (Figure 6A). Δ -FAS with separate Subunit or the following were replaced Recombination of the protein at subunits 114–132: 1 copy of the fluorescent protein UnaG ( -1xUnaG; 30 kDa), 2 contiguous copies of UnaG ( -2xUnaG; 45 kDa), Vibrio harveyi TesA ( -Tes; 50 kDa), and as a control, unintegrated into TesA (Tes) of Vibrio harveyi in a subunit scaffold. Because the molecular weight of these proteins differs significantly from that of γFAS (2.6 MDa), the recombinant mixture was separated by sucrose density centrifugation. Proteins co-precipitated with γFAS into the high molecular weight fraction were considered to have been bound. All integrated into… The proteins in the subunit carrier can all interact with Δ -FAS combines, but it's worth noting that Tes alone cannot combine. (Relative to Δ) -FAS itself exhibits FMN fluorescence, observed -1xUnaG and The fluorescence of -2xUnaG was significantly enhanced. Finally, we verified by LC / MS that the fluorescence was significantly enhanced compared to that of Δ... -FAS or use Δ of subunit recombination -Δ Compared to FAS, using -Tes recombination Δ -FAS showed a spectrum of different acyl-CoA products. It is noteworthy that... -Tes recombination Δ In the acyl-CoA product profile of -FAS, the abundance of C8- and C10-CoA was significantly increased, while the abundance of C12-, C14-, and C16-CoA decreased. This was due to subsequent treatment with Vibrio harveyi TesA. The FAS reaction exhibited a similar product spectrum, which we attributed to the specificity of the thioesterase within the FAS dome. In summary, the inventors have demonstrated that knowledge of yACP structural snapshots can provide a reference for regulating the product spectrum of yFAS in the production of oleochemicals.

[0214] discuss

[0215] Here, the inventors elucidate snapshot structures of enzyme domain-bound ACPs, representing functional intermediates in the yeast fatty acid biosynthesis cycle. These intermediates include ACP states bound to AT-, KS-, MPT-, KR-, and ER-, as well as states bound to substrates, co-substrates, and cofactors. For ACP domains bound to AT-, MPT-, KR-, and ER-, we resolved the functionally indispensable Ppant arm, which shuttles initiating chemical groups, biosynthetic intermediates, and products as covalent adducts from one active site to other active sites. This allows us to investigate the recognition patterns of ACP- and the Ppant arm in different functional states and their resulting structural consequences. In summary, the findings reported in this paper, along with the previously reported results, enable us to reconstruct the complete structural framework of conformational changes in the fatty acid biosynthesis cycle. Figure 7 A).

[0216] These snapshots demonstrate that each FAS dome can accommodate exogenous proteins with a mass of up to 150 kDa. Proof-of-concept experiments show that such exogenous proteins can be used to modulate the product profile of FAS. This framework lays a solid foundation for exploring fatty acid biosynthesis mechanisms at atomic resolution, developing activity-based and biophysical probes, assisting in the design of rational inhibitors, and modifying and utilizing their functions in oleochemical production.

[0217] High-resolution structural determination of type I FAS using cryo-electron microscopy

[0218] To gain functional and mechanistic insights and establish structure-function relationships, a detailed and accurate description of the enzyme's active site and its structural changes across different functional states is essential. This includes the protein backbone and side chains, prosthetic groups, co-substrates, and ordered solvents (water and ions) in macromolecular complexes. While all these features can be identified at different resolution thresholds, ordered solvents can only be accurately described at resolutions better than 2.2 Å. Particularly in cryo-electron microscopy, several factors hinder the acquisition of such fine structures: biochemical, compositional, and conformational heterogeneity; heterogeneity arising from cryo-electron microscopy grid preparation; technical limitations of electron microscopy imaging; and image processing problems caused by low signal-to-noise ratios.

