# Patent

## Title
Engineered biosynthetic pathways for production of histamine by fermentation

## Abstract

The present disclosure describes the engineering of microbial cells for fermentative production of histamine and provides novel engineered microbial cells and cultures, as well as related histamine production methods.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application No. 62/660,875, filed Apr. 20, 2018, which is hereby incorporated by reference in its entirety.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Agreement No. HR0011-15-9-0014, awarded by DARPA. The Government has certain rights in the invention.


INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING
This application includes a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on Apr. 17, 2019, is named ZMGNP011WO_Seq_List_ST25.txt and is 312,107 bytes in size.
FIELD OF THE DISCLOSURE
The present disclosure relates generally to the area of engineering microbes for production of histamine by fermentation.
BACKGROUND
Biogenic amines are organic bases endowed with biological activity, which are frequently found in fermented foods and beverages. Histamine is known to exist in nature in fermented foods such as yogurt (13-36 mg/kg) [1], miso (24 mg/kg) [2], and red wine (24 mg/L) [3]. Some bacteria that live in the human gut also make histamine, and it functions to regulate the immune system by an anti-inflammatory effect [4]. Production of histamine in fermented foods relies on a source of proteins that contain histidine and microbes that histidine decarboxylase. Histamine is the decarboxylation product of histidine that is catalyzed specifically by the enzyme histidine decarboxylase (EC 4.1.1.22). Production of histamine in an industrial fermentation from simple, non-protein, carbon and nitrogen sources requires assembly of a pathway with improved biosynthesis of the amino acid precursor histidine and a highly active histidine decarboxylase.
SUMMARY
The disclosure provides engineered microbial cells, cultures of the microbial cells, and methods for the production of histamine, including the following:
Embodiment 1: An engineered microbial cell that expresses a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine.
Embodiment 2: The engineered microbial cell of embodiment 1, wherein the engineered microbial cell includes increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell.
Embodiment 3: The engineered microbial cell of embodiment 2, wherein the one or more upstream histamine pathway enzyme(s) are selected from the group consisting of an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase.
Embodiment 4: The engineered microbial cell of any one of embodiments 1-3, wherein the engineered microbial cell includes reduced activity of one or more enzyme(s) that consume one or more histamine pathway precursors, said reduced activity being reduced relative to a control cell.
Embodiment 5: The engineered microbial cell of embodiment 4, wherein the one or more enzyme(s) that consume one or more histamine pathway precursors are selected from the group consisting of an enolase, a pyruvate dehydrogenase, a pentose phosphate pathway sugar isomerase, a transaldolase, a transketolase, a ribulose-5-phosphate epimerase, and a ribulose-5-phosphate isomerase.
Embodiment 6: The engineered microbial cell of embodiment 4 or embodiment 5, wherein the reduced activity is achieved by replacing a native promoter of a gene for said one or more enzymes with a less active promoter.
Embodiment 7: The engineered microbial cell of any one of embodiments 1-6, wherein the engineered microbial cell additionally expresses a feedback-deregulated glucose-6-phosphate dehydrogenase or a feedback-deregulated ATP phosphoribosyltransferase.
Embodiment 8: An engineered microbial cell, wherein the engineered microbial cell includes means for expressing a non-native histidine decarboxylase, wherein the engineered microbial cell produces histamine.
Embodiment 9: The engineered microbial cell of embodiment 8, wherein the engineered microbial cell includes means for increasing the activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell.
Embodiment 10: The engineered microbial cell of embodiment 9, wherein the one or more upstream histamine pathway enzyme(s) are selected from the group consisting of an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, a histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase.
Embodiment 11: The engineered microbial cell of any one of embodiments 8-10, wherein the engineered microbial cell includes means for reducing the activity of one or more enzyme(s) that consume one or more histamine pathway precursors, said reduced activity being reduced relative to a control cell.
Embodiment 12: The engineered microbial cell of embodiment 11, wherein the one or more enzyme(s) that consume one or more histamine pathway precursors are selected from the group consisting of an enolase, a pyruvate dehydrogenase, pentose phosphate pathway sugar isomerase, a transketolase, a transaldolase, a ribulose-5-phosphate epimerase, and a ribulose-5-phosphate isomerase.
Embodiment 13: The engineered microbial cell of embodiment 11 or embodiment 12, wherein the reduced activity is achieved by means for replacing a native promoter of a gene for said one or more enzymes with a less active promoter.
Embodiment 14: The engineered microbial cell of any one of embodiments 8-13, wherein the engineered microbial cell additionally includes means for expressing glucose-6-phosphate dehydrogenase or a feedback-deregulated ATP phosphoribosyltransferase.
Embodiment 15: The engineered microbial cell of any one of embodiments 1-14, wherein the engineered microbial cell includes a fungal cell.
Embodiment 16: The engineered microbial cell of embodiment 15, wherein the engineered microbial cell includes a yeast cell.
Embodiment 17: The engineered microbial cell of embodiment 16, wherein the yeast cell is a cell of the genus Saccharomyces or Yarrowia. 
Embodiment 18: The engineered microbial cell of embodiment 17, wherein the yeast cell is a cell of the genus Saccharomyces and of the species cerevisiae.
Embodiment 19: The engineered microbial cell of embodiment 17, wherein the yeast cell is a cell of the genus Yarrowia and of the species lipolytica. 
Embodiment 20: The engineered microbial cell of any one of embodiments 1-19, wherein the non-native histidine decarboxylase includes a histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 or from Acinetobacter baumannii strain AB0057.
Embodiment 21: The engineered microbial cell of any one of embodiments 1 and 16-20, wherein the engineered microbial cell includes increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell, wherein the one or more upstream histamine pathway enzyme(s) comprise an ATP phosphoribosyltransferase.
Embodiment 22: The engineered microbial cell of embodiment 21 wherein the increased activity of the ATP phosphoribosyltransferase is achieved by heterologously expressing it.
Embodiment 23: The engineered microbial cell of embodiment 22, wherein the heterologous ATP phosphoribosyltransferase has at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae. 
Embodiment 24: The engineered microbial cell of any one of embodiments 16-23, wherein the engineered microbial cell includes a feedback-deregulated variant of a Corynebacterium glutamicum ATP phosphoribosyltransferase.
Embodiment 25: The engineered microbial cell of any one of embodiments 1-14, wherein the engineered microbial cell is a bacterial cell.
Embodiment 26: The engineered microbial cell of embodiment 25, wherein the bacterial cell is a cell of the genus Corynebacteria or Bacillus. 
Embodiment 27: The engineered microbial cell of embodiment 26, wherein the bacterial cell is a cell of the genus Corynebacteria and of the species glutamicum. 
Embodiment 28: The engineered microbial cell of embodiment 26, wherein the bacterial cell is a cell of the genus Bacillus and of the species subtilis. 
Embodiment 29: The engineered microbial cell of any one of embodiments 25-28, wherein the non-native histidine decarboxylase includes a histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Acinetobacter baumannii or from Lactobacillus sp. (strain 30a).
Embodiment 30: The engineered microbial cell of any one of embodiments 1 and 25-29, wherein the engineered microbial cell includes increased activity of one or more upstream histamine pathway enzyme(s), said increased activity being increased relative to a control cell, wherein the one or more upstream histamine pathway enzyme(s) comprise an ATP phosphoribosyltransferase and an imidazole-glycerol phosphate dehydratase.
Embodiment 31: The engineered microbial cell of embodiment 30, wherein the increased activity of the ATP phosphoribosyltransferase or the imidazole-glycerol phosphate dehydratase is achieved by heterologously expressing it.
Embodiment 32: The engineered microbial cell of embodiment 31, wherein the heterologous ATP phosphoribosyltransferase has at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c or from Salmonella typhimurium LT2, or the heterologous imidazole-glycerol phosphate dehydratase has at least 70% amino acid sequence identity with an imidazole-glycerol phosphate dehydratase from Corynebacterium glutamicum. 
Embodiment 33: The engineered microbial cell of any one of embodiments 25-32, wherein the engineered microbial cell includes a feedback-deregulated variant of a Salmonella typhimurium ATP phosphoribosyltransferase.
Embodiment 34: The engineered microbial cell of any one of embodiments 1-33, wherein, when cultured, the engineered microbial cell produces histamine at a level of at least 20 mg/L of culture medium.
Embodiment 35: The engineered microbial cell of embodiment 34, wherein, when cultured, the engineered microbial cell produces histamine at a level of at least 300 mg/L of culture medium.
Embodiment 36: A culture of engineered microbial cells according to any one of embodiments 1-35.
Embodiment 37: The culture of embodiment 36, wherein the engineered microbial cells are present in a concentration such that the culture has an optical density at 600 nm of 10-500.
Embodiment 38: The culture of any one of embodiments 36-37, wherein the culture includes histamine.
Embodiment 39: The culture of any one of embodiments 36-38, wherein the culture includes histamine at a level at least 20 mg/L of culture medium.
Embodiment 40: A method of culturing engineered microbial cells according to any one of embodiments 1-35, the method including culturing the cells under conditions suitable for producing histamine.
Embodiment 41: The method of embodiment 40, wherein the method includes fed-batch culture, with an initial glucose level in the range of 1-100 g/L, followed controlled sugar feeding.
Embodiment 42: The method of any one of embodiments 40-41, wherein the fermentation substrate includes glucose and a nitrogen source selected from the group consisting of urea, an ammonium salt, ammonia, and any combination thereof.
Embodiment 43: The method of any one of embodiments 40-42, wherein the culture is pH-controlled during culturing.
Embodiment 44: The method of any one of embodiments 40-43, wherein the culture is aerated during culturing.
Embodiment 45: The method of any one of embodiments 40-44, wherein the engineered microbial cells produce histamine at a level at least 20 mg/L of culture medium.
Embodiment 46: The method of any one of embodiments 40-45, wherein the method additionally includes recovering histamine from the culture.
Embodiment 47: A method for preparing histamine using microbial cells engineered to produce histamine, the method including: (a) expressing a non-native histidine decarboxylase in microbial cells; (b) cultivating the microbial cells in a suitable culture medium under conditions that permit the microbial cells to produce histamine, wherein the histamine is released into the culture medium; and isolating histamine from the culture medium.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Biosynthetic pathway for histamine.
FIG. 2: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Corynebacteria glutamicum. (See also Example 1, Table 1.)
FIG. 3: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Saccharomyces cerevisiae. (See also Example 1, Table 1.)
FIG. 4: Histamine titers measured in the extracellular broth following fermentation by the second-round engineered host Corynebacteria glutamicum. (See also Example 1, Table 2.)
FIG. 5: Histamine titers measured in the extracellular broth following fermentation by the second-round engineered host Saccharomyces cerevisiae. (See also Example 1, Table 2.)
FIG. 6: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Yarrowia lipolytica. (See also Example 2, Table 4.)
FIG. 7: Histamine titers measured in the extracellular broth following fermentation by the first-round engineered host Bacillus subtilis. 
FIG. 8: Histamine acid titers measured in the extracellular broth following fermentation of Saccharomyces cerevisiae expressing the host evaluation designs.
FIG. 9: Histamine acid titers measured in the extracellular broth following fermentation of Corynebacteria glutamicum expressing the host evaluation designs.
FIG. 10: Histamine titers measured in the extracellular broth following fermentation by the third-round engineered host Saccharomyces cerevisiae. (Improvement round.)
FIG. 11: Integration of Promoter-Gene-Terminator into Saccharomyces cerevisiae and Yarrowia lipolytica. 
FIG. 12: Promoter replacement in Saccharomyces cerevisiae and Yarrowia lipolytica. 
FIG. 13: Targeted gene deletion in Saccharomyces cerevisiae and Yarrowia lipolytica. 
FIG. 14: Integration of Promoter-Gene-Terminator into Corynebacteria glutamicum and Bacillus subtilis. 



