Light-operated pyruvate decarboxylase OptoPdc1 and application thereof

Abstract

The invention provides a light-operated pyruvate decarboxylase OptoPdc1 and a saccharomyces cerevisiae light-operated strain constructed on the basis of OptoPdc1. The light-operated pyruvate decarboxylase OptoPdc1 and the saccharomyces cerevisiae light-operated strain disclosed by the invention have the characteristics of safety, no toxicity and easiness in control; the light-operated saccharomyces cerevisiae strain disclosed by the invention can realize bidirectional dynamic regulation and control. The invention further provides application of the light-operated saccharomyces cerevisiae strain in metabolic pathway control.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein engineering or synthetic biology in biotechnology, in particular to a novel light-controlled pyruvate decarboxylase OptoPdc1, a light-controlled Saccharomyces cerevisiae strain constructed by using OptoPdc1, and application of the light-controlled Saccharomyces cerevisiae strain in metabolic engineering. BACKGROUND

[0002] Saccharomyces cerevisiae is a recognized food safety model organism, with clear genetic background, perfect molecular manipulation system, excellent fermentation performance and rich metabolic pathways, and is one of the preferred chassis cells in metabolic engineering, which has a wide range of applications in food, chemical, pharmaceutical, fuel and other fields. Metabolic engineering aims to precisely regulate the distribution of material flow and energy flow in cell metabolism, and to modify and transform the cell network of organisms, so as to realize the directional modification of metabolic characteristics, and to improve the biosynthesis and manufacturing capacity of target products. In the practice of metabolic engineering, improving metabolic imbalance through metabolic pathway regulation is a common strategy, including enhancing precursor supply and substrate or product transport through gene overexpression, knocking out competing pathways and overexpressing related genes of target pathways, etc. These regulation strategies belong to static regulation strategy, which is the most studied and widely used regulation strategy in metabolic engineering. However, at the genetic level, these regulations are permanent and irreversible. Overexpression of target genes at too low or too high levels will cause a series of problems such as insufficient metabolic flow or accumulation of intermediate products, which will have irreversible side effects on cell growth and thus affect the synthesis of target products. In addition, since there is no clear ratio between production pathways and competing pathways, these strategies cannot effectively balance the relationship between competing pathways necessary for cell growth and target product production pathways, so as to make the production of target products reach the optimal level.

[0003] There are various dynamic regulation strategies (RNA, DNA and protein levels) in living organisms in nature, which are used to regulate the cell activities of the organisms themselves. The dynamic regulation process is based on specific signal responses, and the cells can be regulated in real time according to the changes of specific signals to achieve the optimal state of synthesis of target products. According to the types of specific signals, the dynamic regulation strategies are mainly divided into three categories: metabolite response regulation strategy, quorum sensing response regulation strategy and fermentation condition response regulation strategy. The metabolite response and quorum sensing response regulation strategies both face the problems of promoter response threshold and response substance type limitation. In contrast, the fermentation condition response dynamic regulation strategy takes temperature, pH, light and medium composition as input signals, and has the advantages of controllability, universality and reversibility. Among them, the regulation strategy based on light has the advantages of small cell toxicity, fast signal transmission speed, high spatial and temporal resolution and low cost, and thus becomes an ideal regulation strategy. However, the dynamic regulation means in metabolic engineering is mainly concentrated in the RNA and DNA levels, and the dynamic regulation strategy at the protein level is relatively less. SUMMARY

[0004] In view of the above problems that the dynamic regulation strategy at the protein level in the Saccharomyces cerevisiae cell is less, and the existing metabolite response and quorum sensing response regulation strategies both face the problems of promoter response threshold and response substance type limitation, the present application primarily provides a novel light-controlled pyruvate decarboxylase OptoPdc1, and develops a light-controlled Saccharomyces cerevisiae strain based on the OptoPdc1, realizes the dynamic control of the Saccharomyces cerevisiae metabolic pathway by controlling light / dark adjustment, and has excellent characteristics in the synthesis of isobutanol.

[0005] In one aspect, the present application provides a light-controlled pyruvate decarboxylase comprising a pyruvate decarboxylase Pdcl and a LOV domain inserted between adjacent amino acids of Loop-1 or Loop-2 of the Pdcl.

[0006] In certain embodiments, the LOV domain is a LOV2 domain; preferably, the LOV domain is selected from the LOV2 domain of the oat photoreceptor 1 gene (AsLOV2), the photoreceptor protein EL222 from Rhodobacter sphaeroides, the photoreceptor domain LOV2 from Arabidopsis thaliana, or a mutant thereof; preferably, the mutant is a cyclically permuted mutant or a mutant that alters the speed of response; preferably, the LOV domain is AsLOV2, e.g., comprising the sequence set forth in SEQ ID NO: 1; preferably, the LOV domain is cpLOV2, e.g., comprising the sequence set forth in SEQ ID NO: 2; preferably, the LOV domain is cpLOV16, e.g., comprising the sequence set forth in SEQ ID NO: 3; preferably, the LOV domain is cpLOV27, e.g., comprising the sequence set forth in SEQ ID NO: 4.

[0007] In certain embodiments, the insertion position is selected from the following amino acid positions of the Pdc1 protein: between positions 186 and 187, between positions 188 and 189, between positions 189 and 190, between positions 190 and 191, between positions 191 and 192, between positions 192 and 193, between positions 304 and 305, between positions 305 and 306; preferably, the insertion position is between positions 188 and 189, between positions 192 and 193 of the pyruvate decarboxylase.