[0219] For *Saccharomyces cerevisiae* FAS, we addressed the biochemical heterogeneity issue by establishing a mild, chromatography-free purification process. This process enabled the determination of the FAS structure at a resolution better than 3 Å using cryo-electron microscopy and X-ray crystallography. We discovered a weakly bound [structure / organism] in yFAS. The subunits readily dissociate at the concentrations used in cryo-electron microscopy grid preparation. To overcome this problem, we used a genetically deleted subunit. FAS was purified from a *Saccharomyces cerevisiae* strain containing the subunit. We also added saturated concentrations of malonyl-CoA and NADP+ to lock the individual enzyme domains into a homogeneous composition. For cryo-electron microscopy structure resolution, we used a 300 kV Titan KriosMono / BCOR microscope equipped with an optimized electron beam path system. For yFAS, this ultimately achieved a structure resolution of 1.9 Å overall, with an experimental B-factor of 53 Å. 2 The local resolution ranges from 1.9 Å at the center wheel to 2.7 Å at the top of the dome. Figure 1B The B factor of yFAS, obtained using a 300 kV TitanKrios Mono / BCOR microscope, is comparable to that of ferritin imaged using a conventional Titan G3 Krios instrument, see Danev et al., Trends in Biochemical Sciences 44, 837-848, https: / / doi.org / 10.1016 / j.tibs.2019.04.008. This highlights that even for dynamic assemblies like FAS, improvements in electron optics contribute to a resolution increase of nearly 1 Å, which is proportional to the previously described local resolution range. The high-resolution structure confirms the presence of FMN in its reduced form within the ER (…). Figure 1B (i), providing insights into the identification of CoA and revealing the Ser1808-malonyl covalent adduct in MPT ( Figure 1B , v), and the recognition of NADPH / NADP+ by proteins and ordered solvents in KR and ER ( Figure 1B (ii-iv). However, it is noteworthy that the ACP domain was not resolved in high-resolution structures (Fig. 1), while in other cases, the number of partially visible domains exceeded the number expected from its biochemical composition. Figure 2 This can be explained by one or both of the following reasons: 1) insufficient biochemical control to synchronize different functional states; and / or 2) orientation assignment problems in image alignment due to pseudo-symmetric domes, making it difficult to classify conformationally heterogeneous, independent and asynchronous ACP motions, and failing to detect ACPs throughout the entire FAS particle.

[0220] ACP structural snapshots and conformational states in the fatty acid synthesis cycle:

[0221] Standard 3D classification procedures cannot resolve the ACP domain in the full-length FAS. By computationally extracting asymmetric units from the FAS to classify ACP positions and β-subunit conformations, we visualized ACP snapshots representing functional intermediates in the fatty acid synthesis cycle. Two additional heterozygous β-subunit conformations were also identified, representing transitional states from rotational to non-rotational conformations. A comprehensive review of β-subunit conformations and their corresponding ACP positions revealed that the rotational state is exclusive to the AT-bound ACP position. The AT-bound ACP is also compatible with SRot, SNRot, and non-rotational conformations. Therefore, we infer that after the initial acetyl group completes loading of the catalytic AT serine, the ACP moves towards the AT domain and adjusts the position of the Ppant arm to receive the acetyl group. The binding of ACP to AT is likely accompanied by a conformational change from a non-rotational to a rotational state. When the acetyl group is transferred to the catalytic cysteine ​​of KS, the ACP domain moves towards KS and will always adopt a non-rotational state. These changes and ACP movement are sufficient to explain the energy barrier we previously reported (Singh, K. et al., 2020, see above) during the transition from rotational to non-rotational conformation. We had previously hypothesized (Singh, K. et al., 2020, see above) that each functional state of the FAS corresponds to a unique β-subunit conformation. A systematic investigation of the β-subunit conformation and ACP position did not confirm this hypothesis. The binding of the ACP to all other enzyme domains occurs in the non-rotational conformation. This may avoid kinetic barriers in the condensation and elongation cycles and improve synthetic efficiency.