DETAILED DESCRIPTION
This disclosure describes a method for the production of the small molecule histamine via fermentation by a microbial host from simple carbon and nitrogen sources, such as glucose and urea, respectively. This objective can be achieved by introducing a non-native metabolic pathway into a suitable microbial host for industrial fermentation of large-scale chemical products. Illustrative hosts include Saccharomyces cerevisiae, Yarrowia lypolytica, Corynebacteria glutamicum, and Bacillus subtilis. The engineered metabolic pathway links the central metabolism of the host to a non-native pathway to enable the production of histamine. The simplest embodiment of this approach is the expression of an enzyme, a non-native histidine decarboxylase enzyme, in a microbial host strain that can produce histidine. Further engineering of the metabolic pathway by modification of the microbial host central metabolism through overexpression and mutation of a key upstream pathway enzyme, ATP phosphoribosyltransferase, enabled titers of 505 mg/L histamine to be achieved.
The following disclosure describes how to engineer a microbe with the necessary characteristics to produce industrially feasible titers of histamine from simple carbon and nitrogen sources. Active histidine decarboxylases have been identified, and it has been found that feedback-deregulated ATP phosphoribosyltransferase and/or constitutive expression of native ATP phosphoribosyltransferase improve the titers of histidine by fermentation.
Definitions
Terms used in the claims and specification are defined as set forth below unless otherwise specified.
The term “fermentation” is used herein to refer to a process whereby a microbial cell converts one or more substrate(s) into a desired product (such as histamine) by means of one or more biological conversion steps, without the need for any chemical conversion step.
The term “engineered” is used herein, with reference to a cell, to indicate that the cell contains at least one targeted genetic alteration introduced by man that distinguishes the engineered cell from the naturally occurring cell.
The term “native” is used herein to refer to a cellular component, such as a polynucleotide or polypeptide, that is naturally present in a particular cell. A native polynucleotide or polypeptide is endogenous to the cell.
When used with reference to a polynucleotide or polypeptide, the term “non-native” refers to a polynucleotide or polypeptide that is not naturally present in a particular cell.
When used with reference to the context in which a gene is expressed, the term “non-native” refers to a gene expressed in any context other than the genomic and cellular context in which it is naturally expressed. A gene expressed in a non-native manner may have the same nucleotide sequence as the corresponding gene in a host cell, but may be expressed from a vector or from an integration point in the genome that differs from the locus of the native gene.
The term “heterologous” is used herein to describe a polynucleotide or polypeptide introduced into a host cell. This term encompasses a polynucleotide or polypeptide, respectively, derived from a different organism, species, or strain than that of the host cell. In this case, the heterologous polynucleotide or polypeptide has a sequence that is different from any sequence(s) found in the same host cell. However, the term also encompasses a polynucleotide or polypeptide that has a sequence that is the same as a sequence found in the host cell, wherein the polynucleotide or polypeptide is present in a different context than the native sequence (e.g., a heterologous polynucleotide can be linked to a different promotor and inserted into a different genomic location than that of the native sequence). “Heterologous expression” thus encompasses expression of a sequence that is non-native to the host cell, as well as expression of a sequence that is native to the host cell in a non-native context.
As used with reference to polynucleotides or polypeptides, the term “wild-type” refers to any polynucleotide having a nucleotide sequence, or polypeptide having an amino acid, sequence present in a polynucleotide or polypeptide from a naturally occurring organism, regardless of the source of the molecule; i.e., the term “wild-type” refers to sequence characteristics, regardless of whether the molecule is purified from a natural source; expressed recombinantly, followed by purification; or synthesized. The term “wild-type” is also used to denote naturally occurring cells.
A “control cell” is a cell that is otherwise identical to an engineered cell being tested, including being of the same genus and species as the engineered cell, but lacks the specific genetic modification(s) being tested in the engineered cell.
Enzymes are identified herein by the reactions they catalyze and, unless otherwise indicated, refer to any polypeptide capable of catalyzing the identified reaction. Unless otherwise indicated, enzymes may be derived from any organism and may have a native or mutated amino acid sequence. As is well known, enzymes may have multiple functions and/or multiple names, sometimes depending on the source organism from which they derive. The enzyme names used herein encompass orthologs, including enzymes that may have one or more additional functions or a different name.
The term “feedback-deregulated” is used herein with reference to an enzyme that is normally negatively regulated by a downstream product of the enzymatic pathway (i.e., feedback-inhibition) in a particular cell. In this context, a “feedback-deregulated” enzyme is a form of the enzyme that is less sensitive to feedback-inhibition than the native enzyme native to the cell. A feedback-deregulated enzyme may be produced by introducing one or more mutations into a native enzyme. Alternatively, a feedback-deregulated enzyme may simply be a heterologous, native enzyme that, when introduced into a particular microbial cell, is not as sensitive to feedback-inhibition as the native, native enzyme. In some embodiments, the feedback-deregulated enzyme shows no feedback-inhibition in the microbial cell.
The term “histamine” refers to 2-(1I-Imidazol-4-yl)ethanamine (CAS #51-45-6).
The term “sequence identity,” in the context of two or more amino acid or nucleotide sequences, refers to two or more sequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
For sequence comparison to determine percent nucleotide or amino acid sequence identity, typically one sequence acts as a “reference sequence,” to which a “test” sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence relative to the reference sequence, based on the designated program parameters. Alignment of sequences for comparison can be conducted using BLAST set to default parameters.
The term “titer,” as used herein, refers to the mass of a product (e.g., histamine) produced by a culture of microbial cells divided by the culture volume.
As used herein with respect to recovering histamine from a cell culture, “recovering” refers to separating the histamine from at least one other component of the cell culture medium.
Engineering Microbes for Histamine Production
Histamine Biosynthesis Pathway
Histamine is typically derived from the amino acid histidine. The histamine biosynthesis pathway is shown in FIG. 1. The first enzyme of the amino acid biosynthesis pathway, ATP phosphoribosyltransferase, is subject to feedback inhibition by histidine. Histamine production is enabled by the addition of a single non-native enzymatic step in Saccharomyces cerevisiae, Yarrowia lypolytica, Corynebacteria glutamicum, and Bacillus subtilis hosts, which is catalyzed by histidine decarboxylase (EC 4.1.1.22).
Engineering for Microbial Histamine Production
Any histidine decarboxylase that is active in the microbial cell being engineered may be introduced into the cell, typically by introducing and expressing the gene(s) encoding the enzyme(s)s using standard genetic engineering techniques. Suitable histidine decarboxylase may be derived from any source, including plant, archaeal, fungal, gram-positive bacterial, and gram-negative bacterial sources. Exemplary sources include, but are not limited to: Aeromonas salmonicida subsp. pectinolytica 34mel, Acinetobacter baumannii (strain AB0057), Chromobacterium haemolyticum, Chromobacterium sp. LK1, Citrobacter pasteurii, Drosophila melanogaster, Lactobacillus aviarius DSM 20655, Lactobacillus fructivorans, Lactobacillus reuteri, Lactobacillus sp. (strain 30a), Methanosarcina barkeri (strain Fusaro/DSM804), Methanosarcina barkeri str. Wiesmoor, Morganella psychrotolerans, Mus musculus, Oenococcus oeni (Leuconostoc oenos), Pseudomonas putida (Arthrobacter siderocapsulatus), Pseudomonas rhizosphaerae, Pseudomonas sp. bs2935, Solanum lycopersicum, Oryza sativa, Penicillium marneffei, Streptomyces hygroscopicus, Pseudomonas putida, Arabidopsis thaliana (Mouse-ear cress), Glycine soja (Wild soybean), Solanum lycopersicum (Tomato) (Lycopersicon esculentum), Clostridium perfringens, Lactobacillus buchneri, Drosophila melanogaster (Fruit fly), Morganella morganii (Proteus morganii), E. coli, Bos taurus (Bovine), Raoutella planticol (Klebsiella planticola), Acinetobacter baumannii, Acinetobacter haemolyticus, Photobacterium damselae, Tetragenococcus muriaticus, Moritella sp JT01, Streptococcus thermophilus, Enterobacter aerogenes, Citrobacter youngae, Raoultella omithinolytica, and Raoultella planticola. 
One or more copies of histidine decarboxylase gene can be introduced into a selected microbial host cell. If more than one copy of a gene is introduced, the copies can have the same or different nucleotide sequences. In some embodiments, one or both of the heterologous gene(s) is/are expressed from a strong, constitutive promoter. In some embodiments, the heterologous histidine decarboxylase gene(s) is/are expressed from an inducible promoter. The heterologous gene(s) can optionally be codon-optimized to enhance expression in the selected microbial host cell. Illustrative codon-optimization tables for hosts used in the Examples are as follows: Bacillus subtilis Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodin.cgi?species=1423&aa=1&style=N; Yarrowia lipolytica Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodoon/cgi?species=4952&aa=1&style=N; Corynebacteria glutamicum Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodoon/cgi?species=340322&aa=1&style=N; Saccharomyces cerevisiae Kazusa codon table: www.kazusa.or.jp/codon/cgi-bin/showcodoon/cgi?species=4932&aa=1&style=N. Also used, was a modified, combined codon usage scheme for S. cereviae and C. glutamicum, which is reproduced below.