[0008] In certain embodiments, the insertion position is selected from the following amino acid positions of the Pdc1 protein: between positions 188 and 189, between positions 192 and 193.

[0009] In certain embodiments, the Pdc1 protein is wild type; preferably, the wild type Pdc1 protein comprises the sequence set forth in SEQ ID NO: 6.

[0010] In certain embodiments, the N-terminus and / or the C-terminus of the LOV domain is optionally linked to the Pdc1 protein via a peptide linker.

[0011] In certain embodiments, the light-controlled pyruvate decarboxylase comprises the sequence set forth in any one of SEQ ID NOs: 7-22; preferably, the light-controlled pyruvate decarboxylase comprises the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 9 or SEQ ID NO: 20; preferably, the light-controlled pyruvate decarboxylase comprises the sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 20.

[0012] In one aspect, the present application provides a nucleic acid construct comprising a nucleotide sequence encoding a light-controlled pyruvate decarboxylase as described above.

[0013] In certain embodiments, the nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled pyruvate decarboxylase; preferably, the promoter is selected from the group consisting of TEF promoter, GPD promoter, ADH1 promoter. The nucleic acid construct further comprises a terminator operably linked to the nucleotide sequence encoding the light-controlled pyruvate decarboxylase; preferably, the terminator is selected from the group consisting of ACT1 terminator, ADH1 terminator.

[0014] In one aspect, the present application provides a host cell comprising the nucleic acid construct described above; preferably, the host cell is a eukaryotic cell; preferably, the host cell is Saccharomyces cerevisiae.

[0015] In certain embodiments, the host cell is Saccharomyces cerevisiae, which has integrated into the genome an exogenous nucleotide sequence encoding the light-controlled pyruvate decarboxylase described above, preferably, the endogenous Pdcl, Pdc5, Pdc6 genes of the Saccharomyces cerevisiae are disrupted or knocked out; preferably, one copy of the exogenous nucleotide sequence is integrated into the genome of the Saccharomyces cerevisiae; preferably, multiple copies of the exogenous nucleotide sequence are integrated into the genome of the Saccharomyces cerevisiae.

[0016] In certain embodiments, the Saccharomyces cerevisiae has integrated into the genome an exogenous nucleotide sequence encoding the light-controlled pyruvate decarboxylase described above, which is operably linked to a promoter and a terminator at both ends of the exogenous nucleotide sequence; preferably, the promoter is selected from the group consisting of TEF promoter, GPD promoter, ADH1 promoter; preferably, the terminator is selected from the group consisting of ACT1 terminator, ADH1 terminator.

[0017] In certain embodiments, the host cell is a eukaryotic cell; preferably, the host cell is Saccharomyces cerevisiae.

[0018] In one aspect, the present application provides the light-controlled pyruvate decarboxylase, the nucleic acid construct, the host cell described above, for use in a dynamic regulation strategy of Saccharomyces cerevisiae; preferably, the use comprises regulating metabolic pathways and / or biosynthesis.

[0019] The application provides a novel light-controlled pyruvate decarboxylase OptoPdc1, which meets the requirements of single-component regulation of metabolic pathways and can realize high-time and high-spatial resolution activity regulation. The application is based on a light-controlled Saccharomyces cerevisiae strain constructed by the light-controlled pyruvate decarboxylase OptoPdc1, and the production efficiency of a target product can be regulated by changing the frequency and intensity of blue light irradiation. The light-controlled Saccharomyces cerevisiae strain of the application has strict production advantages and less leakage. The light-controlled Saccharomyces cerevisiae strain of the application has excellent application effect in metabolic regulation and has more strict regulation on metabolic pathways. The target product synthesized by dark induction is equivalent to that of a non-light-controlled strain. BRIEF DESCRIPTION OF DRAWINGS

[0020] The application is further described in the following non-limiting drawings:

[0021] Figure 1 : Screening results of light-controllable pyruvate decarboxylase mutants in Loop-1

[0022] Figure 2 : Optimization results of light-controllable pyruvate decarboxylase mutants in Loop-1

[0023] Figure 3 : Screening and optimization results of light-controllable pyruvate decarboxylase mutants in Loop-2

[0024] Figure 4 : Construction process and genomic structure diagram of a light-controlled Saccharomyces cerevisiae strain

[0025] Figure 5 : Growth curve of a light-controlled Saccharomyces cerevisiae strain

[0026] Figure 6 : Fermentation results of a light-controlled Saccharomyces cerevisiae strain for producing isobutanol

[0027] Figure 7 : Fermentation results of a light-controlled Saccharomyces cerevisiae strain for producing isobutanol under different doses of blue light DETAILED DESCRIPTION

[0028] Definitions

[0029] Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. For better understanding of the application, the definitions and explanations of relevant terms are provided as follows.

[0030] As used herein, the term "comprising" and variations thereof as used herein are used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used whilst describing various embodiments, the terms "consisting essentially of and "consisting of can be used in place of "comprising" and "including" to provide for more particular embodiments and are also disclosed.

[0031] As used herein, the articles "a", "an", and "the" refer to "at least one" unless otherwise expressly specified by the context.