[0222] Small-scale conformational changes do indeed occur during condensation and elongation cycles. For fatty acid biosynthesis, the malonyl group must first be activated by the catalytic serine of MPT, and then transferred to the Ppant arm of ACP (…). Figure 1B ,v; Figure 3 , i and ii; and Figure 4 A). When malonyl-CoA binds, the MPT is closed; when CoA is released, the MPT opens; it then closes again to accommodate the Ppant arm of ACP, thus completing the transfer of the malonyl group. The MPT active site bound to ACP is more spacious, capable of accommodating larger fatty acyl chains bound to ACP for release, but how exactly palmitoyl or stearoyl-Ppant-acyl adducts bind is currently unclear. Subsequently, the malonyl-Ppant adduct is carried to KS, where it generates a β-keto acyl intermediate via Claisen condensation. Next, NADPH enters the KR active site, causing the KR ring structure to clamp into NADPH ( Figure 1A and Figure 4 C) thus provides an extended space for the Ppant arm to enter and accommodate the β-ketoacyl intermediate ( Figure 4 C and Figure 4 D). After reduction to the β-hydroxyacyl intermediate, ACP proceeds to DH (see the Lou and Mazhabi-Jafari 2020 literature above), generating the trans-2-enyl acyl intermediate. The dehydration reaction likely constructs the cis or trans configuration of the enyl acyl intermediate and subsequent regiospecificity. However, the published structures have not resolved the Ppant arm, and the inventors have not been able to observe this state in their own experiments to resolve this issue. To complete the fatty acid biosynthesis cycle ( Figure 1A ACP interacts with the reduced FMN in the ER, at which point the Ppant arm becomes kinked, and its binding leads to a narrowing of the NADPH / NADP+ binding gap. Figure 4 E). The binding of the Ppant-acyl adduct of ACP to NADPH / NADP+ is repulsive, suggesting that either NADPH / NADP+ may have left before ACP arrives, or ACP binding may trigger the release of NADPH / NADP+. Subsequently, ACP transfers the Ppant-acyl adduct to the cysteine ​​residue at the KS active site for further cycling, or to the MPT domain, where the Ppant-acyl adduct is transacetylated to the CoA molecule and released. Because ACP binding to KS involves multiple steps (acetyl transfer, condensation of acetyl and malonyl groups, and condensation and transfer of fatty acyl groups of varying lengths with malonyl groups), the biochemical heterogeneity appears too great to resolve the Ppant arm. However, as reported (Singh, K. et al., 2020, see above), when using cyclic FAS, we were able to resolve the Ppant arm by cryo-electron microscopy and X-ray crystallography. Therefore, we hypothesize that KS identification of Ppant may vary significantly depending on the specific properties of the acyl adduct carried by the ACP domain. Resolving a series of high-resolution structures to clearly define the different intermediates involved in KS-catalyzed cysteine ​​binding is crucial to addressing this issue.

[0223] The large ACP domains in type I fungal FAS appear to contribute to the order within the FAS dome.

[0224] A potential mechanistic problem arising from the spatial confinement of the yFAS structure emerges: the universality of the ACP domain and its Ppant arms may lead to recognition confusion. The interaction between bacterial FAS enzymes and ACP depends on a minimal interaction interface containing the Ppant arms and electrostatic interactions (Figures 5 and 6). If ACP binds to the site in a mixed manner, with a sequence that deviates from the strictly required sequence for fatty acid biosynthesis, catalytic efficiency will be reduced. One possible solution to this problem is additional structural domains within yFAS. In fact, we observed associations between the yACP structural domain and structural domains of multiple enzymes (Figures 5 and 6). Figure 7 Based on the data summarized in A, we can conclude that the canonical yACP domain extensively contacts all active sites and is often supplemented by interactions between structural domains. The affinity of yFAS to yACP interactions, as studied by interfacial buried surface area, appears to be conserved compared to the corresponding bacterial system, but its distribution is clearly evident between yACP canonical domain-active site cleft interactions and yACP structural domain-structural fragment interactions (Figs. 5 and 6). Therefore, in type I FAS, ACP recognition may be more dominated by avidity than affinity. Furthermore, the localization pattern of yACP on enzyme domains suggests that structural domains of yACP may play a more active role. yACP binding via steric hindrance is mutually exclusive between: 1) AT and its neighboring MPT and ER domains; 2) MPT and its neighboring AT and ER domains; and 3) DH and its neighboring ER domain (Fig. 7B, ii-vi). In the absence of the yACP domain, this steric hindrance is reduced (Fig. 7B, i). Notably, the "path of least resistance" through which the ACP shuttles along the fatty acid biosynthetic sequence coincides with the functional partitions inferred from γ-subunit binding. Therefore, this shuttle path with minimal hindrance from the yACP domain coincides with the shortest path required to traverse the FAS dome, ultimately improving the efficiency of fatty acid biosynthesis. Another consequence of the steric hindrance generated by the yACP domain is that it ensures that the next enzyme active site to which the ACP will move is vacant. Thus, in addition to minimizing recognition confusion, the yACP domain may promote functional compartmentalization and ensure efficient and orderly sequencing during fatty acid biosynthesis.