Modified Codon Usage Table for Sc and Cg









Amino




Acid
Codon
Fraction












A
GCG
0.22


A
GCA
0.29


A
GCT
0.24


A
GCC
0.25


C
TGT
0.36


C
TGC
0.64


D
GAT
0.56


D
GAC
0.44


E
GAG
0.44


E
GM
0.56


F
TTT
0.37


F
TTC
0.63


G
GGG
0.08


G
GGA
0.19


G
GGT
0.3


G
GGC
0.43


H
CAT
0.32


H
CAC
0.68


I
ATA
0.03


I
ATT
0.38


I
ATC
0.59


K
MG
0.6


K
AAA
0.4


L
TTG
0.29


L
TTA
0.05


L
CTG
0.29


L
CTA
0.06


L
CTT
0.17


L
CTC
0.14


M
ATG
1


N
MT
0.33


N
MC
0.67


P
CCG
0.22


P
CCA
0.35


P
CCT
0.23


P
CCC
0.2


Q
CAG
0.61


Q
CM
0.39


R
AGG
0.11


R
AGA
0.12


R
CGG
0.09


R
CGA
0.17


R
CGT
0.34


R
CGC
0.18


S
AGT
0.08


S
AGC
0.16


S
TCG
0.12


S
TCA
0.13


S
TCT
0.17


S
TCC
0.34


T
ACG
0.14


T
ACA
0.12


T
ACT
0.2


T
ACC
0.53


V
GTG
0.36


V
GTA
0.1


V
GTT
0.26


V
GTC
0.28


W
TGG
1


Y
TAT
0.34


Y
TAC
0.66









Increasing the Activity of Upstream Enzymes
One approach to increasing histamine production in a microbial cell that is capable of such production is to increase the activity of one or more upstream enzymes in the histamine biosynthesis pathway. Upstream pathway enzymes include all enzymes involved in the conversions from a feedstock all the way to into the last native metabolite (histidine, in the illustrative microbial cells described in the Examples below). Such enzymes include an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase. Suitable upstream pathway genes encoding these enzymes may be derived from any source, including, for example, those discussed above as sources for a histidine decarboxylase gene.
In some embodiments, the activity of one or more upstream pathway enzymes is increased by modulating the expression or activity of the native enzyme(s). For example, native regulators of the expression or activity of such enzymes can be exploited to increase the activity of suitable enzymes.
Alternatively, or in addition, one or more promoters can be substituted for native promoters using, for example, a technique such as that illustrated in FIG. 12. In certain embodiments, the replacement promoter is stronger than the native promoter and/or is a constitutive promoter.
In some embodiments, the activity of one or more upstream pathway enzymes is supplemented by introducing one or more of the corresponding genes into the histidine decarboxylase-expressing microbial host cell. An introduced upstream pathway gene may be from an organism other than that of the host cell or may simply be an additional copy of a native gene. In some embodiments, one or more such genes are introduced into a microbial host cell capable of histamine production and expressed from a strong constitutive promoter and/or can optionally be codon-optimized to enhance expression in the selected microbial host cell.
Example 1 describes the successful engineering of C. glutamicum to express a heterologous histamine decarboxylase from Acinetobacter baumannii (SEQ ID NO:1) and to constitutively express a heterologous C. glutamicum imidazoleglycerol-phosphate dehydratase (SEQ ID NO:2). This strain resulted from two rounds of genetic engineering and produced histamine at a titer of 24 mg/L of culture medium. This titer was increased to 68 mg/L in a C. glutamicum strain engineered to express a histamine decarboxylase from Acinetobacter baumannii (strain AB0057) (SEQ ID NO:1) and an ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3).
Example 2 describes the successful engineering of Y. lypolytica to express a histidine decarboxylase from Acinetobacter baumannii (strain AB0057) (SEQ ID NO:1) and an ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3) to give a histamine titer of 505 mg/L. Example 2 also describes the engineering B. subtilis to express a histamine decarboxylase from Lactobacillus sp. (strain 30a) (SEQ ID NO:4) and an ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (SEQ ID NO:5) to give a histamine titer of 18 mg/L. Also in Example 2, S. cerevisiae was engineered to express a histamine decarboxylase from Chromobacterium sp. LK1 (SEQ ID NO:6) and an ATP phosphoribosyltransferase S. cerevisiae S288c (SEQ ID NO:3) to give a histamine titer of 111 mg/L.
In various embodiments, the engineering of a histamine-producing microbial cell to increase the activity of one or more upstream pathway enzymes increases the histamine titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in histamine titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. (Ranges herein include their endpoints.) These increases are determined relative to the histamine titer observed in a histamine-producing microbial cell that lacks any increase in activity of upstream pathway enzymes. This reference cell may have one or more other genetic alterations aimed at increasing histamine production, e.g., the cell may express a feedback-deregulated enzyme.
In various embodiments, the histamine titers achieved by increasing the activity of one or more upstream pathway genes are at least 1, 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or 10 gm/L. In various embodiments, the titer is in the range of 10 mg/L to 10 gm/L, 20 mg/L to 5 gm/L, 50 mg/L to 4 gm/L, 100 mg/L to 3 gm/L, 500 mg/L to 2 gm/L or any range bounded by any of the values listed above.
Introduction of Feedback-Deregulated Enzymes
Since histidine biosynthesis is subject to feedback inhibition, another approach to increasing histamine production in a microbial cell engineered to produce histamine is to introduce feedback-deregulated forms of one or more enzymes that are normally subject to feedback regulation. Examples of such enzymes include glucose-6-phosphate dehydrogenase and ATP phosphoribosyltransferase. A feedback-deregulated form can be a heterologous, native enzyme that is less sensitive to feedback inhibition than the native enzyme in the particular microbial host cell. Alternatively, a feedback-deregulated form can be a variant of a native or heterologous enzyme that has one or more mutations or truncations rendering it less sensitive to feedback inhibition than the corresponding native enzyme. Examples of the latter include a variant ATP phosphoribosyltransferase (from C. glutamicum) containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO:7) and a variant ATP phosphoribosyltransferase (from Salmonella typhimurium) containing the deletion of amino acids Q207 and E208 (SEQ ID NO:5).
In various embodiments, the engineering of a histamine-producing microbial cell to express a feedback-deregulated enzymes increases the histamine titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in histamine titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. These increases are determined relative to the histamine titer observed in a histamine-producing microbial cell that does not express a feedback-deregulated enzyme. This reference cell may (but need not) have other genetic alterations aimed at increasing histamine production, i.e., the cell may have increased activity of an upstream pathway enzyme resulting from some means other than feedback-insensitivity.
In various embodiments, the histamine titers achieved by using a feedback-deregulated enzyme to increase flux though the histamine biosynthetic pathway are at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L, or at least 1, 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 g/L. In various embodiments, the titer is in the range of 50 μg/L to 50 g/L, 75 μg/L to 20 g/L, 100 μg/L to 10 g/L, 200 μg/L to 5 g/L, 500 μg/L to 4 g/L, 1 mg/L to 3 g/L, 500 mg/L to 2 g/L or any range bounded by any of the values listed above.
The approaches of supplementing the activity of one or more native enzymes and/or introducing one or more feedback-deregulated enzymes can be combined in histamine decarboxylase-expressing microbial cells to achieve even higher histamine production levels. For example, a histamine titer of 385 mg/L was achieved in S. cerevisiae in two rounds of engineering from the introduction of three genes: a histidine decarboxylase gene (from Chromobacterium sp. LK1) (SEQ ID NO:6), an ATP phosphoribosyltransferase (from C. glutamicum) containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO:7), and a constitutively expressed ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3). (Example 1.)
Reduction of Precursor Consumption
Another approach to increasing histamine production in a microbial cell that is capable of such production is to decrease the activity of one or more enzymes that consume one or more histamine pathway precursors. In some embodiments, the activity of one or more such enzymes is reduced by modulating the expression or activity of the native enzyme(s). Illustrative enzymes of this type include an enolase, a pyruvate dehydrogenase, a pentose phosphate pathway sugar isomerase, a transaldolase, a transketolase, a ribulose-5-phosphate epimerase, and a aribulose-5-phosphate isomerase. The activity of such enzymes can be decreased, for example, by substituting the native promoter of the corresponding gene(s) with a less active or inactive promoter or by deleting the corresponding gene(s). See FIGS. 12 and 13 for examples of schemes for promoter replacement and targeted gene deletion, respectively, in S. cervisiae and Y. lipolytica. 
In various embodiments, the engineering of a histamine-producing microbial cell to reduce precursor consumption by one or more side pathways increases the histamine titer by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent or by at least 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold. In various embodiments, the increase in histamine titer is in the range of 10 percent to 100-fold, 2-fold to 50-fold, 5-fold to 40-fold, 10-fold to 30-fold, or any range bounded by any of the values listed above. These increases are determined relative to the histamine titer observed in a histamine-producing microbial cell that does not include genetic alterations to reduce precursor consumption. This reference cell may (but need not) have other genetic alterations aimed at increasing histamine production, i.e., the cell may have increased activity of an upstream pathway enzyme.
In various embodiments, the histamine titers achieved by reducing precursor consumption by one or more side pathways are at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L, or at least 1, 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 g/L. In various embodiments, the titer is in the range of 50 μg/L to 50 g/L, 75 μg/L to 20 g/L, 100 μg/L to 10 g/L, 200 μg/L to 5 g/L, 500 μg/L to 4 g/L, 1 mg/L to 3 g/L, 500 mg/L to 2 g/L or any range bounded by any of the values listed above.
The approaches of increasing the activity of one or more native enzymes and/or introducing one or more feedback-deregulated enzymes and/or reducing precursor consumption by one or more side pathways can be combined to achieve even higher histamine production levels.
Microbial Host Cells
Any microbe that can be used to express introduced genes can be engineered for fermentative production of histamine as described above. In certain embodiments, the microbe is one that is naturally incapable of fermentative production of histamine. In some embodiments, the microbe is one that is readily cultured, such as, for example, a microbe known to be useful as a host cell in fermentative production of compounds of interest. Bacteria cells, including gram positive or gram negative bacteria can be engineered as described above. Examples include, in addition to C. glutamicum cells, Bacillus subtilus, B. lichenformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., P. alcaligenes, P. citrea, Lactobacilis spp. (such as L. lactis, L. plantarum), L. grayi, E. coli, E. faecium, E. gallinarum, E. casseliflavus, and/or E. faecalis cells.
There are numerous types of anaerobic cells that can be used as microbial host cells in the methods described herein. In some embodiments, the microbial cells are obligate anaerobic cells. Obligate anaerobes typically do not grow well, if at all, in conditions where oxygen is present. It is to be understood that a small amount of oxygen may be present, that is, there is some level of tolerance level that obligate anaerobes have for a low level of oxygen. Obligate anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes.
Alternatively, the microbial host cells used in the methods described herein can be facultative anaerobic cells. Facultative anaerobes can generate cellular ATP by aerobic respiration (e.g., utilization of the TCA cycle) if oxygen is present. However, facultative anaerobes can also grow in the absence of oxygen. Facultative anaerobes engineered as described above can be grown under substantially oxygen-free conditions, wherein the amount of oxygen present is not harmful to the growth, maintenance, and/or fermentation of the anaerobes, or can be alternatively grown in the presence of greater amounts of oxygen.
In some embodiments, the microbial host cells used in the methods described herein are filamentous fungal cells. (See, e.g., Berka & Barnett, Biotechnology Advances, (1989), 7(2):127-154). Examples include Trichoderma longibrachiatum, T. viride, T. koningii, T. harzianum, Penicillium sp., Humicola insolens, H. lanuginose, H. grisea, Chrysosporium sp., C. lucknowense, Gliocladium sp., Aspergillus sp. (such as A. oryzae, A. niger, A. sojae, A. japonicus, A. nidulans, or A. awamori), Fusarium sp. (such as F. roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora sp. (such as N. crassa or Hypocrea sp.), Mucor sp. (such as M. miehei), Rhizopus sp., and Emericella sp. cells. In particular embodiments, the fungal cell engineered as described above is A. nidulans, A. awamori, A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F. oxysporum, or F. solani. Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Patent Pub. No. 2011/0045563.
Yeasts can also be used as the microbial host cell in the methods described herein. Examples include: Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Hansenula polymorpha, Pichia stipites, Kluyveromyces marxianus, Kluyveromyces spp., Yarrowia lipolytica and Candida sp. In some embodiments, the Saccharomyces sp. is S. cerevisiae (See, e.g., Romanos et al., Yeast, (1992), 8(6):423-488). Illustrative plasmids or plasmid components for use with such hosts include those described in U.S. Pat. No. 7,659,097 and U.S. Patent Pub. No. 2011/0045563.
In some embodiments, the host cell can be an algal cell derived, e.g., from a green algae, red algae, a glaucophyte, a chlorarachniophyte, a euglenid, a chromista, or a dinoflagellate. (See, e.g., Saunders & Warmbrodt, “Gene Expression in Algae and Fungi, Including Yeast,” (1993), National Agricultural Library, Beltsville, Md.). Illustrative plasmids or plasmid components for use in algal cells include those described in U.S. Patent Pub. No. 2011/0045563.
In other embodiments, the host cell is a cyanobacterium, such as cyanobacterium classified into any of the following groups based on morphology: Chlorococcales, Pleurocapsales, Oscillatoriales, Nostocales, Synechosystic or Stigonematales (See, e.g., Lindberg et al., Metab. Eng., (2010) 12(1):70-79). Illustrative plasmids or plasmid components for use in cyanobacterial cells include those described in U.S. Patent Pub. Nos. 2010/0297749 and 2009/0282545 and in Intl. Pat. Pub. No. WO 2011/034863.
Genetic Engineering Methods
Microbial cells can be engineered for fermentative histamine production using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature, see e.g., “Molecular Cloning: A Laboratory Manual,” fourth edition (Sambrook et al., 2012); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications” (R. I. Freshney, ed., 6th Edition, 2010); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction,” (Mullis et al., eds., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994).
Vectors are polynucleotide vehicles used to introduce genetic material into a cell. Vectors useful in the methods described herein can be linear or circular. Vectors can integrate into a target genome of a host cell or replicate independently in a host cell. For many applications, integrating vectors that produced stable transformants are preferred. Vectors can include, for example, an origin of replication, a multiple cloning site (MCS), and/or a selectable marker. An expression vector typically includes an expression cassette containing regulatory elements that facilitate expression of a polynucleotide sequence (often a coding sequence) in a particular host cell. Vectors include, but are not limited to, integrating vectors, prokaryotic plasmids, episomes, viral vectors, cosmids, and artificial chromosomes.
Illustrative regulatory elements that may be used in expression cassettes include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods In Enzymology 185, Academic Press, San Diego, Calif. (1990).
In some embodiments, vectors may be used to introduce systems that can carry out genome editing, such as CRISPR systems. See U.S. Patent Pub. No. 2014/0068797, published 6 Mar. 2014; see also Jinek M., et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science 337:816-21, 2012). In Type II CRISPR-Cas9 systems, Cas9 is a site-directed endonuclease, namely an enzyme that is, or can be, directed to cleave a polynucleotide at a particular target sequence using two distinct endonuclease domains (HNH and RuvC/RNase H-like domains). Cas9 can be engineered to cleave DNA at any desired site because Cas9 is directed to its cleavage site by RNA. Cas9 is therefore also described as an “RNA-guided nuclease.” More specifically, Cas9 becomes associated with one or more RNA molecules, which guide Cas9 to a specific polynucleotide target based on hybridization of at least a portion of the RNA molecule(s) to a specific sequence in the target polynucleotide. Ran, F. A., et al., (“In vivo genome editing using Staphylococcus aureus Cas9,” Nature 520(7546):186-91, 2015 Apr. 9], including all extended data) present the crRNA/tracrRNA sequences and secondary structures of eight Type II CRISPR-Cas9 systems. Cas9-like synthetic proteins are also known in the art (see U.S. Published Patent Application No. 2014-0315985, published 23 Oct. 2014).
Example 1 describes illustrative integration approaches for introducing polynucleotides and other genetic alterations into the genomes of C. glutamicum and S. cerevisiae cells.
Vectors or other polynucleotides can be introduced into microbial cells by any of a variety of standard methods, such as transformation, conjugation, electroporation, nuclear microinjection, transduction, transfection (e.g., lipofection mediated or DEAE-Dextrin mediated transfection or transfection using a recombinant phage virus), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. Transformants can be selected by any method known in the art. Suitable methods for selecting transformants are described in U.S. Patent Pub. Nos. 2009/0203102, 2010/0048964, and 2010/0003716, and International Publication Nos. WO 2009/076676, WO 2010/003007, and WO 2009/132220.
Engineered Microbial Cells
The above-described methods can be used to produce engineered microbial cells that produce, and in certain embodiments, overproduce, histamine. Engineered microbial cells can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more genetic alterations, such as 30-100 alterations, as compared to a native microbial cell, such as any of the microbial host cells described herein. Engineered microbial cells described in the Example below have one, two, or three genetic alterations, but those of skill in the art can, following the guidance set forth herein, design microbial cells with additional alterations. In some embodiments, the engineered microbial cells have not more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 genetic alterations, as compared to a native microbial cell. In various embodiments, microbial cells engineered for histamine production can have a number of genetic alterations falling within the any of the following illustrative ranges: 1-10, 1-9, 1-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-7, 3-6, 3-5, 3-4, etc.
In some embodiments, an engineered microbial cell expresses at least one heterologous histamine decarboxylase, such as in the case of a microbial host cell that does not naturally produce histamine. In various embodiments, the microbial cell can include and express, for example: (1) a single heterologous histamine decarboxylase gene, (2) two or more heterologous histamine decarboxylase genes, which can be the same or different (in other words, multiple copies of the same heterologous histamine decarboxylase genes can be introduced or multiple, different heterologous histamine decarboxylase genes can be introduced), (3) a single heterologous histamine decarboxylase gene that is not native to the cell and one or more additional copies of an native histamine decarboxylase gene, or (4) two or more non-native histamine decarboxylase genes, which can be the same or different, and one or more additional copies of an native histamine decarboxylase gene.
This engineered host cell can include at least one additional genetic alteration that increases flux through the pathway leading to the production of histidine (the immediate precursor of histamine). These “upstream” enzymes in the pathway include: an ATP phosphoribosyltransferase, a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase, including any isoforms, paralogs, or orthologs having these enzymatic activities (which as those of skill in the art readily appreciate may be known by different names). The at least one additional alteration can increase the activity of the upstream pathway enzyme(s) by any available means, e.g., by: (1) modulating the expression or activity of the native enzyme(s), (2) expressing one or more additional copies of the genes for the native enzymes, and/or (3) expressing one or more copies of the genes for one or more non-native enzymes.