[0032] As used herein, the terms "pyruvate decarboxylase 1" or "Pdcl protein" have the same meaning and are used interchangeably. The sequence of the Pdcl protein is well known to those skilled in the art and can be found in various public databases, for example, GenBank: GHM88437.1. In this document, when referring to the amino acid position of the Pdcl protein, the reference is made to the sequence set forth in SEQ ID NO: 6. For example, the expression "the amino acid residue at position 188 of the Pdcl protein" refers to the amino acid residue at position 188 of the sequence set forth in SEQ ID NO: 6 and its corresponding position. The corresponding position refers to the position in the sequence to be compared that is equivalent to the particular amino acid position in SEQ ID NO: 6 upon optimal alignment of the sequence to be compared with SEQ ID NO: 6 (i.e., to obtain the highest percentage identity).

[0033] As used herein, the term "LOV domain" refers to a light- oxygen-voltage (LOV) domain, typically derived from a photoreceptor protein of a plant, alga or bacterium. Preferably, the LOV domain is a LOV2 domain, preferably from Avena sativa, Rhodobacter sphaeroides or Arabidopsis thaliana. In certain exemplary embodiments, the LOV domain has the amino acid sequence set forth in SEQ ID NO: 1, 23 or 24 or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0034] As used herein, the term "circular permutation" refers to the linking of the original N- and C-termini of a protein by a linker and the introduction of new termini by cleavage of the original peptide bond of the protein. The new protein thus formed is called a circularly permuted mutant.

[0035] As used herein, the term "gene" is used broadly to refer to a DNA nucleic acid associated with a biological function.

[0036] As used herein, the term "operably linked" refers to the functional linkage of an element to another, wherein the elements are in a relationship that allows them to function in the intended manner. For example, with respect to a promoter, the functional linkage between the promoter and a gene of interest such that the promoter sequence is capable of initiating transcription of the gene of interest.

[0037] As used herein, the term "promoter" refers to an expression control element that permits binding of RNA polymerase and initiation of transcription.

[0038] As used herein, the term "host cell" refers to a cell that can be used for the introduction of a vector, including but not limited to, eukaryotic cells, prokaryotic cells, insect cells, mammalian cells, and plant cells.

[0039] Light-controlled pyruvate decarboxylase

[0040] Based on the following insights and findings, the present application was completed through the inventors' research, and specifically provides the following aspects.

[0041] Pyruvate decarboxylase (Pdcl) is a key enzyme in the process of alcohol fermentation, which is involved in the non-oxidative conversion of pyruvate to acetaldehyde and carbon dioxide. Pdcl has a homotetramer structure, each monomer consists of 563 amino acid residues, including α, β and γ three functional domains, the domains are connected by long flexible loop region, the α domain at the N terminal and the γ domain at the C terminal are involved in the binding of ThDP, Mg2+ and NADH, and the β domain is involved in the binding of the substrate pyruvate. 2+and the beta domain of the core region is a regulatory domain, containing the binding site of activators such as pyruvamide, and is involved in the activation of the enzyme by substrates. The light-oxygen-voltage sensing domain 2 (LOV2) domain has a small molecular weight (100-140 amino acids), and its chromophore flavin mononucleotide (FMN) is ubiquitous in various types of cells, so LOV2 is a widely used light control element. In various types of cells, the LOV2 domain has been applied to the reversible, spatial-temporal manipulation of cell signaling and behavior, including the reversible control of intracellular protein degradation, protein-protein interaction, and protein localization. The C-terminus of the LOV2 domain contains a Ja helix, which forms a stable conformation between the Ja helix and the beta sheet of the Per-Arnt-Sim (PAS) core domain under dark conditions; under blue light irradiation, covalent binding occurs between the conserved cysteine (Cysteine) and FMN in the LOV2 domain, causing the rotation of the conserved glutamine (Glutamine) residue, ultimately leading to the change of Ja helix conformation and the separation between the central beta sheet. Based on the pyruvate decarboxylase (Pdcl) and the light control element LOV2, we have the possibility to fuse them and construct a new light-controllable pyruvate decarboxylase, which can realize the dynamic regulation of carbon flux from ethanol synthesis to target product synthesis by controlling the activity of PDC.

[0042] In one aspect, the present application provides a light-controllable pyruvate decarboxylase comprising a Pdcl protein and a LOV domain inserted between adjacent amino acids of Loop-1 or Loop-2 region of the Pdcl protein.

[0043] In certain embodiments, the Loop-1 region of the Pdcl protein refers to amino acid positions 186-192; and the Loop-2 region refers to amino acid positions 304-305. In this context, when referring to the amino acid positions of the Pdcl protein, reference is made to the sequence shown in SEQ ID NO: 6.

[0044] In certain embodiments, the N-terminus and / or C-terminus of the LOV domain is directly linked to the Pdcl protein.

[0045] In certain embodiments, the N-terminus and / or C-terminus of the LOV domain is linked to the Pdcl protein via a peptide linker (e.g., a peptide linker comprising one or several flexible amino acids).

[0046] LOV domain

[0047] Any LOV domain known in the art can be used in the present application. In certain embodiments, the LOV domain is a LOV2 domain. LOV2 domains from different species are all photoreceptor domains that play the same role in different species; and, the three-dimensional structures of LOV2 domains from different species are highly similar in structure and the corresponding allosteric mechanisms are the same. Therefore, LOV2 domains from different species are all suitable for use in the present application.

[0048] In certain embodiments, the LOV domain is selected from the LOV2 domain of the oat photoreceptor 1 gene (AsLOV2), the photoreceptor EL222 from Rhodobacter sphaeroides, the photoreceptor domain LOV2 from Arabidopsis thaliana, or a mutant thereof.