Claims

1. A protein involved in fatty acid synthesis, said protein comprising one or more polypeptide chains, wherein said polypeptide chains contain... i) One or more subunits comprising the following amino acid sequence: SEQ ID No. 7 (ACP domain); SEQ ID No. 1 (ER domain); SEQ ID No. 2 (AT domain); SEQ ID No. 3 (KS domain); SEQ ID No. 4 (DH domain); SEQ ID No. 5 (KR domain); and / or SEQ ID No. 6 (MPT domain); ii) In the ACP domain, at least one amino acid substitution is present at the positions of amino acids 5-11, 20-25, 34-105, and 135-147 corresponding to SEQ ID No. 7; and / or in the ER domain, at least one amino acid substitution is present at the positions of amino acids 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 corresponding to SEQ ID No. 1; and / or in the AT domain, at least one amino acid substitution is present at the positions of amino acids 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 corresponding to SEQ ID No. 2; and / or in the KS domain, at least one amino acid substitution is present at the positions of amino acids 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 corresponding to SEQ ID No.

7. 3 has at least one amino acid substitution at amino acid positions 68-131; and / or in the DH domain, has an amino acid substitution at amino acid positions 189-310 corresponding to SEQ ID No. 4; and / or in the KR domain, has an amino acid substitution at amino acid positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, 602-643 corresponding to amino acid positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, 602-643 corresponding to amino acid positions 36-66, 176-187, 353-417, 242-248, 293-306, 325-335, 353-358, 396-415 corresponding to amino acid positions 1-30, 48-170, 242-248, 293-306, 325-335, 353-358, 396-415 corresponding to amino acid positions 396-415; and / or in, The amino acid sequence containing the at least one amino acid substitution has at least 70%, or preferably at least 80%, 90%, or 95% sequence identity with the corresponding amino acid sequences of SEQ ID No. 1 and / or SEQ ID No. 2 and / or SEQ ID No. 3 and / or SEQ ID No. 4 and / or SEQ ID No. 5 and / or SEQ ID No. 6 and / or SEQ ID No. 7; thereby resulting in an alteration in fatty acid synthesis activity, wherein the positions shown in ii) indicate the amino acids in each domain involved in the interaction between ACP and type I FAS proteins or peptides, and the amino acid substitutions of the type I FAS proteins or peptides are not located within the catalytic center of the corresponding type I FAS proteins or peptides.

2. The protein according to claim 1, wherein the ACP domain contains at least one amino acid substitution at amino acid positions 40-49, 51-70, and 72-105 corresponding to SEQ ID No. 7; and / or the ER domain contains at least one amino acid substitution at amino acid positions 40-45, 65-70, 92-109, 126-140, 160-170, 175-188, 314-324, 290-301, and 467-477 corresponding to SEQ ID No. 1; and / or the AT domain contains at least one amino acid substitution at amino acid positions 163, 274, 364, 401, 403, 405, 406, and 428 corresponding to SEQ ID No. 2; and / or the KS domain contains at least one amino acid substitution at amino acid positions 95-102 and 109-130 corresponding to SEQ ID No. 3; and / or the DH domain contains at least one amino acid substitution corresponding to SEQ ID No.

7. The 198th amino acid position of 4 contains an amino acid substitution; and / or the KR domain contains an amino acid substitution at the positions of amino acids 39-62, 178-184, 354-364, 372-392, 409-413, 545-557, and 603-642 corresponding to SEQ ID No. 5; and / or the MPT domain contains at least one amino acid substitution at the positions of amino acids 20-28, 48-64, 81-96, and 102-110 corresponding to SEQ ID No. 6; and / or.

3. The protein according to claim 1 or 2, wherein the amino acid substitutions are as shown in Table 1.

4. A nucleic acid molecule encoding a protein or polypeptide according to any one of claims 1 to 3, preferably further comprising a vector nucleic acid sequence, more preferably comprising an expression vector sequence and / or comprising a promoter nucleic acid sequence and a terminator nucleic acid sequence, and / or comprising other regulatory nucleic acid sequences, and / or preferably comprising dsDNA, ssDNA, cDNA, LNA, PNA, CNA, RNA or mRNA or combinations thereof in the nucleic acid molecule.

5. A host cell containing the nucleic acid molecule of claim 4 and preferably expressing the nucleic acid molecule, wherein the host cell is preferably selected from bacterial cells, fungal cells, algal cells, actinomycete cells or eubacterial cells.