In some embodiments, increased flux through the pathway can be achieved by expressing one or more genes encoding a feedback-deregulated enzyme, as discussed above. For example, the engineered host cell can include and express one or more feedback-deregulated ATP phosphoribosyltransferase genes.
The engineered microbial cells can contain introduced genes that have a native nucleotide sequence or that differ from native. For example, the native nucleotide sequence can be codon-optimized for expression in a particular host cell. The amino acid sequences encoded by any of these introduced genes can be native or can differ from native. In various embodiments, the amino acid sequences have at least 60 percent, 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with a native amino acid sequence.
In some embodiments, increased availability of precursors to histamine can be achieved by reducing the expression or activity of enzymes that consume one or more histamine pathway precursors, such as an enolase, a pyruvate dehydrogenase, a pentose phosphate pathway sugar isomerase, a transaldolase, a transketolase, a ribulose-5-phosphate epimerase, and a aribulose-5-phosphate isomerase. For example, the engineered host cell can include one or more promoter swaps to down-regulate expression of any of these enzymes and/or can have their genes deleted to eliminate their expression entirely.
The approach described herein has been carried out in bacterial cells, namely C. glutamicum and B. subtilis (prokaryotes) and in fungal cells, namely the yeasts S. cerevisiae and Y. lypolytica (eukaryotes). (See Examples 1 and 2.)
Illustrative Engineered Yeast Cells
In certain embodiments, the engineered yeast (e.g., S. cerevisiae) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Chromobacterium sp. LK1 (e.g., SEQ ID NO:6). In particular embodiments, the Chromobacterium sp. LK1 histamine decarboxylase can include SEQ ID NO:6. The engineered yeast (e.g., S. cerevisiae) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae (SEQ ID NO:3). In particular embodiments, the S. cerevisiae ATP phosphoribosyltransferase includes SEQ ID NO:3.
In certain embodiments, the engineered yeast (e.g., Y. lipolytica) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Acinetobacter baumannii strain AB0057 (e.g., SEQ ID NO:1). In particular embodiments, the Acinetobacter baumannii strain AB0057 histamine decarboxylase can include SEQ ID NO:1. The engineered yeast (e.g., Y. lipolytica) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c (SEQ ID NO:3). In particular embodiments, the S. cerevisiae S288c ATP phosphoribosyltransferase includes SEQ ID NO:3.
These may be the only genetic alterations of the engineered yeast cell, or the yeast cell can include one or more additional genetic alterations, as discussed more generally above.
For example, in particular embodiments, the engineered yeast S. cerevisiae cell described above additionally expresses a feedback deregulated variant of a C. glutamicum ATP phosphoribosyltransferase, which typically has at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, or 95 percent amino acid sequence identity to a variant of a C. glutamicum ATP phosphoribosyltransferase containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO:7) In particular embodiments, the C. glutamicum ATP phosphoribosyltransferase variant can include SEQ ID NO:7.
Illustrative Engineered Bacterial Cells
In certain embodiments, the engineered bacterial (e.g., C. glutamicum) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Acinetobacter baumannii (e.g., SEQ ID NO:1). In particular embodiments, the Acinetobacter baumannii histamine decarboxylase can include SEQ ID NO:1. The engineered bacterial (e.g., C. glutamicum) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c (SEQ ID NO:3). In particular embodiments, the S. cerevisiae S288c ATP phosphoribosyltransferase includes SEQ ID NO:3. In some embodiments, the engineered bacterial (e.g., C. glutamicum) cell expresses, instead of the ATP phosphoribosyltransferase, an imidazole-glycerol phosphate dehydratase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to an imidazole-glycerol phosphate dehydratase from C. glutamicum (SEQ ID NO:2). In particular embodiments, the C. glutamicum imidazole-glycerol phosphate dehydratase includes SEQ ID NO:2.
In certain embodiments, the engineered bacterial (e.g., B. subtilis) cell expresses a heterologous histamine decarboxylase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity to a histamine decarboxylase from Lactobacillus sp. (strain 30a) (e.g., SEQ ID NO:4). In particular embodiments, the Lactobacillus sp. (strain 30a) histamine decarboxylase can include SEQ ID NO:4. The engineered bacterial (e.g., B. subtilis) cell can also express a heterologous ATP phosphoribosyltransferase having at least 70 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent or 100 percent amino acid sequence identity with an ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (SEQ ID NO:5). In particular embodiments, the Salmonella typhimurium LT2 ATP phosphoribosyltransferase includes SEQ ID NO:5.
Culturing of Engineered Microbial Cells
Any of the microbial cells described herein can be cultured, e.g., for maintenance, growth, and/or histamine production.
In some embodiments, the cultures are grown to an optical density at 600 nm of 10-500, such as an optical density of 50-150.
In various embodiments, the cultures include produced histamine at titers of at least 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg/L, or at least 1, 10, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, or 900 mg/L or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 g/L. In various embodiments, the titer is in the range of 10 μg/L to 10 g/L, 25 μg/L to 20 g/L, 100 μg/L to 10 g/L, 200 μg/L to 5 g/L, 500 μg/L to 4 g/L, 1 mg/L to 3 g/L, 500 mg/L to 2 g/L or any range bounded by any of the values listed above.
Culture Media
Microbial cells can be cultured in any suitable medium including, but not limited to, a minimal medium, i.e., one containing the minimum nutrients possible for cell growth. Minimal medium typically contains: (1) a carbon source for microbial growth; (2) salts, which may depend on the particular microbial cell and growing conditions; and (3) water. Suitable media can also include any combination of the following: a nitrogen source for growth and product formation, a sulfur source for growth, a phosphate source for growth, metal salts for growth, vitamins for growth, and other cofactors for growth.
Any suitable carbon source can be used to cultivate the host cells. The term “carbon source” refers to one or more carbon-containing compounds capable of being metabolized by a microbial cell. In various embodiments, the carbon source is a carbohydrate (such as a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide), or an invert sugar (e.g., enzymatically treated sucrose syrup). Illustrative monosaccharides include glucose (dextrose), fructose (levulose), and galactose; illustrative oligosaccharides include dextran or glucan, and illustrative polysaccharides include starch and cellulose. Suitable sugars include C6 sugars (e.g., fructose, mannose, galactose, or glucose) and C5 sugars (e.g., xylose or arabinose). Other, less expensive carbon sources include sugar cane juice, beet juice, sorghum juice, and the like, any of which may, but need not be, fully or partially deionized.
The salts in a culture medium generally provide essential elements, such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize proteins and nucleic acids.
Minimal medium can be supplemented with one or more selective agents, such as antibiotics.
To produce histamine, the culture medium can include, and/or is supplemented during culture with, glucose and/or a nitrogen source such as urea, an ammonium salt, ammonia, or any combination thereof.
Culture Conditions
Materials and methods suitable for the maintenance and growth of microbial cells are well known in the art. See, for example, U.S. Pub. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2004/033646, WO 2009/076676, WO 2009/132220, and WO 2010/003007, Manual of Methods for General Bacteriology Gerhardt et al., eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass.
In general, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as about 20° C. to about 37° C., about 6% to about 84% CO2, and a pH between about 5 to about 9). In some aspects, cells are grown at 35° C. In certain embodiments, such as where thermophilic bacteria are used as the host cells, higher temperatures (e.g., 50° C.-75° C.) may be used. In some aspects, the pH ranges for fermentation are between about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about 6.5 to about 7.0). Cells can be grown under aerobic, anoxic, or anaerobic conditions based on the requirements of the particular cell.
Standard culture conditions and modes of fermentation, such as batch, fed-batch, or continuous fermentation that can be used are described in U.S. Publ. Nos. 2009/0203102, 2010/0003716, and 2010/0048964, and International Pub. Nos. WO 2009/076676, WO 2009/132220, and WO 2010/003007. Batch and Fed-Batch fermentations are common and well known in the art, and examples can be found in Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc.
In some embodiments, the cells are cultured under limited sugar (e.g., glucose) conditions. In various embodiments, the amount of sugar that is added is less than or about 105% (such as about 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%) of the amount of sugar that can be consumed by the cells. In particular embodiments, the amount of sugar that is added to the culture medium is approximately the same as the amount of sugar that is consumed by the cells during a specific period of time. In some embodiments, the rate of cell growth is controlled by limiting the amount of added sugar such that the cells grow at the rate that can be supported by the amount of sugar in the cell medium. In some embodiments, sugar does not accumulate during the time the cells are cultured. In various embodiments, the cells are cultured under limited sugar conditions for times greater than or about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours or even up to about 5-10 days. In various embodiments, the cells are cultured under limited sugar conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 95, or 100% of the total length of time the cells are cultured. While not intending to be bound by any particular theory, it is believed that limited sugar conditions can allow more favorable regulation of the cells.
In some aspects, the cells are grown in batch culture. The cells can also be grown in fed-batch culture or in continuous culture. Additionally, the cells can be cultured in minimal medium, including, but not limited to, any of the minimal media described above. The minimal medium can be further supplemented with 1.0% (w/v) glucose (or any other six-carbon sugar) or less. Specifically, the minimal medium can be supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose. In some cultures, significantly higher levels of sugar (e.g., glucose) are used, e.g., at least 10% (w/v), 20% (w/v), 30% (w/v), 40% (w/v), 50% (w/v), 60% (w/v), 70% (w/v), or up to the solubility limit for the sugar in the medium. In some embodiments, the sugar levels falls within a range of any two of the above values, e.g.: 0.1-10% (w/v), 1.0-20% (w/v), 10-70% (w/v), 20-60% (w/v), or 30-50% (w/v). Furthermore, different sugar levels can be used for different phases of culturing. For fed-batch culture (e.g., of S. cerevisiae or C. glutamicum), the sugar level can be about 100-200 g/L (10-20% (w/v)) in the batch phase and then up to about 500-700 g/L (50-70% in the feed).
Additionally, the minimal medium can be supplemented 0.1% (w/v) or less yeast extract. Specifically, the minimal medium can be supplemented with 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), 0.02% (w/v), or 0.01% (w/v) yeast extract. Alternatively, the minimal medium can be supplemented with 1% (w/v), 0.9% (w/v), 0.8% (w/v), 0.7% (w/v), 0.6% (w/v), 0.5% (w/v), 0.4% (w/v), 0.3% (w/v), 0.2% (w/v), or 0.1% (w/v) glucose and with 0.1% (w/v), 0.09% (w/v), 0.08% (w/v), 0.07% (w/v), 0.06% (w/v), 0.05% (w/v), 0.04% (w/v), 0.03% (w/v), or 0.02% (w/v) yeast extract. In some cultures, significantly higher levels of yeast extract can be used, e.g., at least 1.5% (w/v), 2.0% (w/v), 2.5% (w/v), or 3% (w/v). In some cultures (e.g., of S. cerevisiae or C. glutamicum), the yeast extract level falls within a range of any two of the above values, e.g.: 0.5-3.0% (w/v), 1.0-2.5% (w/v), or 1.5-2.0% (w/v).
Illustrative materials and methods suitable for the maintenance and growth of the engineered microbial cells described herein can be found below in Example 1.
Histamine Production and Recovery
Any of the methods described herein may further include a step of recovering histamine. In some embodiments, the produced histamine contained in a so-called harvest stream is recovered/harvested from the production vessel. The harvest stream may include, for instance, cell-free or cell-containing aqueous solution coming from the production vessel, which contains histamine as a result of the conversion of production substrate by the resting cells in the production vessel. Cells still present in the harvest stream may be separated from the histamine by any operations known in the art, such as for instance filtration, centrifugation, decantation, membrane crossflow ultrafiltration or microfiltration, tangential flow ultrafiltration or microfiltration or dead end filtration. After this cell separation operation, the harvest stream is essentially free of cells.
Further steps of separation and/or purification of the produced histamine from other components contained in the harvest stream, i.e., so-called downstream processing steps may optionally be carried out. These steps may include any means known to a skilled person, such as, for instance, concentration, extraction, crystallization, precipitation, adsorption, ion exchange, and/or chromatography. Any of these procedures can be used alone or in combination to purify histamine. Further purification steps can include one or more of, e.g., concentration, crystallization, precipitation, washing and drying, treatment with activated carbon, ion exchange, nanofiltration, and/or re-crystallization. The design of a suitable purification protocol may depend on the cells, the culture medium, the size of the culture, the production vessel, etc. and is within the level of skill in the art.
The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. Changes therein and other uses which are encompassed within the spirit of the disclosure, as defined by the scope of the claims, will be identifiable to those skilled in the art.
Example 1—Construction and Selection of Strains of Corynebacteria glutamicum and Saccharomyces cerevisiae Engineered to Produce Histamine
Plasmid/DNA Design
All strains tested for this work were transformed with plasmid DNA designed using proprietary software. Plasmid designs were specific to each of the host organisms engineered in this work. The plasmid DNA was physically constructed by a standard DNA assembly method. This plasmid DNA was then used to integrate metabolic pathway inserts by one of two host-specific methods, each described below.
C. glutamicum Pathway Integration
A “loop-in, single-crossover” genomic integration strategy has been developed to engineer C. glutamicum strains. FIG. 14 illustrates genomic integration of loop-in only and loop-in/loop-out constructs and verification of correct integration via colony PCR. Loop-in only constructs (shown under the heading “Loop-in”) contained a single 2-kb homology arm (denoted as “integration locus”), a positive selection marker (denoted as “Marker”)), and gene(s) of interest (denoted as “promoter-gene-terminator”). A single crossover event integrated the plasmid into the C. glutamicum chromosome. Integration events are stably maintained in the genome by growth in the presence of antibiotic (25p g/ml kanamycin). Correct genomic integration in colonies derived from loop-in integration were confirmed by colony PCR with UF/IR and DR/IF PCR primers.
Loop-in, loop-out constructs (shown under the heading “Loop-in, loop-out) contained two 2-kb homology arms (5′ and 3′ arms), gene(s) of interest (arrows), a positive selection marker (denoted “Marker”), and a counter-selection marker. Similar to “loop-in” only constructs, a single crossover event integrated the plasmid into the chromosome of C. glutamicum. Note: only one of two possible integrations is shown here. Correct genomic integration was confirmed by colony PCR and counter-selection was applied so that the plasmid backbone and counter-selection marker could be excised. This results in one of two possibilities: reversion to wild-type (lower left box) or the desired pathway integration (lower right box). Again, correct genomic loop-out is confirmed by colony PCR. (Abbreviations: Primers: UF=upstream forward, DR=downstream reverse, IR=internal reverse, IF=internal forward.)
S. cerevisiae Pathway Integration
A “split-marker, double-crossover” genomic integration strategy has been developed to engineer S. cerevisiae strains. FIG. 11 illustrates genomic integration of complementary, split-marker plasmids and verification of correct genomic integration via colony PCR in S. cerevisiae. Two plasmids with complementary 5′ and 3′ homology arms and overlapping halves of a URA3 selectable marker (direct repeats shown by the hashed bars) were digested with meganucleases and transformed as linear fragments. A triple-crossover event integrated the desired heterologous genes into the targeted locus and re-constituted the full URA3 gene. Colonies derived from this integration event were assayed using two 3-primer reactions to confirm both the 5′ and 3′ junctions (UF/IF/wt-R and DR/IF/wt-F). For strains in which further engineering is desired, the strains can be plated on 5-FOA plates to select for the removal of URA3, leaving behind a small single copy of the original direct repeat. This genomic integration strategy can be used for gene knockout, gene knock-in, and promoter titration in the same workflow.
Cell Culture
The workflow established for S. cerevisiae involved a hit-picking step that consolidated successfully built strains using an automated workflow that randomized strains across the plate. For each strain that was successfully built, up to four replicates were tested from distinct colonies to test colony-to-colony variation and other process variation. If fewer than four colonies were obtained, the existing colonies were replicated so that at least four wells were tested from each desired genotype.
The colonies were consolidated into 96-well plates with selective medium (SD-ura for S. cerevisiae) and cultivated for two days until saturation and then frozen with 16.6% glycerol at −80° C. for storage. The frozen glycerol stocks were then used to inoculate a seed stage in minimal media with a low level of amino acids to help with growth and recovery from freezing. The seed plates were grown at 30° C. for 1-2 days. The seed plates were then used to inoculate a main cultivation plate with minimal medium and grown for 48-88 hours. Plates were removed at the desired time points and tested for cell density (OD600), viability and glucose, supernatant samples stored for LC-MS analysis for product of interest.
Cell Density
Cell density was measured using a spectrophotometric assay detecting absorbance of each well at 600 nm. Robotics were used to transfer fixed amounts of culture from each cultivation plate into an assay plate, followed by mixing with 175 mM sodium phosphate (pH 7.0) to generate a 10-fold dilution. The assay plates were measured using a Tecan M1000 spectrophotometer and assay data uploaded to a LIMS database. A non-inoculated control was used to subtract background absorbance. Cell growth was monitored by inoculating multiple plates at each stage, and then sacrificing an entire plate at each time point.
To minimize settling of cells while handling large number of plates (which could result in a non-representative sample during measurement) each plate was shaken for 10-15 seconds before each read. Wide variations in cell density within a plate may also lead to absorbance measurements outside of the linear range of detection, resulting in underestimate of higher OD cultures. In general, the tested strains so far have not varied significantly enough for this be a concern.
Liquid-Solid Separation
To harvest extracellular samples for analysis by LC-MS, liquid and solid phases were separated via centrifugation. Cultivation plates were centrifuged at 2000 rpm for 4 minutes, and the supernatant was transferred to destination plates using robotics. 75 μL of supernatant was transferred to each plate, with one stored at 4° C., and the second stored at 80° C. for long-term storage.
First-Round Genetic Engineering Results in Corynebacteria glutamicum and Saccharomyces cerevisiae 
A library approach was taken to screen heterologous pathway enzymes to establish the histamine pathway. For histidine decarboxylase, 18 heterologous sequences were tested from Bacteria, Archaea, Viridiplantae, Vertebrata, Metazoa, and Arthropoda sources listed in Table 1. The histidine decarboxylases were codon-optimized and expressed in both Saccharomyces cerevisiae and Corynebacteria glutamicum hosts.
Histidine biosynthesis is subject to feedback inhibition, therefore a feedback deregulated ATP phosphoribosyltransferase was tested with the histidine decarboxylases to improve production of histidine, the substrate for histidine decarboxylase. The ATP phosphoribosyltransferases tested were from Salmonella typhimurium and Corynebacteria glutamicum, harboring known deletions and point mutations that render them resistant to feedback inhibition.
First-round genetic engineering results are shown in Table 1 and FIGS. 2 (C. glutamicum) and 3 (S. cerevisiae).