[0049] In certain embodiments, the mutant is a circularly permuted mutant. In this context, "circularly permuted" has its meaning well known to those skilled in the art, which refers to the original N-terminus and C-terminus of a protein being linked by a linker and new termini being introduced by cleaving the original peptide bond of the protein.

[0050] In certain embodiments, the mutant is a mutant that changes the speed of response. Such mutants are known to those skilled in the art and typically can comprise a mutation selected from the group consisting of V461I, V74I, L165I, the amino acid positions referring to positions in AsLOV2, the corresponding positions in other LOV domains can be determined by sequence alignment (see, e.g., Zoltowski, B. D., B. Vaccaro, and B. R. Crane, Mechanism-based tuning of a LOV domain photoreceptor. Nature Chemical Biology, 5(11): 827-834 (2009)). Introducing mutations into LOV2 that change the speed of response does not change the mechanism and fact that LOV2 responds to blue light with an allosteric change, and thus these mutants are also suitable for use in the present application.

[0051] In certain embodiments, the LOV domain is AsLOV2. In certain embodiments, the AsLOV2 comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0052] In certain embodiments, the LOV domain is a circularly permuted mutant of AsLOV2. In certain embodiments, the circularly permuted mutant of AsLOV2 is cpLOV2, cpLOV16, or cpLOV27, which comprises the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0053] In certain embodiments, the LOV domain is a light-sensitive protein EL222 of Rhodopseudomonas palustris, for example comprising the amino acid sequence set forth in SEQ ID NO: 23.

[0054] In certain embodiments, the LOV domain is a light-sensing domain LOV2 of Arabidopsis thaliana, for example comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0055] Pdc1 protein

[0056] In certain embodiments, the Pdc1 protein is a wild-type Pdc1 protein. In certain embodiments, the wild-type Pdc1 protein comprises the sequence set forth in SEQ ID NO: 6.

[0057] The light-controlled pyruvate decarboxylase of the present application can be produced by various methods known in the art, for example, by a genetic engineering method (recombinant technology), or by a chemical synthesis method (for example, Fmoc solid phase method). The light-controlled pyruvate decarboxylase of the present application is not limited by the way it is produced.

[0058] Nucleic acid construct

[0059] In another aspect, the present application provides a nucleic acid construct comprising a nucleotide sequence encoding the light-controlled repressor protein of the present application.

[0060] In certain embodiments, the nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controlled pyruvate decarboxylase of the present application. In certain embodiments, the promoter comprised by the nucleic acid construct can be any promoter recognized by the RNA polymerase encoded by the RNAP gene comprised in the host chromosome, that is, as long as it uses the RNA polymerase of the host cell. In certain embodiments, the promoter is selected from the group consisting of TEF promoter, GPD promoter, ADH1 promoter.

[0061] In certain embodiments, the nucleic acid construct further comprises a terminator operably linked to the nucleotide sequence encoding the light-controlled pyruvate decarboxylase, the terminator being selected from the group consisting of ACT1 terminator, ADH1 terminator.

[0062] Host cell

[0063] In another aspect, the present application provides a host cell comprising the nucleic acid construct as described above.

[0064] In certain embodiments, the host cell is a eukaryotic cell.

[0065] In certain embodiments, the host cell is S. cerevisiae.

[0066] In certain embodiments, the host cell is S. cerevisiae, which has integrated into its genome an exogenous nucleotide sequence encoding a light-controlled pyruvate decarboxylase of the present application.

[0067] In certain embodiments, the endogenous Pdcl gene of the S. cerevisiae is disrupted. In certain embodiments, the disruption comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases), is deleted, or is replaced with an exogenous nucleotide sequence. A "loss-of-function mutation" refers to a mutation that results in a protein encoded and expressed by the mutated gene losing its biological functional activity. A loss-of-function mutation includes, but is not limited to, missense mutation, nonsense mutation, frameshift mutation, base deletion, base substitution, base addition, and any combination thereof (e.g., deletion or replacement or addition of a gene fragment), as long as the gene comprising the loss-of-function mutation cannot produce or express a protein with biological functional activity.

[0068] In certain embodiments, the endogenous Pdcl, Pdcs, Pdc6 genes of the S. cerevisiae are all knocked out, and one or more copies of the exogenous nucleotide sequence are integrated into its genome.

[0069] In certain embodiments, the integration of the exogenous nucleotide sequence and / or the disruption of the endogenous gene by the host cell can be achieved by any gene editing system well known to one skilled in the art. Exemplary gene editing systems include CRISPR / Cas, ZFN, TALEN, and the like.

[0070] In certain embodiments, the S. cerevisiae having integrated into its genome an exogenous nucleotide sequence encoding a light-controlled pyruvate decarboxylase of the present application can further comprise the nucleic acid construct described above.

[0071] Methods for metabolic pathway control

[0072] In another aspect, the present application provides the use of the light-controlled pyruvate decarboxylase, the nucleic acid construct, the host cell of the present application for modulating a metabolic pathway. The modulating a metabolic pathway is performed in vitro. The use is for non-therapeutic purposes.

[0073] It is known to one skilled in the art that S. cerevisiae can produce some important compounds through anabolism, and thus by modulating the anabolic pathway in S. cerevisiae cells, the amount of synthesis of the metabolite can be changed (e.g., increased). Therefore, the light-controlled pyruvate decarboxylase, the nucleic acid construct, and the host cell of the present application can be used for modulating a metabolic pathway.