6. The host cell according to claim 5, wherein the host cell is a non-oil-producing yeast or an oil-producing yeast cell, preferably, the host cell is an oil-producing yeast genus selected from Candida, Rhodosporidium, Yarrowia, Cryptococcus, Rhodotorula, Lipomyces, and Trichosporon, such as Yarrowialipolytica; or a non-oil-producing yeast genus selected from Saccharomyces, Schizosaccharomyces, Pichia pastoris, Kluyveromyces lactis, and Ustilago maydis.

7. A method for producing fatty acids, comprising the step of expressing the nucleic acid molecule of claim 4, preferably in the host cell of claim 5 or 6, or expressing the protein or polypeptide of any one of claims 1 to 3.

8. The method according to claim 7, for producing short-chain (C6 to C12) fatty acids, wherein the protein or polypeptide comprises at least one of the following substitutions: Met56Val, Met56Ile, Val26Leu, Val26Ile, Lys62Arg, Gln58Glu, Lys64Arg, Lys83Arg, Asn326Asp, Asn326Gln, Asn326Glu, Asn328Asp, Asn328Gln, Asn328Glu, Gln333His, Gln333Glu, Arg398Glu, Arg398His, His413Asp, His413Asn, His413Glu, His413Gln, His413Tyr, His413Arg; or in SEQ ID No. 6 Lys37Arg, Lys37Glu, Lys37Gln, Gly42His, Gly42Tyr, Gly42Phe, Asn48Asp, Asn48Gln, Asn48Glu, Asn48His, Asn4 in No.7 8Arg, Asn48Tyr, Thr45Met, Thr45His, Thr45Asn, Thr45Asp, Thr45Gln, Thr45Glu, Leu51Arg, Leu51Tyr, Lys64Glu, Lys64A rg, Glu67Asn, Glu67Asp, Glu67Gln, Glu67His, Glu67Tyr, Ser98Tyr, Ser98His, Ser98Glu, Ser98Gln, Ser98Arg, Ser98Lys, Ser99Tyr, Ser99His, Ser99Glu, Ser99Gln, Ser99Arg, Ser99Lys; or expressing a nucleic acid molecule encoding the protein or polypeptide, preferably expressed in the host cell of any one of claims 5 or 6.

9. Use of the protein according to any one of claims 1 to 3, the nucleic acid according to claim 4, or the host cell according to claim 5 or 6: Producing biofuels; Production of fine chemicals; Determination of biological agents used to kill actinomycetes; Determine reagents that reduce the production of mycotoxins by actinomycetes; Determining the effectiveness of fungicides.

10. A computer simulation method for identifying molecules capable of selectively altering steps in type I FAS-catalyzed fatty acid synthesis, the method comprising: Identify and simulate at least one amino acid in the ACP domain corresponding to amino acid positions 5-11, 20-25, 34-105, and 135-147 of SEQ ID No. 7; and / or in the ER domain corresponding to amino acid positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 of SEQ ID No. 1; and / or in the AT domain corresponding to amino acid positions 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 of SEQ ID No. 2; and / or in the KS domain corresponding to amino acid positions 68-131 of SEQ ID No. 3; and / or in the DH domain... A molecule in which at least one amino acid in the domains corresponds to the amino acid positions 189-310 of SEQ ID No. 4; and / or in the KR domain, corresponds to the amino acid positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, 602-643 of SEQ ID No. 5; and / or in the MPT domain, corresponds to the amino acid positions 1-30, 48-170, 242-248, 293-306, 325-335, 353-358, 396-415 of SEQ ID No. 6; wherein the positions shown in ii) indicate the amino acid in each domain that participates in the interaction between ACP and type I FAS protein or polypeptide, and the amino acid substitution of the type I FAS protein or polypeptide is not located within the catalytic center of the corresponding type I FAS protein or polypeptide.

11. The computer simulation method according to claim 10, comprising determining and simulating at least one amino acid substitution at positions 40-49, 51-70, and 72-105 of SEQ ID No. 7 in the ACP domain under physiological conditions; and / or at least one amino acid substitution at positions 40-45, 65-70, 92-109, 126-140, 160-170, 175-188, 314-324, 290-301, and 467-477 of SEQ ID No. 1 in the ER domain; and / or at least one amino acid substitution at positions 163, 274, 364, 401, 403, 405, 406, and 428 of SEQ ID No. 2 in the AT domain; and / or at least one amino acid substitution at positions SEQ ID No. 7 in the KS domain. The molecule comprising: at least one amino acid substitution at positions 95-102 and 109-130 of SEQ ID No. 3; and / or an amino acid substitution in the DH domain corresponding to position 198 of SEQ ID No. 4; and / or an amino acid substitution in the KR domain corresponding to positions 39-62, 178-184, 354-364, 372-392, 409-413, 545-557, and 603-642 of SEQ ID No. 5; and / or an amino acid substitution in the MPT domain corresponding to positions 20-28, 48-64, 81-96, and 102-110 of SEQ ID No.