TABLE 1







First-round genetic engineering results in Corynebacteria glutamicum and Saccharomyces cerevisiae






















E1 




E2 





Enzyme 
Enzyme 
Codon

Enzyme 

Enzyme 
Codon




E1
1—
1—
Optimi-
E2
2—
E2
2—
Optimi-


Strain
Titer
Uniprot
activity 
source
zation
Uniprot
activity 
Modifi-
source
zation


name
(μg/L)
ID
name
organism
Abbrev.
ID
name
cations
organism
Abbrev.










Corynebacterium glutamicum

















CgHISM
985.0
E3QMN8
histidine
Methanosarcina
Cg







N_06


decarboxylase
barkeri str.












Wiesmoor








CgHISM
695.7
Q467R8
histidine
Methanosarcina
Cg







N_07


decarboxylase
barkeri (strain












Fusaro / 












DSM 804)








CgHISM
385.0
P00862
histidine
Lactobacillus sp.
Cg







N_08


decarboxylase
(strain 30a)








CgHISM
14370.2
B71459
histidine
Acinetobacter
Cg







N_11


decarboxylase
baumannii












(strain












AB0057)








CgHISM
401.1
E3QMN8
histidine
Methanosarcina
Cg







N_13


decarboxylase
barkeri str.












Wiesmoor








CgHISM
7529.8
P00862
histidine
Lactobacillus
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_15


decarboxylase
sp.


phosphoribosyl-
Q207-E208
typhimurium







(strain 30a)


transferase





CgHISM
4.0
P00862
histidine
Lactobacillus
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_16


decarboxylase
sp.


phosphoribosyl-
Q207-E208
typhimurium







(strain 30a)


transferase





CgHISM
3.9
P23738
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_17


decarboxylase
barkeri (strain


phosphoribosyl-
Q207-E208
typhimurium







Fusaro / 


transferase









DSM 804)








CgHISM
75.0
Q05733
histidine
Drosophila
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_19


decarboxylase
melanogaster


phosphoribosyl-
Q207-E208
typhimurium










transferase





CgHISM
3.8
J6KM89
histidine
Chromo-
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_24


decarboxylase
bacterium


phosphoribosyl-
Q207-E208
typhimurium







sp. LK1


transferase





CgHISM
1.7
E3QMN8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_25


decarboxylase
barkeri str.


phosphoribosyl-
Q207-E208
typhimurium







Wiesmoor


transferase





CgHISM
458.5
E3QMN8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_26


decarboxylase
barkeri str.


phosphoribosyl-
Q207-E208
typhimurium







Wiesmoor


transferase





CgHISM
462.1
Q467R8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_27


decarboxylase
barkeri (strain


phosphoribosyl-
Q207-E208
typhimurium







Fusaro / 


transferase









DSM 804)








CgHISM
1258.2
Q467R8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_28


decarboxylase
barkeri (strain


phosphoribosyl-
Q207-E208
typhimurium







Fusaro / 


transferase









DSM 804)








CgHISM
2126.4
P00862
histidine
Lactobacillus sp.
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_30


decarboxylase
(strain 30a)


phosphoribosyl-
Q207-E208
typhimurium










transferase





CgHISM
234.7
Q05733
histidine
Drosophila
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_33


decarboxylase
melanogaster


phosphoribosyl-
Q207-E208
typhimurium










transferase





CgHISM
11905.3
B71459
histidine
Acinetobacter
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_35


decarboxylase
baumannii (strain


phosphoribosyl-
Q207-E208
typhimurium







AB0057)


transferase





CgHISM
615.0
E3QMN8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_39


decarboxylase
barkeri str.


phosphoribosyl-
Q207-E208
typhimurium







Wiesmoor


transferase










Saccharomyces cerevisiae

















ScHISM
36145.0
P00862
histidine
Lactobacillus 
Sc
Q9Z472
ATP
N215K,
Coryne-
Sc


N_16


decarboxylase
sp.


phosphoribosyl-
L231F, 
bacterium







(strain 30a)


transferase
T235A
glutamicum



ScHISM
2369.9
P54772
histidine
Solanum
Sc
P00499
ATP
Deletion of
Salmonella 
Sc


N_17


decarboxylase
lycopersicum


phosphoribosyl-
Q207-E208
typhimurium










transferase





ScHISM
1747.7
P23738
histidine
Mus musculus
Sc
P00499
ATP
Deletion of
Salmonella 
Cg


N_18


decarboxylase



phosphoribosyl-
Q207-E208
typhimurium










transferase





ScHISM
2432.4
Q05733
histidine
Drosophila
Sc
P00499
ATP
Deletion of
Salmonella 
Sc


N_20


decarboxylase
melanogaster


phosphoribosyl-
Q207-E208
typhimurium










transferase





ScHISM
43606.9
J6KM89
histidine
Chromo-
Sc
P00499
ATP
Deletion of
Salmonella 
Cg


N_21


decarboxylase
bacterium


phosphoribosyl-
Q207-E208
typhimurium







sp. LK1


transferase





ScHISM
43021.9
E3QMN8
histidine
Methanosarcina
Sc
Q9Z472
ATP
N215K,
Coryne-
Sc


N_22


decarboxylase
barkeri str.


phosphoribosyl-
L231F, 
bacterium







Wiesmoor


transferase
T235A
glutamicum



ScHISM
36145.8
Q467R8
histidine
Methanosarcina
Sc
P00499
ATP
Deletion of
Salmonella 
Sc


N_23


decarboxylase
barkeri (strain


phosphoribosyl-
Q207-E208
typhimurium







Fusaro / 


transferase









DSM 804)








ScHISM
47208.0
P00862
histidine
Lactobacillus
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_24


decarboxylase
sp.


phosphoribosyl-
Q207-E208
typhimurium







(strain 30a)


transferase





ScHISM
3130.1
P23738
histidine
Mus musculus
Cg
Q9Z472
ATP
N215K,
Coryne-
Sc


N_25


decarboxylase



phosphoribosyl-
L231F, 
bacterium










transferase
T235A
glutamicum



ScHISM
3262.5
Q05733
histidine
Drosophila
Cg
P00499
ATP
Deletion of
Salmonella 
Sc


N_26


decarboxylase
melanogaster


phosphoribosyl-
Q207-E208
typhimurium










transferase





ScHISM
90811.0
J6KM89
histidine
Chromo-
Cg
Q9Z472
ATP
N215K,
Coryne-
Sc


N_28


decarboxylase
bacterium


phosphoribosyl-
L231F, T235A
bacterium







sp. LK1


transferase

glutamicum



ScHISM
42708.8
E3QMN8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Sc


N_29


decarboxylase
barkeri str.


phosphoribosyl-
Q207-E208
typhimurium







Wiesmoor


transferase





ScHISM
27660.1
Q467R8
histidine
Methanosarcina
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_30


decarboxylase
barkeri 


phosphoribosyl-
Q207-E208
typhimurium







(strain


transferase









Fusaro /












DSM 804)








ScHISM
33356.6
P00862
histidine
Lactobacillus 
Sc
Q9Z472
ATP
N215K,
Coryne-
Sc


N_31


decarboxylase
sp.


phosphoribosyl-
L231F, 
bacterium







(strain 30a)


transferase
T235A
glutamicum



ScHISM
711.5
P54772
histidine
Solanum
Sc
P00499
ATP
Deletion of
Salmonella 
Sc


N_32


decarboxylase
lycopersicum


phosphoribosyl-
Q207-E208
typhimurium










transferase





ScHISM
1523.1
P23738
histidine
Mus musculus
Sc
P00499
ATP
Deletion of
Salmonella 
Cg


N_33


decarboxylase



phosphoribosyl-
Q207-E208
typhimurium










transferase





ScHISM
43170.7
E3QMN8
histidine
Methanosarcina
Sc
Q9Z472
ATP
N215K,
Coryne-
Sc


N_37


decarboxylase
barkeri str.


phosphoribosyl-
L231F, 
bacterium







Wiesmoor


transferase
T235A
glutamicum



ScHISM
30675.5
Q467R8
histidine
Methanosarcina
Sc
P00499
ATP
Deletion of
Salmonella 
Sc


N_38


decarboxylase
barkeri (strain


phosphoribosyl-
Q207-E208
typhimurium







Fusaro / 


transferase









DSM 804)








ScHISM
38293.2
P00862
histidine
Lactobacillus 
Cg
P00499
ATP
Deletion of
Salmonella 
Cg


N_39


decarboxylase
sp.


phosphoribosyl-
Q207-E208
typhimurium







(strain 30a)


transferase





Note:


“Cg” refers to codon optimization for Corynebacterium glutamicum; “Sc” refers to codon optimization for Saccharomyces cerevisiae.






Second-Round Genetic Engineering Results in Corynebacteria glutamicum and Saccharomyces cerevisiae 
A library approach was taken to improve histamine production by separately expressing each upstream pathway enzyme with a constitutive promoter to screen for the rate-limiting step. The histidine pathway enzymes screened are listed in Table 2. In addition, the enzymes in Table 2, the strains contained the best enzymes from first round: the Corynebacteria glutamicum host contains histidine decarboxylase (UniProt ID B7I459) (SEQ ID NO: 1) and ATP phosphoribosyltransferase (UniProt ID P00499) (SEQ ID NO: 5) containing the deletion Q207-E208, and the Saccharomyces cerevisiae host contains histidine decarboxylase (UniProt ID J6KM89)(SEQ ID NO: 6) and ATP phosphoribosyltransferase (UniProt ID Q9Z472) (SEQ ID NO: 7) containing the amino acid substitutions N215K, L231F and T235A.
Second-round genetic engineering results are shown in Table 2 and FIGS. 4 (C. glutamicum) and 5 (S. cerevisiae).
In C. glutamicum, a titer of 24 mg/L was achieved after two rounds of engineering from the integration of two genes: a histidine decarboxylase gene from Acinetobacter baumannii, and constitutive expression of an imidazoleglycerol-phosphate dehydratase from C. glutamicum. 
In S. cerevisiae, a titer of 385 mg/L was achieved in two rounds of engineering from the integration of three genes: a histidine decarboxylase gene from Chromobacterium sp. LK1 (SEQ ID NO: 6), an ATP phosphoribosyltransferase from C. glutamicum containing the amino acid substitutions N215K, L231F, and T235A (SEQ ID NO: 7), and a constitutively expressed ATP phosphoribosyltransferase from S. cerevisiae (SEQ ID NO: 3).







TABLE 2







Second-round genetic engineering results in genetic engineering results in


Corynebacteria glutamicum and Saccharomyces cerevisiae

















E1 Codon



Titer
E1

Enzyme 1-
Optimization


Strain name
(μg/L)
Uniprot ID
Enzyme 1-activity name
source organism
Abbrev.





Corynebacteria







glutamicum







CgHISMN_41
13702.1
O68602
1-(5-phosphoribosyl)5[(5-
Corynebacterium
Native





phosphoribosylamino)
glutamicum






methylideneamino]







imidazole-4-







carboxamide isomerase




CgHISMN_42
12671.2
Q9KJU3
Imidazoleglycerol-
Corynebacterium
Native





phosphate dehydratase
glutamicum



CgHISMN_43
11800.4
Q9KJU4
Histidinol-phosphate
Corynebacterium
Native





aminotransferase
glutamicum



CgHISMN_44
8667.2
Q8NNT5
Histidinol dehydrogenase
Corynebacterium
Native






glutamicum



CgHISMN_45
12375.3
Q9Z471
Phosphoribosyl-
Corynebacterium
Native





ATP pyrophosphatase
glutamicum



CgHISMN_46
10963.6
O31139
Imidazole glycerol
Corynebacterium
Native





phosphate synthase subunit
glutamicum



CgHISMN_47
16246.0
O69043
Imidazole glycerol
Corynebacterium
Native





phosphate synthase subunit
glutamicum



CgHISMN_48
13038.8
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium
Native






glutamicum



CgHISMN_49
10749.0
Q8NNT9
phosphoribosyl-
Corynebacterium
Native





AMP cyclohydrolase
glutamicum



CgHISMN_50
12960.8
O68602
1-(5-phosphoribosyl)5[(5-
Corynebacterium
Native





phosphoribosylamino)
glutamicum






methylideneamino]







imidazole-4-







carboxamide isomerase




CgHISMN_51
9958.4
Q9KJU3
Imidazoleglycerol-
Corynebacterium
Native





phosphate dehydratase
glutamicum



CgHISMN_52
18963.0
Q9KJU4
Histidinol-phosphate
Corynebacterium
Native





aminotransferase
glutamicum



CgHISMN_53
20328.9
Q8NNT5
Histidinol dehydrogenase
Corynebacterium
Native






glutamicum



CgHISMN_54
20051.4
O31139
Imidazole glycerol phosphate
Corynebacterium
Native





synthase subunit
glutamicum



CgHISMN_55
15070.9
O69043
Imidazole glycerol phosphate
Corynebacterium
Native





synthase subunit
glutamicum



CgHISMN_56
12799.1
O68602
1-(5-phosphoribosyl)5[(5-
Corynebacterium
Native





phosphoribosylamino)
glutamicum






methylideneamino]







imidazole-4-







carboxamide isomerase




CgHISMN_57
24773.6
Q9KJU3
Imidazoleglycerol-
Corynebacterium
Native





phosphate dehydratase
glutamicum



CgHISMN_58
15268.6
Q9KJU4
Histidinol-phosphate
Corynebacterium
Native





aminotransferase
glutamicum



CgHISMN_59
12555.0
Q8NNT5
Histidinol dehydrogenase
Corynebacterium
Native






glutamicum



CgHISMN_60
17725.6
Q9Z471
Phosphoribosyl-
Corynebacterium
Native





ATP pyrophosphatase
glutamicum



CgHISMN_61
18777.4
O69043
Imidazole glycerol
Corynebacterium
Native





phosphate synthase subunit
glutamicum



CgHISMN_62
19782.8
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium
Native






glutamicum



CgHISMN_63
15092.7
Q8NNT9
phosphoribosyl-
Corynebacterium
Native





AMP cyclohydrolase
glutamicum



Saccharomyces







cerevisiae







ScHISMN_41
385518.2
P00498
ATP phosphoribosyltransferase
Saccharomyces
Native






cerevisiae



ScHISMN_42
70003.1
P00815
histidinol dehydrogenase,
Saccharomyces
Native





phosphoribosyl-AMP cyclohydrolase,
cerevisiae






phosphoribosyl-ATP diphosphatase




ScHISMN_43
75039.5
P33734
Imidazole glycerol phosphate
Saccharomyces
Native





synthase subunit HisF
cerevisiae



ScHISMN_44
71402.5
P07172
histidinol-phosphate transaminase
Saccharomyces
Native






cerevisiae



ScHISMN_46
64866.5
P06633
Imidazoleglycerol-
Saccharomyces
Native





phosphate dehydratase
cerevisiae



ScHISMN_48
113026.6
P00498
ATP phosphoribosyltransferase
Saccharomyces
Native






cerevisiae



ScHISMN_49
79488.5
P00815
histidinol dehydrogenase,
Saccharomyces
Native





phosphoribosyl-AMP cyclohydrolase,
cerevisiae






phosphoribosyl-ATP diphosphatase




ScHISMN_50
92719.6
P33734
Imidazole glycerol phosphate
Saccharomyces
Native





synthase subunit HisF
cerevisiae



ScHISMN_51
88847.1
P07172
histidinol-phosphate transaminase
Saccharomyces
Native






cerevisiae



ScHISMN_52
70650.9
P38635
histidinol-phosphatase
Saccharomyces
Native






cerevisiae



ScHISMN_53
74127.8
P06633
Imidazoleglycerol-
Saccharomyces
Native





phosphate dehydratase
cerevisiae



ScHISMN_56
73080.2
P00815
histidinol dehydrogenase,
Saccharomyces
Native





phosphoribosyl-AMP cyclohydrolase,
cerevisiae






phosphoribosyl-ATP diphosphatase




ScHISMN_57
78656.1
P33734
Imidazole glycerol phosphate
Saccharomyces
Native





synthase subunit HisF
cerevisiae



ScHISMN_58
69769.0
P07172
histidinol-phosphate transaminase
Saccharomyces
Native






cerevisiae



ScHISMN_59
59139.1
P38635
histidinol-phosphatase
Saccharomyces
Native






cerevisiae



ScHISMN_60
65506.7
P06633
Imidazoleglycerol-
Saccharomyces
Native





phosphate dehydratase
cerevisiae









Third-Round Genetic Engineering Results in Saccharomyces cerevisiae 
Histamine production was further pursued in S. cerevisiae, and we designed plasmids to integrate additional copies of upstream pathway genes expressed by a strong constitutive promoter to avoid native regulation of a gene (Table 3). An expanded search was undertaken to test additional histidine decarboxylases that have similar sequences to the enzymes initially identified as active (Table 3).
In parallel we pursued modulating native gene expression to further improve histamine production. Our engineering approach was to take a best S. cerevisiae strain from the second round and test either a strong or weak constitutive promoter in place of the native promoter. Gene targets for promoter changes were selected to redirect flux supply precursors to histidine. Strain designs being tested include designs for increasing pentose phosphate pathway flux by expressing a non-native feedback deregulated glucose-6-phosphate dehydrogenase (zwf) and decreasing the “lower” pentose phosphate pathway flux thru the sugar isomerase enzymes.
Promoter replacements for lower expression of genes that are thought to be essential (i.e., cannot be deleted), but were expected to increase the upper glycolysis metabolite pool available for histamine production, targeted: 1) enolase (Eno2), to reduce flux through lower glycolysis, 2) pyruvate dehydrogenase (PDH, Lpd1) for lower flux through the C3/C2 node, and 3) pentose phosphate pathway sugar isomerases, which use the histamine metabolite precursor ribose-5-phosphate (Tall). An illustrative list of promoter-swap (“proswap”) and deletion (“knockout”) targets in S. cerevisiae includes:
