[0074] In certain embodiments, the metabolic pathway can be endogenous or exogenous to the S. cerevisiae, including but not limited to the production pathway of isobutanol. Thus, in certain embodiments, the metabolic pathway involved in the present application also includes metabolic pathways involved in the biosynthesis or microbial synthesis of a compound.

[0075] In another aspect, the present application provides a method for regulating a metabolic pathway, comprising:

[0076] (1) providing a light-controllable pyruvate decarboxylase for regulating a metabolic pathway of the present application in a host cell;

[0077] (2) culturing the host cell under conditions allowing expression of the metabolic pathway, and regulating the activity of the light-controllable pyruvate decarboxylase.

[0078] In certain embodiments, step (1) comprises: providing a nucleic acid construct comprising the system; introducing the plasmid containing the target metabolic pathway into a host cell, wherein the host cell comprises an exogenous nucleotide sequence encoding the light-controllable pyruvate decarboxylase of the present application integrated in its genome.

[0079] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell is S. cerevisiae. In certain embodiments, the host cell is S. cerevisiae having the exogenous nucleotide sequence encoding the light-controllable pyruvate decarboxylase expression cassette integrated in its genome. In certain embodiments, the endogenous Pdc 1, Pdc5 or Pdc6 gene of the S. cerevisiae is disrupted. In certain embodiments, the endogenous Pdc 1 gene of the S. cerevisiae is replaced by the exogenous nucleotide sequence. In certain embodiments, one copy of the endogenous Pdc 1 gene of the S. cerevisiae is replaced by the exogenous nucleotide sequence.

[0080] In certain embodiments, the host cell is a eukaryotic cell. In certain embodiments, the host cell is S. cerevisiae.

[0081] In certain embodiments, the nucleic acid construct and the genes of the target metabolic pathway are located in different expression cassettes. In certain embodiments, the nucleic acid construct further comprises a promoter operably linked to the nucleotide sequence encoding the light-controllable pyruvate decarboxylase; preferably, the promoter is any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, i.e. as long as it uses the RNA polymerase of the host cell, preferably the promoter is selected from the group consisting of TEF promoter, GPD promoter, ADH1 promoter.

[0082] In certain embodiments, the step of introducing the plasmid containing the metabolic pathway of interest into the host cell described in any of the above embodiments can be performed by any means known in the art, such as transfection, transformation or transduction.

[0083] In certain embodiments, the plasmid containing the metabolic pathway of interest described in any of the above embodiments, wherein the promoter is any promoter recognized by the RNA polymerase encoded by the RNAP gene contained in the host chromosome, i.e. as long as it uses the RNA polymerase of the host cell, preferably selected from TEF promoter, GPD promoter, ADH1 promoter.

[0084] In certain embodiments, the light-controllable pyruvate decarboxylase is selected from the light-controllable pyruvate decarboxylases OptoPdc1 Dark-V1 or light-controllable pyruvate decarboxylase OptoPdc1 Dark-V2 ; the regulatory conditions in step (2) comprise culturing the host cell under dark condition to accumulate the biomass of the host cell, and fermenting under blue light / dark pulse or blue light condition.

[0085] In certain embodiments, the blue light described herein refers to light with wavelength between 430-495 nm (e.g. 450-480 nm). The blue light described herein can be emitted by a light source, including LED light board, light strip, light tube, etc.

[0086] Examples

[0087] Those skilled in the art will appreciate that the examples describe the application in terms of preferred embodiments, and that the application is not intended to be limited to the preferred embodiments. The experimental methods in the examples are conventional unless otherwise specified. The specific conditions in the examples are conventional or as recommended by the manufacturer unless otherwise specified. The reagents or instruments used are conventional products available on the market unless otherwise specified.

[0088] Example 1: Screening of light-controllable pyruvate decarboxylase mutants in Loop-1

[0089] To facilitate the screening of light-controllable pyruvate decarboxylases, we used a strain (YZM-1) with deletion of pyruvate decarboxylases (Δpdc1, Δpdc5, Δpdc6) as the chassis strain. Restriction enzymes XmaI and AscI were used to perform double digestion on plasmids pYZ285 and pYZ125, respectively (Table 1), to obtain linearized P TEF1 -PDC1-T ACT1The expression cassette gene fragment and linearized vector fragment were ligated using T4 DNA ligase (Table 2), and after identification by bacterial liquid PCR and sequencing verification, the plasmid pML1 carrying the wild-type PDC1 expression cassette was successfully obtained. Using this plasmid as the starting plasmid, the Gibson assembly technique was used to insert the light control element AsLOV2 or cpLOV2 sequence at the candidate site, thereby obtaining a series of plasmids carrying PDC1-LOV2 / cpLOV2 mutants. Among them, the candidate site was obtained by comprehensive analysis of the structure, function, sequence conservation and solvent accessibility of Pdc1 protein. Both the insertion site of the light control element cannot destroy the structure folding and catalytic function region of Pdc1 itself, and it needs to be located at a site with poor sequence conservation and good solvent accessibility.

[0090] The plasmid containing the PDC1 mutant was transformed into a pyruvate decarboxylase-deficient Saccharomyces cerevisiae strain (Δpdc1, Δpdc5, Δpdc6), and cultured in a medium containing glycerol (SCGE-URA), and the obtained clones were cultured in SCGE-URA liquid medium to ensure that the strain could grow normally. The OD600 of the bacterial liquid was diluted by gradient dilution to 1, 10 600 , 10 -1 , 10 -2 , 10 -3 , 10 -4 μL of the diluted bacterial liquid was spotted on two solid media containing glucose as the carbon source (SC-URA), and the obtained two plates were cultured under blue light irradiation conditions (40 μmol / m 2 / s) and dark conditions. By comparing the growth performance of the colonies under light and dark conditions, positive clones with growth differences were selected. At the same time, we took 3 μL of gradient-diluted bacterial liquid and spotted it on plates containing glycerol and ethanol (SCGE-URA) as a control group.