6.

12. A computer simulation method for identifying molecules capable of selectively altering the activity of enzyme domains present in FAS, wherein the identified and modeled molecules, under physiological conditions, exhibit at least one amino acid corresponding to amino acid positions 32-140, 159-190, 211-220, 239-250, 288-329, 381-390, 450-480, and 500-505 of SEQ ID No. 1 in the ER domain; and / or at least one amino acid corresponding to amino acid positions 5-141, 162-166, 273-277, 362-368, 291-430, and 542-554 of SEQ ID No. 2 in the AT domain; and / or at least one amino acid corresponding to amino acid positions 68-131 of SEQ ID No. 3 in the KS domain; and / or at least one amino acid corresponding to amino acid positions 4 of SEQ ID No. 5 in the DH domain. The interaction of the amino acids at positions 189-310; and / or at positions 36-66, 176-187, 353-417, 442-460, 498-520, 543-560, and 602-643 of SEQ ID No. 5 in the KR domain; and / or at least one amino acid at positions 1-30, 48-170, 242-276, 293-306, 325-335, 353-358, and 396-415 of SEQ ID No. 6 in the MPT domain; and / or at least one amino acid at positions 5-11, 20-25, 34-105, and 135-147 of SEQ ID No. 7 in the ACP domain; thereby resulting in at least a change in the enzyme activity in the FAS.

13. The computer simulation method according to claim 12 for identifying molecules capable of selectively altering the activity of enzyme domains present in FAS, wherein the identified and modeled molecules exhibit, under physiological conditions, at least one amino acid substitution at positions 40-49, 51-70, and 72-105 of SEQ ID No. 7 in the ACP domain; and / or at least one amino acid substitution at positions 40-45, 65-70, 92-109, 126-140, 160-170, 175-188, 314-324, 290-301, and 467-477 of SEQ ID No. 1 in the ER domain; and / or at least one amino acid substitution at positions 163, 274, 364, 401, 403, 405, 406, and 428 of SEQ ID No. 2 in the AT domain; and / or at least one amino acid substitution at positions 163, 274, 364, 401, 403, 405, 406, and 428 of SEQ ID No. 7 in the KS domain. The interaction of at least one amino acid substitution at positions 95-102 and 109-130 of SEQ ID No. 3; and / or in the DH domain, an amino acid substitution corresponding to position 198 of SEQ ID No. 4; and / or in the KR domain, an amino acid substitution corresponding to positions 39-62, 178-184, 354-364, 372-392, 409-413, 545-557, and 603-642 of SEQ ID No. 5; and / or in the MPT domain, an interaction of at least one amino acid substitution corresponding to positions 20-28, 48-64, 81-96, and 102-110 of SEQ ID No.

6.

14. A method for identifying compounds capable of altering the activity of at least one enzyme in FAS, the method comprising contacting a candidate compound with FAS and determining whether the candidate compound (i) interacts with at least one amino acid as defined in any one of claims 10 to 13; and / or (ii) is a competitor of ACP in terms of interacting with at least one amino acid in SEQ ID No. 1 to 6 as defined in any one of claims 10 to 14.

15. A protein having the following characteristics: (i) a mutation of at least one amino acid at the positions of amino acids 5-11, 20-25, 34-105, and 135-147 corresponding to SEQ ID No. 7; or (ii) not containing the amino acid sequence of positions 1 to 79 of SEQ ID No.

7.

16. The protein according to claim 15, characterized by: i) at least one amino acid substitution mutation occurring at the positions of amino acids 40-49, 51-70, and 72-105 of SEQ ID No. 7 in the ACP domain; or ii) consisting of amino acids 80 to 169 of SEQ ID No.

7.

17. The protein according to claim 15 or 16, which is used as a fungicide or actinomycete biocide, or for inhibiting the enzymatic activity of at least one enzyme in fungal FAS.

18. The method according to any one of claims 11 to 15, used for determining fungicides.

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