Annotation_name
Type
Promoter_name
Gene_name









YDR380W
knockout

Aro10



YDL047W
knockout

Sit4



YML035C
knockout

Amd1



YMR020W
knockout

Fms1



YNL229C
knockout

Ure2



YJL052W
proswap
pRnr1
Tdh1



YJR009C
proswap
pRnr1
Tdh2



YGR192C
proswap
pRnr1
Tdh3



YFL018C
proswap
pRnr1
Lpd1



YHR174W
proswap
pRnr1
Eno2



YNR001C
proswap
pRnr1
Cit1



YCR012W
proswap
pRnr1
Pgk1



YLR354C
proswap
pRnr1
Tal1



YBR117C
proswap
pRnr1
Tkl2



YPR074C
proswap
pRnr1
Tkl1



YML035C
proswap
pRev1
Amd1



YHR216W
proswap
pRev1
Imd2



YOR155C
proswap
pRev1
Isn1



YNL229C
proswap
pRev1
Ure2



YER086W
proswap
pRnr1
Ilv1



YDR380W
proswap
pRnr1
Aro10



YEL009C
proswap
pRev1
Gcn4










Promoters were selected based on expression data from Lee et al [7].
Additional genetic engineering results for S. cerevisiae are shown in Table 3 and FIG. 10. The parent strain for the strain designs shown in Table 3 (also the reference strain, ScHISMN_41) contained a histidine decarboxylase (UniProt ID J6KM89) and an ATP phosphoribosyltransferase (UniProt ID Q9Z472) harboring the amino acid substitutions N215K, L231F and T235A, and the ATP phosphoribosyltransferase from S. cerevisiae. The reference strain had a histamine titer of 131 mg/L.
Improved titer was observed in strains that expressed each of the following enzymes from a strong constitutive promoter:
1. Transketolase (EC 2.2.1.1) (SEQ ID NO: 27), which catalyzes the interconversion of sugars in the pentose phosphate pathway and produces ribose-5-phosphate, which is a precursor to PPRP, the initial metabolite in the histidine biosynthesis pathway.
2. Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) (SEQ ID NO: 28) (highest titer: 191 mg/L relative to control in experiment 131 mg/L).
3. ATP phosphoribosyltransferase (EC 2.4.2.17) (SEQ ID NO: 3).
4. Trifunctional histidinol dehydrogenase (EC 1.1.1.23)/phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19)/phosphoribosyl-ATP diphosphatase (EC 3.6.1.31) (SEQ ID NO: 29).
5. Histidinol-phosphate aminotransferase (EC 2.6.1.9) (SEQ ID NO: 14).
6. 5′ProFAR isomerase (EC 5.3.1.16) (SEQ ID NO: 31).
7. Imidazole glycerol phosphate synthase (EC 4.3.1.B2) (SEQ ID NO: 21).
8. Triose-phosphate isomerase (EC 5.3.1.1), harboring the amino acid substitutions harboring the amino acid substitutions I170V (SEQ ID NO: 32) or I170T [8].
9. Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49), harboring the amino acid substitution A243T (SEQ ID NO: 26).
10. Various histidine decarboxylases (EC 4.1.1.22):

    
    
        a. UniProt ID A0A089YPE5 (SEQ ID NO: 33)
        b. UniProt ID A0A126S6G9 (SEQ ID NO: 34)
        c. UniProt ID A0A0A1R6V3 (SEQ ID NO: 35)
        d. UniProt ID A0A1W0CM88 (SEQ ID NO: 36)
        e. UniProt ID P00862 (SEQ ID NO: 4)
        f. UniProt ID A0A0K6GJ74 (SEQ ID NO: 37)
        g. UniProt ID T0QL99 (SEQ ID NO: 38)
        h. UniProt ID A0A1B8HLR1 (SEQ ID NO: 39)
    
    









TABLE 3







Third-round genetic engineering results in Saccharomyces cerevisiae


Built and tested strain designs:






















Enzyme 
E1
Enzyme 

Enzyme 
Enzyme

Enzyme 
E3
Enzyme 




E1
1—
Modi-
1—
E2
2—
2—
E3
3—
Modi-
3—


Strain
Titer
Uniprot
activity 
fica-
source
Uniprot
activity 
source
Uniprot
activity 
fica-
source


name
(μg/L)
ID
name
tions
organism
ID
name
organism
ID
name
tions
organism





Sc
39059
A0A0C
Histidine

Lactobacillus









HISM

1PR48
decarboxy-

fructivorans









N_100


lase











ScHIS
144871
T0QL99
Histidine

Aeromonas









MN_


decarboxy-

salmonicida









101


lase

subsp.














pectinolytica














34mel









ScHIS
143763
A0A1B8
Histidine

Morganella









MN_

HLR1
decarboxy-

psychrotoler-









102


lase

ans









ScHIS
155931
Q9Z472
ATP

Coryne-
P00815
tri-
Saccharo-
P0A717
Ribose-

Escheri-


MN_


phosphori-

bacterium

functional
myces

phos-

chia


l03


bosyl-

glutamicum

histidinol
cerevisiae

phate

coli





transferase

(strain 

dehydro-
S288c

pyro-

(strain







ATCC

genase/


phos-

K12)







13032 / 

phos-


pho-









DSM

phoribo-


kinase









20300 / 

syl-AMP












JCM

cyclo-












1318 / 

hydrolase/












LMG

phos-












3730 / 

phoribo-












NCIMB

syl-ATP












10025)

diphos-














phatase







ScHIS
151846
P0A717
Ribose-

Escherichia









MN_


phosphate

coli 









104


pyrophos-

(strain












pho-

K12)












kinase











ScHIS
191110
Q12265
Ribose-

Saccharo-
P38620
Ribose-
Saccharo-
Q680A5
Ribose-

Arabi-


MN_


phosphate

myces

phosphate
myces

phos-

dopsis


105


pyropho-

cerevisiae

pyrophos-
cerevisiae

phate

thaliana





spho-

(strain 

phokinase
S288c

pyro-

(Mouse-





kinase

ATCC




phos-

ear







204508 /




pho-

cress)







S288c)




kinase









(Baker's














yeast)









ScHIS
160586
P32895
Ribose-

Saccharo-
P38689
Ribose-
Saccharo-






MN_


phosphate

myces

phosphate
myces






106


pyrophos-

cerevisiae

pyrophos-
cerevisiae









pho-

(strain 

phokinase
S288c









kinase

ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
157191
P23254
Trans-

Saccharo-
P32895
Ribose-
Saccharo-
P38689
Ribose-

Saccharo-


MN_


ketolase

myces

phosphate
myces

phos-

myces


107




cerevisiae

pyrophos-
cerevisiae

phate

cerevisiae







(strain 

phokinase
S288c

pyro-

S288c







ATCC




phos-









204508 /




pho-









S288c)




kinase









(Baker's














yeast)









ScHIS
168183
P23254
Trans-

Saccharo-
Q12265
Ribose-
Saccharo-
P38620
Ribose-

Saccharo-


MN_


ketolase

myces

phosphate
myces

phos-

myces


108




cerevisiae

pyrophos-
cerevisiae

phate

cerevisiae







(strain 

phokinase
S288c

pyro-

S288c







ATCC




phos-









204508 /




pho-









S288c)




kinase









(Baker's














yeast)









ScHIS
125249
P23254
Trans-

Saccharo-
Q12265
Ribose-
Saccharo-
Q680A5
Ribose-

Arabi-


MN_


ketolase

myces

phosphate
myces

phos-

dopsis


109




cerevisiae

pyrophos-
cerevisiae

phate

thaliana







(strain 

phokinase
S288c

pyro-

(Mouse-







ATCC




phos-

ear







204508 /




pho-

cress)







S288c)




kinase









(Baker's














yeast)









ScHIS
157653
P23254
Trans-

Saccharo-
P0A717
Ribose-
Escherichia






MN_


ketolase

myces

phosphate
coli






110




cerevisiae

pyrophos-
(strain











(strain 

phokinase
K12)











ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
136093
P23254
Trans-

Saccharo-
P15019
Trans-
Saccharo-
P0A717
Ribose-

Escheri-


MN_


ketolase

myces

aldolase
myces

phos-

chia


111




cerevisiae


cerevisiae

phate

coli







(strain 


S288c

pyro-

(strain







ATCC




phos-

K12)







204508 /




pho-









S288c)




kinase









(Baker's














yeast)









ScHIS

P06775
Histidine

Saccharo-









MN_


permease

myces









112




cerevisiae














(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
160417
P00815
trifunctional

Saccharo-
P40545
5'ProFAR
Saccharo-
O59667
Bifunc-

Schizo-


MN_


histidinol

myces

isomerase
myces

tional

saccharo-


113


dehydro-

cerevisiae


cerevisiae

phos-

myces





genase/

(strain 


S288c

phoribo-

pombe





phos-

ATCC




syl-AMP

ATCC





phoribo-

204508 /




cyclo-

24843





syl-AMP

S288c)




hydrolase 







cyclo-

(Baker's




and







hydro-

yeast)




phos-







lase/






phoribo-







phos-






syl-ATP







phoribo-






pyrophos-







syl-ATP






phatase







diphos-














phatase











ScHIS
116907
P06633
Imidazole-

Saccharo-
P07172
Histidinol-
Saccharo-
P38635
Histidi-

Saccharo-


MN_


glycerol-

myces

phosphate
myces

nol-

myces


114


phosphate

cerevisiae

amino-
cerevisiae

phos-

cerevisiae





dehydratase

(strain 

trans-
S288c

phatase

S288c







ATCC

ferase












204508 /














S288c)














(Baker's














yeast)









ScHIS
131308













MN_














41














ScHIS
123614
P00815
trifunctional

Saccharo-









MN_


histidinol

myces









73


dehydro-

cerevisiae












genase/

(strain 












phos-

ATCC












phoribo-

204508 /












syl-AMP

S288c)












cyclohydro-

(Baker's












lase/

yeast)












phos-














phoribo-














syl-ATP














diphos-














phatase











ScHIS
129393
P06633
Imidazole-

Saccharo-









MN_


glycerol-

myces









74


phosphate

cerevisiae












dehydratase

(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
151455
P07172
Histidinol-

Saccharo-









MN_


phosphate

myces









75


aminotrans-

cerevisiae












ferase

(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
138833
P00498
ATP

Saccharo-









MN_


phosphori-

myces









76


bosyl-

cerevisiae












transferase

(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
164217
P40545
5′ProFAR

Saccharo-









MN_


isomerase

myces









77




cerevisiae














(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
159871
P33734
Imidazole

Saccharo-









MN_


glycerol

myces









78


phosphate

cerevisiae












synthase

(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
145179
P00942
Triosephos-
I170V
Saccharo-









MN_


phate

myces









79


isomerase

cerevisiae














(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
137192
P00942
Triosephos-
I170T
Saccharo-









MN_


phate

myces









80


isomerase

cerevisiae














(strain 














ATCC














204508 /














S288c)














(Baker's














yeast)









ScHIS
139699
Q9Z472
ATP

Coryne-









MN_


phosphori-

bacterium









81


bosyl-

glutamicum












transferase

(strain 














ATCC














13032 / 














DSM














20300 / 














JCM














1318 /














LMG














3730 / 














NCIMB














10025)









ScHIS
148665
Q9Z472
ATP
N215K,
Coryne-









MN_


phosphori-
L231F,
bacterium









82


bosyl-
T235A
glutamicum












transferase

(strain 














ATCC














13032 / 














DSM














20300 / 














JCM














1318 /LMG














3730 / 














NCIMB














10025)









ScHIS
109350
Q9Z472
ATP
N215K,
Coryne-
P00815
tri-
Saccharo-






MN_


phosphori-
L231F,
bacterium

functional
myces






84


bosyl-
T235A
glutamicum

histidinol
cerevisiae









transferase

(strain 

dehydro-
S288c











ATCC

genase/












13032 / 

phos-












DSM

phoribo-












20300 / 

syl-AMP












JCM

cyclo-












1318 / 

hydro-












LMG

lase/












3730 / 

phos-












NCIMB

phoribo-












10025)

syl-ATP














diphos-














phatase







ScHIS
144154
Q9Z472
ATP

Coryne-
P00815
tri-
Saccharo-
P00942
Triose-
I170V
Saccharo-


MN_


phosphori-

bacterium

functional
myces

phos-

myces


85


bosyl-

glutamicum

histidinol
cerevisiae

phate

cerevisiae





transferase

(strain 

dehydro-
S288c

iso-

S288c







ATCC

genase/


merase









13032 / 

phos-












DSM

phoribo-












20300 / 

syl-AMP












JCM

cyclo-












1318 / 

hydro-












LMG

lase/












3730 / 

phos-












NCIMB

phoribo-












10025)

syl-ATP














diphos-














phatase







ScHIS
145171
Q9Z472
ATP
N215K,
Coryne-
P00815
tri-
Saccharo-
P00942
Triose-
I170V
Saccharo-


MN_


phosphori-
L231F,
bacterium

functional
myces

phos-

myces


86


bosyl-
T235A
glutamicum

histidinol
cerevisiae

phate

cerevisiae





transferase

(strain 

dehydro-
S288c

iso-

S288c







ATCC

genase/


merase









13032 / 

phos-












DSM

phoribo-












20300 / 

syl-AMP












JCM

cyclo-












1318 / 

hydrolase/












LMG

phos-












3730 / 

phoribo-












NCIMB

syl-ATP












10025)

diphos-














phatase







ScHIS
166497
Q9Z472
ATP

Coryne-
P00815
tri-
Saccharo-
A4QEF2
Glucose-
A243T
Coryne-


MN_


phosphori-

bacterium

functional
myces

6-phos-

bacterium


87


bosyl-

glutamicum

histidinol
cerevisiae

phate

glutami-





transferase

(strain 

dehydro-
S288c

1-

cum







ATCC

genase/


dehydro-

(strain R)







13032 / 

phos-


genase









DSM

phoribo-












20300 /

syl-AMP












JCM

cyclo-












1318 / 

hydro-












LMG

lase/












3730 / 

phos-












NCIMB

phoribo-












10025)

syl-ATP














diphos-














phatase







ScHIS
152555
Q9Z472
ATP
N215K,
Coryne-
P00815
tri-
Saccharo-
A4QEF2
Glucose-
A243T
Coryne-


MN_


phosphori-
L231F,
bacterium

functional
myces

6-phos-

bacterium


88


bosyl-
T235A
glutamicum

histidinol
cerevisiae

phate 

glutami-





transferase

(strain 

dehydro-
S288c

1-

cum







ATCC

genase/


dehydro-

(strain R)







13032 / 

phos-


genase









DSM

phoribo-












20300 / 

syl-AMP












JCM

cyclo-












1318 / 

hydro-












LMG

lase/












3730 / 

phos-












NCIMB

phoribo-












10025)

syl-ATP














diphos-














phatase







ScHIS
143866
O66000
Histidine

Oenococcus









MN_


decarboxy-

oeni









89


lase

(Leuconostoc














oenos)









ScHIS
124157
A0A0R
Histidine

Lactobacillus









MN_

1Y874
decarboxy-

aviarius









90


lase

subsp.














aviarius 














DSM














20655









ScHIS
68849
A0A1H
Histidine
S9R
Pseudo-









MN_

1TEB8
decarboxy-

monas









92


lase

sp. bs2935









ScHIS
157127
A0A089
Histidine

Pseudo-









MN_

YPE5
decarboxy-

monas









93


lase

rhizosphaerae









ScHIS
175497
A0A126
Histidine

Pseudo-









MN_

S6G9
decarboxy-

monas putida









94


lase

(Arthrobacter














siderocap-














sulatus)









ScHIS
116642
A0A0J6
Histidine

Chromo-









MN_

KM89
decarboxy-

bacterium sp.