[0091] Table 1, double enzyme digestion system component table (50 μl)

[0092] Reagent Volume (μl) 10X CutSmart Buffer 5 pYZ285 / pYZ125 (1.5 μg each) X XmaI 1 AscI 1 Add ddH2O To 50

[0093] Table 2, T4 DNA ligation system component table (10 μl)

[0094]

[0095]

[0096] The results show that there is no significant difference in the growth of Pdc1 in SC-URA and SCGE-URA medium without blue light irradiation; in SC-URA medium, the growth of Pdc1 is almost unaffected whether or not irradiated with blue light Figure 1). This result indicates that the wild type Pdc1 catalytic activity is not affected by blue light, and the S. cerevisiae with wild type Pdc1 catalytic activity can grow normally in the medium with glucose as carbon source, which verifies the feasibility of the screening method based on growth phenotype.

[0097] In addition, we successfully screened a series of strains with response to blue light, and the growth of these strains under dark condition is better than that under blue light illumination Figure 1 ), and we collectively call these Pdc1 mutants with better growth under dark condition than that under blue light illumination as OptoPdc1 Dark In loop1, we successfully achieved the light control effect by introducing AsLOV2, and the effective insertion sites include D186, S188, L189, K190, P191 and N192. In particular, the light control effect of S188, L189 and N192 sites inserted with AsLOV2 is particularly significant.

[0098] Example 2: Optimization of the light-controllable pyruvate decarboxylase mutants in Loop-1

[0099] In order to obtain pyruvate decarboxylase mutants with better light control performance, we carried out a series of optimization experiments based on the light-controllable pyruvate decarboxylase mutants screened in Loop-1, including the optimization of Linker, i.e. adding glycine (G), glycine-serine (GS), glycine-serine-glycine (GSG) between the light control element and Pdc1 in S188-AsLOV2 mutant or L189-AsLOV2 mutant, and the optimization of the type of light control element, i.e. replacing AsLOV2 with cyclic permutation mutants at the corresponding insertion sites, including cp2, cp6, cp16 and cp27. The optimized plasmids were transformed into pyruvate decarboxylase deficient S. cerevisiae strains (Δpdc1, Δpdc5, Δpdc6) respectively, and the same screening method as in Example 1 was used for testing. The results show that compared with S188, the overall growth ability of the mutants S188-G, S188-GS and S188-GSG with added linker in SC-URA medium under dark condition is improved, and with the increase of the length of the linker, the growth ability is also gradually enhanced, but at the same time, the “leakage” phenomenon (i.e. the phenomenon that the growth under blue light condition which should inhibit the growth) is also increasingly obvious Figure 2 ). The experimental results show that a linker with appropriate length is beneficial to the optimization of light control effect. In summary, the S188-GS mutant exhibits better light control performance.

[0100] In addition, based on the S188-GS mutant, we tested the light control effect of different cpLOV2 mutants (Figure 2 ). Different cpLOV2 mutants showed significant differences in light control ability at this site. Based on the mutant L189-AsLOV2, we tested the light control effect of different cpLOV2 mutants. The experimental results showed that the strain carrying the L189-cp2 mutant showed more obvious growth difference and light control ability under dark and blue light conditions.

[0101] Example 3: Screening and optimization of light-controllable pyruvate decarboxylase mutants in Loop-2

[0102] In order to screen pyruvate decarboxylase mutants with good light control performance, we made AsLOV2 insertion and linker optimization in the amino acid sites of Loop-2. The experimental results showed that after inserting AsLOV2 at K304 and N305 amino acid sites, the strain could not grow at all on SC-URA medium. After adding serine-glycine (SG) and glycine-serine (GS) at both ends of AsLOV2, the mutant strain showed obvious growth difference between dark and blue light conditions Figure 3 ), which again verified the benefit of appropriate length of linker for optimizing light control effect.

[0103] In summary, we screened 17 mutants with light response effect. These mutants showed higher catalytic activity under dark conditions than under blue light irradiation, so they were collectively referred to as OptoPdc1 Dark Among them, S188-GS and L189-cp2 are two mutants with better light control effect, which are named OptoPdc1-V1 and OptoPdc1-V2, respectively, for subsequent research.

[0104] Example 4: Construction of light-controllable Saccharomyces cerevisiae strains

[0105] In order to ensure the genetic stability of the light-controllable Saccharomyces cerevisiae strain and avoid the loss and instability of the plasmid, we integrated the expression cassettes of the two mutants OptoPdc1-V1 and OptoPdc1-V2 and the wild-type Pdc1 into the HIS3 site of the Saccharomyces cerevisiae genome Figure 4). The gene integration adopted a homologous recombination strategy, and the specific steps were as follows: (1) amplification of gene integration fragments: using plasmids pML370 (carrying wild-type Pdc1-CgHIS expression cassette), pML371 (carrying OptoPdc1-V1-CgHIS expression cassette) and pML372 (carrying OptoPdc1-V2-CgHIS expression cassette) as templates, primers MZL_Oli_691 and MZL_Oli_692 were used for PCR amplification to obtain the expression frame required for gene integration with 500 bp homologous arms at both ends. (2) Transformation and selection: the amplified integration fragments were transformed into YMZ-1 strain, and uniformly coated on SCGE-HIS plates. Single colonies were picked, and the genome was extracted and identified by PCR to obtain gene integration successful strains YMZ-2, YMZ-3 and YMZ-4. (3) Screening marker loop-out: the plasmid B222 with Cre recombinase was transformed into YMZ-2, YMZ-3 and YMZ-4 strains, cultured on SCGE-URA plates, and further verified the screening marker loop-out condition using SCGE and SCGE-HIS plates to obtain strains YMZ-5, YMZ-6 and YMZ-7. (4) Eliminate recombinant plasmid: two rounds of streak culture were performed on SCGE plates containing 5-FOA, and SCGE and SCGE-URA plates were used for verification. Strains that only grew on SCGE plates but not on SCGE-URA plates were the desired positive strains. (5) Sequencing verification: the genome of the positive strain was used as a template, and primers MZL_Oli_691 and MZL_Oli_685 were used for PCR amplification and sequencing. The correct sequencing result was the target strain. Finally, the control Saccharomyces cerevisiae strain YMZ-8 and the light-controlled Saccharomyces cerevisiae strains YMZ-9 and YMZ-10 were obtained.

[0106] Example 5: Characterization of growth characteristics of light-controlled Saccharomyces cerevisiae strains

[0107] To further analyze the growth characteristics of the light-controlled strains, we characterized their growth curves in SC medium. The control strains YMZ-8 (Pdc1), YMZ-9 (OptoPdc1-V1) and YMZ-10 (OptoPdc1-V2) were measured for their growth curves. After being cultured in 24-well plates (1 mL, SCGE medium) to the late logarithmic growth phase, they were then transferred to two new 24-well plates (1 mL, SC medium) at an inoculation amount of 2%, and placed in blue light and darkness, respectively, at 30°C, 200 rpm, and continued to culture for 48 h. The cell density OD 600. The results show that there is no significant difference in the growth of the control strain YMZ-8 under blue light and dark conditions, while the growth trends of the light-controlled strains YMZ-9 and YMZ-10 under blue light and dark conditions show obvious differences, and the growth trend of YMZ-9 and YMZ-10 in the dark environment is always better than that under blue light Figure 5 ), which is consistent with the initial screening results, further confirming that OptoPdc1 has the ability to regulate the growth of S. cerevisiae through a light response mechanism, which has potential application value for regulating the metabolic activity of S. cerevisiae.

[0108] Example 6: Application of light-controlled S. cerevisiae strains in isobutanol synthesis

[0109] In S. cerevisiae, the biosynthetic pathway of isobutanol is a naturally occurring metabolic pathway that can be divided into two main parts: the de novo synthesis pathway in mitochondria and the Ehrlich pathway in cytoplasm. We used the mitochondrial compartmentalization strategy to locate the enzymes in the Ehrlich pathway to mitochondria, so that the entire isobutanol synthesis pathway is concentrated in one organelle, thereby reducing the transport and loss of intermediate metabolites. The plasmid pJA182 carries the key genes of the isobutanol synthesis pathway, and all have mitochondrial targeting signal peptides. We transformed the plasmid pJA182 into three different chassis strains YMZ-8, YMZ-9 and YMZ-10 to obtain strains Ycy6, Ycy7 and Ycy8 for testing the application of light-controlled strains in controlling isobutanol production. First, single colonies of fermentation strains were picked on plates and inoculated into liquid culture medium of SCGE-URA for overnight culture. The next day, we transferred the cultured bacterial liquid to two 24-well plates containing 1 mL of fresh culture medium, adjusted the initial OD 600 to 0.15, and cultured in the dark for 24 h. Then the two well plates were placed in blue light and dark conditions respectively for continuous culture for 4 h. After that, we collected the bacterial cells by centrifugation and resuspended them in 1 mL of fresh SC-URA (containing 15% glucose) medium, and used Sealplate film (MKCT0105) sealing tape to seal the well plates to maintain anaerobic conditions. The well plates were placed in blue light and dark, respectively, to start fermentation, and after 60 h of fermentation, the culture was centrifuged and the supernatant was collected for high performance liquid chromatography (HPLC) analysis. The fermentation results show that blue light has relatively little effect on the fermentation of non-light-controlled Ycy6, with a 1.21-fold difference in isobutanol production between blue light and dark conditions. The light-controlled strain Ycy7 (OptoPdc1-V1) has an isobutanol production in the dark that is 8.50 times that in blue light, and the light-controlled strain Ycy8 (OptoPdc1-V2) has an isobutanol production in the dark that is 4.09 times that in blue light Figure 6). In summary, Ycy7 strain has better photoregulated stringency, while Ycy8 strain has stronger overall production capacity. The results show that the photoregulated strain exhibits significant photosensitivity in isobutanol production, fully verifying the feasibility of using photoregulated strains to control metabolic pathways.