95


lase

LK1









ScHIS
171681
A0A0A1
Histidine

Citrobacter









MN_

R6V3
decarboxy-

pasteurii









96


lase











ScHIS
171393
A0A1W
Histidine

Chromo-









MN_

0CM88
decarboxy-

bacterium









97


lase

haemolyticum









ScHIS
152065
P00862
Histidine

Lactobacillus









MN_


decarboxy-

sp. (strain









98


lase

30a)









ScHIS
148362
A0A0K6
Histidine

Lactobacillus









MN_

GJ74
decarboxy-

reuteri









99


lase





Note:


E1, E2, and E3 genes were codon-optimized according to modified codon usage for Cg and Sc






Example 2—Host Evaluation for Histamine Production
Histamine production was also tested in two additional hosts, Bacillus subtilus and Yarrowia lipolytica, which were engineered to express the enzymes from the best-performing Corynebacterium glutamicum and Saccharomyces cerevisiae strains.
Host evaluation designs were selected to express 1-3 enzymes and, each design was tested with four different codon optimizations based on the host organisms C. glutamicum, S. cerevisiae, B. subtilis, and Y. lipolytica. The codon optimizations tested were based on the Kazusa codon usage tables tabulated for each host for gene codon optimization (www.kazusa.or.jp/codon/).
Histamine production was demonstrated in Y. lipolytica (FIG. 6) and B. subtilis (FIG. 7) and further improved in C. glutamicum (FIG. 9) and S. cerevisiae (FIG. 8).
In Y. lipolytica (FIG. 6, Table 4, below) the best performing strain produced 505 mg/L histamine and expressed the histidine decarboxylase from Acinetobacter baumannii strain AB0057 (UniProt ID B7I459), where the DNA sequence was codon-optimized for Y. lipolytica, and the ATP phosphoribosyltransferase from S. cerevisiae S288c (UniProt ID P00498), where the DNA sequence was codon optimized for Y. lipolytica. The same two genes were also tested where the DNA sequence was codon-optimized for B. subtilis and S. cerevisiae and the resulting strains produced no histamine titer.
The second best-performing strain in Y. lipolytica also expressed the histidine decarboxylase from Acinetobacter baumannii strain AB0057 (UniProt ID B7I459), where the DNA sequence was codon-optimized for Y. lipolytica, and the ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (UniProt ID P00499), where the DNA was codon optimized for Y. lipolytica. Versions of these two genes were also tested where the DNA sequence was codon optimized for B. subtilis (which produced 0 titer), codon-optimized for S. cerevisiae (which produced 33 micrograms histamine) and codon-optimized using a combined codon table for S. cerevisiae and C. glutamicum (produced 97 mg/L histamine).
The third best-performing strain in Y. lipolytica produced 258 mg/L histamine and expressed the histidine decarboxylase from Chromobacterium sp. LK1 (UniProt ID A0A0J6KM89), where the DNA sequence was codon optimized for Y. lipolytica, and the ATP phosphoribosyltransferase from C. glutamicum ATCC 13032 (UniProt ID Q9Z472) harboring the amino acid substitutions N215K, L231F, T235A (SEQ ID NO: 7), where the DNA sequence was codon-optimized for Y. lipolytica (SEQ ID NO: 64). Versions of these two genes were also tested where the DNA sequences were codon-optimized for S. cerevisiae (SEQ ID NO: 65, 66) or B. subtilis (SEQ ID NO: 67, 68), and these Y. lipolytica strains produced 1.8 mg/L and 0.3 mg/L, respectively. Accordingly, codon-optimization of genes affects expression in Y. lipolytica. 
In B. subtilis (FIG. 7, Table 5, below) the best performing strain produced 18 mg/L histamine and expressed the histamine decarboxylase from Lactobacillus sp. (strain 30a) (UniProt ID P00862)(SEQ ID NO: 4) with the ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (UniProt ID P00499)(SEQ ID NO: 5) where the DNA sequence was codon optimized for Bacillus subtilis (SEQ ID NO: 69, 59). The same two genes were also tested where the DNA sequence was codon-optimized for S. cerevisiae (SEQ ID NO: 70, 60) or modified codon usage table for C. glutamicum and S. cerevisiae (SEQ ID NO: 71, 62), and these strains produced 6.7 mg/L or 0 mg/L histamine, respectively.
The host evaluation designs were also tested in S. cerevisiae and C. glutamicum. In S. cerevisiae (FIG. 8, Table 6, below) the best-performing strain produced 111 mg/L histamine and expressed the histamine decarboxylase from Chromobacterium sp. LK1 (UniProt ID A0A0J6KM89)(SEQ ID NO: 51) and the ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c (UniProt ID P00498)(SEQ ID NO: 3), where the DNA sequences were codon-optimized for Y. lipolytica (SEQ ID NO: 63, 53). The same two genes were also tested where the DNA sequences were codon optimized for S. cerevisiae (SEQ ID NO: 65, 57) and B. subtilis (SEQ ID NO: 67, 55) produced 86 mg/L and 101 mg/L, respectively.
In C. glutamicum (FIG. 9, Table 7), the best-performing strain produced 68 mg/L histamine and expressed the histamine decarboxylase from Acinetobacter baumannii (strain AB0057) (UniProt ID B7I459) (SEQ ID NO: 1) with the ATP phosphoribosyltransferase from Saccharomyces cerevisiae S288c (UniProt ID P00498) (SEQ ID NO: 3) where the DNA sequences were codon-optimized using a modified codon usage table for C. glutamicum and S. cerevisiae (SEQ ID NO: 72, 73). The same two genes were also tested where the DNA sequence was codon-optimized for Y. lipolytica (SEQ ID NO: 52, 53) or S. cerevisiae (SEQ ID NO: 56, 57), and these strains produced 16 mg/L and 18 microgram/L histamine, respectively.
The second best-performing strain in C. glutamicum produced 15 mg/L histamine and also expressed a histidine decarboxylase from Acinetobacter baumannii strain AB0057 (UniProt ID B7I459) (SEQ ID NO: 1), where the DNA sequence was codon optimized for Y. lipolytica (SEQ ID NO: 52), and an ATP phosphoribosyltransferase from Salmonella typhimurium LT2 (UniProt ID P00499) (SEQ ID NO: 5), where the DNA was codon optimized for Y. lipolytica (SEQ ID NO: 58). These same two genes were also tested, where the DNA sequences were codon-optimized for B. subtilis (SEQ ID NO: 54, 59) (which produced 8 mg/L histamine) or codon-optimized for S. cerevisiae (SEQ ID NO: 56, 60)(which produced 9.3 mg/L histamine).
Since the best performing strain is in the host Y. lipolytica, further strain improvements can be pursued in this host organism. Designs that can further enhance histamine production in Y. lipolytica include:
1. Transketolase (EC 2.2.1.1) (SEQ ID NO: 27), which catalyzes the interconversion of sugars in the pentose phosphate pathway and produces ribose-5-phosphate, which is a precursor to PPRP, the initial metabolite in the histidine biosynthesis pathway.
2. Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) (SEQ ID NO: 28).
3. ATP phosphoribosyltransferase (EC 2.4.2.17) (SEQ ID NO: 5).
4. Trifunctional histidinol dehydrogenase (EC 1.1.1.23)/phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19)/phosphoribosyl-ATP diphosphatase (EC 3.6.1.31) (SEQ ID NO: 20).
5. Histidinol-phosphate aminotransferase (EC 2.6.1.9) (SEQ ID NO: 14).
6. 5′ProFAR isomerase (EC 5.3.1.16) (SEQ ID NO: 31).
7. Imidazole glycerol phosphate synthase (EC 4.3.1.B2) (SEQ ID NO: 21).
8. Triose-phosphate isomerase (EC 5.3.1.1) harboring the amino acid substitution I170V (SEQ ID NO: 32).
9. Glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49) harboring the amino acid substitution A243T (SEQ ID NO: 26).
10. Various histidine decarboxylases:

    
    
        a. UniProt ID A0A089YPE5 (SEQ ID NO: 33)
        b. UniProt ID A0A126S6G9 (SEQ ID NO: 34)
        c. UniProt ID A0A0A1R6V3 (SEQ ID NO: 35)
        d. UniProt ID A0A1W0CM88 (SEQ ID NO: 36)
        e. UniProt ID P00862 (SEQ ID NO: 4)
        f. UniProt ID A0A0K6GJ74 (SEQ ID NO: 37)
        g. UniProt ID T0QL99 (SEQ ID NO: 38)
        h. UniProt ID A0A1B8HLR1 (SEQ ID NO: 39)
    
    


Example 3—Improvement of Histamine Production in Yarrowia lipolytica Engineered to Produce Histamine
Three improvement rounds of genetic engineering were carried out in Yarrowia lipolytica. 
First-Improvement Round Genetic Engineering in Yarrowia lipolytica 
Strategy: Improve flux into histidine and then histamine by overexpression of two enzymes.
















UniProt


Codon



Enzyme Name
ID
Organism
Description
optmization
Mutation







Histidine
B71459
Acinetobacter
Last decarboxylation
Yarrowia
None


decarboxylase

baumannii
step of histamine
lypolytica



(HDC)


biosynthesis




ATP
P00498
Saccharomyces
Upstream step of
Yarrowia
None


phosphoribosyltransferase

cerevisiae
histidine biosynthesis.
lypolytica



(ATP-PRase)


Utilization of ATP







to covert PRPP to







PR-ATP









Summary: ATP phosphoribosyltransferase catalyzes the first committed step of histidine biosynthesis pathway. This enzyme would be allosterically feedback-inhibited by histidine and competitively inhibited by AMP and ADP. The results did not indicate activity and/or inhibition of P00498.
Second-Improvement Round Genetic Engineering in Yarrowia lipolytica 
Strategy: Overexpression of one enzyme. The final step of histamine biosynthesis was enhanced by utilizing the best first-round histidine decarboxylase which was modified to include a solubility tag to improve protein folding.
















UniProt


Codon



Enzyme Name
ID
Organism
Description
optmization
Mutation







Histidine
B71459
Acinetobacter
Last decarboxylation
Yarrowia
None


decarboxylase

baumannii
step of histamine
lypolytica



(HDC)


biosynthesis









Summary: The histidine decarboxylase used for the second round of genetic engineering was the same as for the first round, although the codon optimization was different. Furthermore, an N-terminal solubility tag (MQYKLALNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFT VT, SEQ ID NO:142) was included in the second-round enzyme.
Third-Improvement Round Genetic Engineering in Yarrowia lipolytica 
Strategy: Overexpression of two enzymes in pathways upstream of histidine biosynthesis to improve flux into phosphoribosyl pyrophosphate (PRPP).
















UniProt


Codon



Enzyme Name
ID
Organism
Description
optmization
Mutation







Ribose-phosphate
E7EAU9
Bacillus
ATP dependent step for
Yarrowia
L135I


pyrophosphokinase

amyloliquefaciens
synthesis of PRPP
lypolytica



(RPPK)







Glocose-6-phosphate
A4QEF2
Corynebacterium
Upstream pathway to
Yarrowia
A243T


1-dehydrogenase

glutamicum
push carbon flux into
lypolytica



(G6PDH)


ribose-5-phosphate









Summary: Ribose-phosphate pyrophosphokinase is competitively inhibited ADP. The L135I mutation at the ATP binding site on the enzyme relieves ADP inhibition. This strain expressed histamine at a titer of 1.68 g/L of culture medium.







TABLE 4







First-round results for histamine production in Yarrowia lipolytica



















E1
Enzyme 1
Enzyme 1
E1 Codon
E2
Enzyme 2
E2
Enzyme 2
E2 Codon


Strain
Titer
Uniprot
activity
source
Optimization
Uniprot
activity
Modifi-
source
Optimization


name
(μg/L)
ID
name
organism
Abbrev.
ID
name
cations
organism
Abbrev.




















Yarrowia












lipolytica












YIHISMN_
   0
B71459
Histidine
Acinetobacter
Bacillus
P00498
ATP

Saccharomyces
Bacillus


01


decarboxylase
baumannii
subtilis

phosphoribosyl-

cerevisiae
subtilis






(strain


transferase

S288c







AB0057)








YIHISMN_
   0
B71459
Histidine
Acinetobacter
Saccharo-
P00498
ATP

Saccharomyces
Saccharo-


02


decarboxylase
baumannii
myces

phosphoribosyl-

cerevisiae
myces






(strain
cerevisiae

transferase

S288c
cerevisiae






AB0057)








YIHISMN_
505019
B71459
Histidine
Acinetobacter
Yarrowia
P00498
ATP

Saccharomyces
Yarrowia


03


decarboxylase
baumannii
lipolytica

phosphoribosyl-

cerevisiae
lipolytica






(strain


transferase

S288c







AB0057)








YIHISMN_
   0
P00862
Histidine
Lactobacillus
Bacillus
P00499
ATP

Salmonella
Bacillus


04


decarboxylase
sp.
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain 30a)


transferase

(strain LT2/












SGSC1412/












ATCC 700720)



YIHISMN_
 32011
P00862
Histidine
Lactobacillus
Saccharo-
P00499
ATP

Salmonella
Saccharo-


05


decarboxylase
sp.
myces

phosphoribosyl-

typhimurium
myces






(strain 30a)
cerevisiae

transferase

(strain LT2/
cerevisiae











SGSC1412/












ATCC 700720)



YIHISMN_
  833
P00862
Histidine
Lactobacillus
Yarrowia
P00499
ATP

Salmonella
Yarrowia


06


decarboxylase
sp.
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain 30a)


transferase

(strain LT2/












SGSC1412/












ATCC 700720)



YIHISMN_
  299
A0A0J6
Histidine
Chromo-
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


07

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






sp. LK1


transferase
T235A
ATCC 13032



YIHISMN_
 1778
A0A0J6
Histidine
Chromo-
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


08

KM89
decarboxylase
bacterium
myces

phosphoribosyl-
L231F,
glutamicum
myces






sp. LK1
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


YIHISMN_
257949
A0A0J6
Histidine
Chromo-
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


09

KM89
decarboxylase
bacterium
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






sp. LK1


transferase
T235A
ATCC 13032



YIHISMN_
   0
B71459
Histidine
Acinetobacter
Bacillus
P00499
ATP

Salmonella
Bacillus


10


decarboxylase
baumannii
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain


transferase

LT2







AB0057)








YIHISMN_
 96836
B71459
Histidine
Acinetobacter
modified
P00499
ATP

Salmonella
modified


11


decarboxylase
baumannii
codon

phosphoribosyl-

typhimurium
codon






(strain
usage for 

transferase

LT2
usage for 






AB0057)
Cg and Sc




Cg and Sc


YIHISMN_
  33
B71459
Histidine
Acinetobacter
Saccharo-
P00499
ATP

Salmonella
Saccharo-


12


decarboxylase
baumannii
myces

phosphoribosyl-

typhimurium
myces






(strain
cerevisiae

transferase

LT2
cerevisiae






AB0057)








YIHISMN_
366139
B71459
Histidine
Acinetobacter
Yarrowia
P00499
ATP

Salmonella
Yarrowia


13


decarboxylase
baumannii
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain


transferase

LT2







AB0057)








YIHISMN_
  23
P00862
Histidine
Lactobacillus
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


14


decarboxylase
sp.
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






(strain 30a)


transferase
T235A
ATCC 13032



YIHISMN_
  26
P00862
Histidine
Lactobacillus
modified
Q9Z472
ATP
N215K,
Corynebacterium
modified


15


decarboxylase
sp.
codon

phosphoribosyl
L231F,
glutamicum
codon






(strain 30a)
usage for 

transferase
T235A
ATCC 13032
usage for 







Cg and Sc




Cg and Sc


YIHISMN_
  56
P00862
Histidine
Lactobacillus
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


16


decarboxylase
sp.
myces

phosphoribosyl-
L231F,
glutamicum
myces






(strain 30a)
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


YIHISMN_
 1406
P00862
Histidine
Lactobacillus
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


17


decarboxylase
sp.
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






(strain 30a)


transferase
T235A
ATCC 13032



YIHISMN_

A0A0J6
Histidine
Chromo-
Bacillus
P00498
ATP

Saccharomyces
Bacillus


18

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-

cerevisiae
subtilis






sp. LK1


transferase

S288c



YIHISMN_
 90046
A0A0J6
Histidine
Chromo-
modified
P00498
ATP

Saccharomyces
modified


19

KM89
decarboxylase
bacterium
codon

phosphoribosyl-

cerevisiae
codon






sp. LK1
usage for 

transferase

S288c
usage for 







Cg and Sc




Cg and Sc


YIHISMN_
 1639
A0A0J6
Histidine
Chromo-
Saccharo-
P00498
ATP

Saccharomyces
Saccharo-


20

KM89
decarboxylase
bacterium
myces

phosphoribosyl-

cerevisiae
myces






sp. LK1
cerevisiae

transferase

S288c
cerevisiae
















TABLE 5







First-round results for production of histamine in Bacillus subtilis



















E1
Enzyme 1
Enzyme 1
E1 Codon
E2
Enzyme 2
E2
Enzyme 2
E2 Codon


Strain
Titer
Uniprot
activity
source
Optimization
Uniprot
activity
Modifi-
source
Optimization


name
(μg/L)
ID
name
organism
Abbrev.
ID
name
cations
organism
Abbrev.




