[0110] Example 7: Effect of different doses of blue light on isobutanol synthesis of photoregulated strains

[0111] To characterize the regulatory effect of different doses of blue light on isobutanol synthesis of photoregulated strains, we tested the effect of light pulses of different light cycles on the fermentation of photoregulated strain Ycy8. First, the monoclonal of Ycy8 was inoculated into liquid SCGE-URA medium for overnight culture, and then transferred to a 24-well plate containing 1 mL of fresh SCGE-URA medium for further culture. After 28 h of culture, we collected the bacterial cells by centrifugation and resuspended them in 1 mL of SC-URA (containing 15% glucose) medium. Then, we placed the bacterial solution under different light conditions for fermentation. The fermentation conditions included: (1) 1 s of blue light irradiation per 1000 s, denoted as 1 s ON (1000 s); (2) 10 s of blue light irradiation per 1000 s, denoted as 10 s ON (1000 s); (3) 100 s of blue light irradiation per 1000 s, denoted as 100 s ON (1000 s); (4) continuous blue light irradiation, denoted as Blue; (5) continuous darkness, denoted as Dark. After 72 h of fermentation, the fermentation broth was collected and the isobutanol yield was detected Figure 7 ). The results showed that under continuous blue light irradiation, the isobutanol yield of Ycy8 was the lowest (116 mg / L); under continuous darkness, the yield increased to 319 mg / L.

[0112] When the light condition was 1 s ON (1000 s), the Ycy8 strain produced the most isobutanol (349 mg / L), and as the pulse condition blue light time was extended to 10 s and 100 s, the isobutanol yield showed a downward trend. Especially when the blue light irradiation time was 100 s, the isobutanol yield was even lower than that under continuous darkness. The experimental results show that by adjusting the ratio of blue light and darkness duration, effective regulation of isobutanol yield can be achieved.

[0113] The above has described various embodiments of the present application, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and these changes are all within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A light-controlled pyruvate decarboxylase comprising a pyruvate decarboxylase Pdcl and a LOV domain inserted between adjacent amino acids of Loop-1 or Loop-2 of said pyruvate decarboxylase Pdcl.

2. The light-controlled pyruvate decarboxylase of claim 1, wherein, said LOV domain is a LOV2 domain; preferably, said LOV domain is selected from the LOV2 domain of the oat photoreceptor 1 gene AsLOV2, the photoreceptor protein EL222 from Rhodobacter sphaeroides, the photoreceptor domain LOV2 from Arabidopsis thaliana, or a mutant thereof; preferably, said mutant is a cyclically permuted mutant or a mutant that alters the speed of response; preferably, said LOV domain is AsLOV2, such as comprising the sequence of SEQ ID NO: 1; preferably, said LOV domain is cpLOV2, such as comprising the sequence of SEQ ID NO: 2; preferably, said LOV domain is cpLOV16, such as comprising the sequence of SEQ ID NO: 3; preferably, said LOV domain is cpLOV27, such as comprising the sequence of SEQ ID NO:

4.

3. The light-controlled pyruvate decarboxylase of claim 1 or 2, wherein, said insertion position is selected from the following amino acid positions of a pyruvate decarboxylase: between positions 186 and 187, between positions 188 and 189, between positions 189 and 190, between positions 190 and 191, between positions 191 and 192, between positions 192 and 193, between positions 304 and 305, between positions 305 and 306; preferably, said insertion position is between positions 188 and 189, between positions 192 and 193 of a pyruvate decarboxylase.

4. The light-controlled pyruvate decarboxylase of any one of claims 1-3, wherein, said pyruvate decarboxylase is wild-type; preferably, said wild-type pyruvate decarboxylase comprises the sequence of SEQ ID NO:

6.

5. The light-controlled pyruvate decarboxylase of claim 4, wherein, the N-terminus and / or the C-terminus of said LOV domain is optionally linked to said Pdcl by a peptide linker; said peptide linker is selected from glycine (G), glycine-serine (GS), serine-glycine (SG), glycine-serine-glycine (GSG), or any two combinations thereof.

6. The light-controlled pyruvate decarboxylase of claim 4, comprising the sequence of any one of SEQ ID NOs: 7-22; preferably, said light-controlled pyruvate decarboxylase comprises the sequence of SEQ ID NO: 14 (S188-GS) or SEQ ID NO: 9 (L189-AsLOV2) or SEQ ID NO: 20 (L189-cp2); preferably, said light-controlled pyruvate decarboxylase comprises the sequence of SEQ ID NO: 14 (S188-GS) or SEQ ID NO: 20 (L189-cp2).

7. A nucleic acid construct comprising a nucleotide sequence encoding the light- controlled pyruvate decarboxylase of any one of claims 1-6; optionally, further comprising a promoter operably linked to said nucleotide sequence encoding the light-controlled pyruvate decarboxylase; preferably, said promoter is selected from a TEF promoter, a GPD promoter, an ADH1 promoter. optionally, a terminator operably linked thereto; Preferably, the terminator is selected from the group consisting of ACT1 terminator, ADH1 terminator.

8. An expression vector comprising the nucleic acid construct of claim 7.

9. A host cell, characterized in that, comprising the nucleic acid construct of claim 7 or the vector of claim 8; Preferably, the host cell is a eukaryotic cell; Preferably, the host cell is Saccharomyces cerevisiae; More preferably, the host cell is Saccharomyces cerevisiae, which has integrated into its genome one or more copies of an exogenous nucleotide sequence encoding the light-controlled pyruvate decarboxylase of any one of claims 1-6; preferably, the exogenous nucleotide sequence encoding the light-controlled pyruvate decarboxylase is operably linked at both ends with a promoter and a terminator. Preferably, the endogenous Pdcl, Pdc5, Pdc6 genes of the Saccharomyces cerevisiae are disrupted or knocked out.

10. Use of the host cell of claim 9 in the regulation of metabolic pathways and / or biosynthesis, in particular by culturing under blue light irradiation to achieve the regulation.

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