BsHISMN_

B71459
Histidine
Acinetobacter
Yarrowia
P00498
ATP

Saccharomyces
Yarrowia


01


decarboxylase
baumannii
lipolytica

phosphoribosyl-

cerevisiae
lipolytica






(strain


transferase

S288c







AB0057)








BsHISMN_
919.7
P00862
Histidine
Lactobacillus
Yarrowia
P00499
ATP

Salmonella
Yarrowia


02


decarboxylase
sp.
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain 30a)


transferase

LT2



BsHISMN_
2.4
A0A0J6
Histidine
Chromo-
modified
Q9Z472
ATP
N215K,
Corynebacterium
modified


03

KM89
decarboxylase
bacterium
codon

phosphoribosyl-
L231F,
glutamicum
codon






sp. LK1
usage for 

transferase
T235A
ATCC 13032
usage for 







Cg and Sc




Cg and Sc


BsHISMN_
9156.1
P00862
Histidine
Lactobacillus
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


04


decarboxylase
sp.
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






(strain 30a)


transferase
T235A
ATCC 13032



BsHISMN_
5057.2
P00862
Histidine
Lactobacillus
modified
Q9Z472
ATP
N215K,
Corynebacterium
modified


05


decarboxylase
sp.
codon

phosphoribosyl
L231F,
glutamicum
codon






(strain 30a)
usage for 

transferase
T235A
ATCC 13032
usage for 







Cg and Sc




Cg and Sc


BsHISMN_

P00862
Histidine
Lactobacillus
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


06


decarboxylase
sp.
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






(strain 30a)


transferase
T235A
ATCC 13032



BsHISMN_
2532.4
B71459
Histidine
Acinetobacter
Bacillus
P00498
ATP

Saccharomyces
Bacillus


07


decarboxylase
baumannii
subtilis

phosphoribosyl-

cerevisiae
subtilis






(strain


transferase

S288c







AB0057)








BsHISMN_
13183.4
B71459
Histidine
Acinetobacter
modified
P00498
ATP

Saccharomyces
modified


08


decarboxylase
baumannii
codon

phosphoribosyl-

cerevisiae
codon






(strain
usage for 

transferase

S288c
usage for 






AB0057)
Cg and Sc




Cg and Sc


BsHISMN_
114.3
B71459
Histidine
Acinetobacter
Saccharo-
P00498
ATP

Saccharomyces
Saccharo-


09


decarboxylase
baumannii
myces

phosphoribosyl-

cerevisiae
myces






(strain
cerevisiae

transferase

S288c
cerevisiae






AB0057)








BsHISMN_
18336.5
P00862
Histidine
Lactobacillus
Bacillus
P00499
ATP

Salmonella
Bacillus


10


decarboxylase
sp.
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain 30a)


transferase

LT2



BsHISMN_
0
P00862
Histidine
Lactobacillus
modified
P00499
ATP

Salmonella
modified


11


decarboxylase
sp.
codon

phosphoribosyl-

typhimurium
codon






(strain 30a)
usage for 

transferase

LT2
usage for 







Cg and Sc




Cg and Sc


BsHISMN_
6778.2
P00862
Histidine
Lactobacillus
Saccharo-
P00499
ATP

Salmonella
Saccharo-


12


decarboxylase
sp.
myces

phosphoribosyl-

typhimurium
myces






(strain 30a)
cerevisiae

transferase

LT2
cerevisiae


BsHISMN_

A0A0J6
Histidine
Chromo-
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


13

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






sp. LK1


transferase
T235A
ATCC 13032



BsHISMN_
1071.1
A0A0J6
Histidine
Chromo-
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


14

KM89
decarboxylase
bacterium
myces

phosphoribosyl-
L231F,
glutamicum
myces






sp. LK1
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


BsHISMN_

A0A0J6
Histidine
Chromo-
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


15

KM89
decarboxylase
bacterium
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






sp. LK1


transferase
T235A
ATCC 13032



BsHISMN_
233.4
B71459
Histidine
Acinetobacter
Bacillus
P00499
ATP

Salmonella
Bacillus


16


decarboxylase
baumannii
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain


transferase

LT2







AB0057)








BsHISMN_
16.2
B71459
Histidine
Acinetobacter
modified
P00499
ATP

Salmonella
modified


17


decarboxylase
baumannii
codon

phosphoribosyl-

typhimurium
codon






(strain
usage for 

transferase

LT2
usage for 






AB0057)
Cg and Sc




Cg and Sc


BsHISMN_
61
B71459
Histidine
Acinetobacter
Saccharo-
P00499
ATP

Salmonella
Saccharo-


18


decarboxylase
baumannii
myces

phosphoribosyl-

typhimurium
myces






(strain
cerevisiae

transferase

LT2
cerevisiae






AB0057)








BsHISMN_
1413.5
B71459
Histidine
Acinetobacter
Yarrowia
P00499
ATP

Salmonella
Yarrowia


19


decarboxylase
baumannii
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain


transferase

LT2







AB0057)








BsHISMN_
6630.6
P00862
Histidine
Lactobacillus
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


20


decarboxylase
sp.
myces

phosphoribosyl-
L231F,
glutamicum
myces






(strain 30a)
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


BsHISMN_
43.8
A0A0J6
Histidine
Chromo-
Bacillus
P00498
ATP

Saccharomyces
Bacillus


21

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-

cerevisiae
subtilis






sp. LK1


transferase

S288c



BsHISMN_

A0A0J6
Histidine
Chromo-
modified
P00498
ATP

Saccharomyces
modified


22

KM89
decarboxylase
bacterium
codon

phosphoribosyl-

cerevisiae
codon






sp. LK1
usage for 

transferase

S288c
usage for 







Cg and Sc




Cg and Sc


BsHISMN_
529
A0A0J6
Histidine
Chromo-
Saccharo-
P00498
ATP

Saccharomyces
Saccharo-


23

KM89
decarboxylase
bacterium
myces

phosphoribosyl-

cerevisiae
myces






sp. LK1
cerevisiae

transferase

5288c
cerevisiae


BsHISMN_
15026.1
A0A0J6
Histidine
Chromo-
Yarrowia
P00498
ATP

Saccharomyces
Yarrowia


24

KM89
decarboxylase
bacterium
lipolytica

phosphoribosyl-

cerevisiae
lipolytica






sp. LK1


transferase

S288c



BsHISMN_

A0A0J6
Histidine
Chromo-
modified
Q9Z472
ATP

Corynebacterium
modified


25

KM89
decarboxylase
bacterium
codon

phosphoribosyl-

glutamicum
codon






sp. LK1
usage for 

transferase

ATCC 13032
usage for 







Cg and Sc




Cg and Sc
















TABLE 6







Host evaluation designs for production of histamine tested in Saccharomyces cerevisiae



















E1
Enzyme 1
Enzyme 1
E1 Codon
E2
Enzyme 2
E2
Enzyme 2
E2 Codon


Strain
Titer
Uniprot
activity
source
Optimization
Uniprot
activity
Modifi-
source
Optimization


name
(μg/L)
ID
name
organism
Abbrev.
ID
name
cations
organism
Abbrev.




















Saccharomyces












cerevisiae












ScHISMN_
 17466
P00862
Histidine
Lactobacillus
Bacillus
P00499
ATP

Salmonella
Bacillus


116


decarboxylase
sp.
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain 30a)


transferase

LT2



ScHISMN_
 28646
P00862
Histidine
Lactobacillus
Saccharo-
P00499
ATP

Salmonella
Saccharo-


117


decarboxylase
sp.
myces

phosphoribosyl-

typhimurium
myces






(strain 30a)
cerevisiae

transferase

LT2
cerevisiae


ScHISMN_
 48150
P00862
Histidine
Lactobacillus
Yarrowia
P00499
ATP

Salmonella
Yarrowia


118


decarboxylase
sp.
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain 30a)


transferase

LT2



ScHISMN_
 59265
A0A0J6
Histidine
Chromo-
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


119

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






sp. LK1


transferase
T235A
ATCC 13032



ScHISMN_
 72566
A0A0J6
Histidine
Chromo-
modified
Q9Z472
ATP
N215K,
Corynebacterium
modified


120

KM89
decarboxylase
bacterium
codon

phosphoribosyl
L231F,
glutamicum
codon






sp. LK1
usage for 

transferase
T235A
ATCC 13032
usage for 







Cg and Sc




Cg and Sc


ScHISMN_
 46418
A0A0J6
Histidine
Chromo-
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


121

KM89
decarboxylase
bacterium
myces

phosphoribosyl-
L231F,
glutamicum
myces






sp. LK1
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


ScHISMN_
 64087
A0A0J6
Histidine
Chromo-
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


122

KM89
decarboxylase
bacterium
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






sp. LK1


transferase
T235A
ATCC 13032



ScHISMN_
 80704
B71459
Histidine
Acinetobacter
Bacillus
P00499
ATP

Salmonella
Bacillus


123


decarboxylase
baumannii
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain


transferase

LT2







AB0057)








ScHISMN_
 70043
B71459
Histidine
Acinetobacter
Yarrowia
P00499
ATP

Salmonella
Yarrowia


124


decarboxylase
baumannii
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain


transferase

LT2







AB0057)








ScHISMN_
 25331
P00862
Histidine
Lactobacillus
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


125


decarboxylase
sp.
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






(strain 30a)


transferase
T235A
ATCC 13032



ScHISMN_
 33970
P00862
Histidine
Lactobacillus
modified
Q9Z472
ATP
N215K,
Corynebacterium
modified


126


decarboxylase
sp.
codon

phosphoribosyl
L231F,
glutamicum
codon






(strain 30a)
usage for 

transferase
T235A
ATCC 13032
usage for 







Cg and Sc




Cg and Sc


ScHISMN_
 21402
P00862
Histidine
Lactobacillus
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


127


decarboxylase
sp.
myces

phosphoribosyl-
L231F,
glutamicum
myces






(strain 30a)
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


ScHISMN_
 41854
P00862
Histidine
Lactobacillus
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


128


decarboxylase
sp.
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






(strain 30a)


transferase
T235A
ATCC 13032



ScHISMN_
101496
A0A0J6
Histidine
Chromo-
Bacillus
P00498
ATP

Saccharomyces
Bacillus


129

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-

cerevisiae
subtilis






sp. LK1


transferase

S288c



ScHISMN_
 85546
A0A0J6
Histidine
Chromo-
Saccharo-
P00498
ATP

Saccharomyces
Saccharo-


130

KM89
decarboxylase
bacterium
myces

phosphoribosyl-

cerevisiae
myces






sp. LK1
cerevisiae

transferase

S288c
cerevisiae


ScHISMN_
111109
A0A0J6
Histidine
Chromo-
Yarrowia
P00498
ATP

Saccharomyces
Yarrowia


131

KM89
decarboxylase
bacterium
lipolytica

phosphoribosyl-

cerevisiae
lipolytica






sp. LK1


transferase

S288c
















TABLE 7







Host evaluation designs for production of histamine tested in Corynebacterium glutamicum



















E1
Enzyme 1
Enzyme 1
E1 Codon
E2
Enzyme 2
E2
Enzyme 2
E2 Codon


Strain
Titer
Uniprot
activity
source
Optimization
Uniprot
activity
Modifi-
source
Optimization


name
(μg/L)
ID
name
organism
Abbrev.
ID
name
cations
organism
Abbrev.





CgHISMN_

B71459
Histidine
Acinetobacter
Bacillus
P00498
ATP

Saccharomyces
Bacillus


70


decarboxylase
baumannii
subtilis

phosphoribosyl-

cerevisiae
subtilis






(strain


transferase

S288c







AB0057)








CgHISMN_
68395.9
B71459
Histidine
Acinetobacter
modified
P00498
ATP

Saccharomyces
modified


71


decarboxylase
baumannii
codon

phosphoribosyl-

cerevisiae
codon






(strain
usage for 

transferase

S288c
usage for 






AB0057)
Cg and Sc




Cg and Sc


CgHISMN_
18
B71459
Histidine
Acinetobacter
Saccharo-
P00498
ATP

Saccharomyces
Saccharo-


72


decarboxylase
baumannii
myces

phosphoribosyl-

cerevisiae
myces






(strain
cerevisiae

transferase

S288c
cerevisiae






AB0057)








CgHISMN_
16325.5
B71459
Histidine
Acinetobacter
Yarrowia
P00498
ATP

Saccharomyces
Yarrowia


73


decarboxylase
baumannii
lipolytica

phosphoribosyl-

cerevisiae
lipolytica






(strain


transferase

S288c







AB0057)








CgHISMN_
4883.6
P00862
Histidine
Lactobacillus
Bacillus
P00499
ATP

Salmonella
Bacillus


74


decarboxylase
sp.
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain 30a)


transferase

LT2



CgHISMN_

P00862
Histidine
Lactobacillus
Saccharo-
P00499
ATP

Salmonella
Saccharo-


75


decarboxylase
sp.
myces

phosphoribosyl-

typhimurium
myces






(strain 30a)
cerevisiae

transferase

LT2
cerevisiae


CgHISMN_

P00862
Histidine
Lactobacillus
Yarrowia
P00499
ATP

Salmonella
Yarrowia


76


decarboxylase
sp.
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain 30a)


transferase

LT2



CgHISMN_
5.4
A0A0J6
Histidine
Chromo-
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


77

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






sp. LK1


transferase
T235A
ATCC 13032



CgHISMN_
88.6
A0A0J6
Histidine
Chromo-
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


78

KM89
decarboxylase
bacterium
myces

phosphoribosyl-
L231F,
glutamicum
myces






sp. LK1
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


CgHISMN_

A0A0J6
Histidine
Chromo-
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


79

KM89
decarboxylase
bacterium
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






sp. LK1


transferase
T235A
ATCC 13032



CgHISMN_
8368.2
B71459
Histidine
Acinetobacter
Bacillus
P00499
ATP

Salmonella
Bacillus


80


decarboxylase
baumannii
subtilis

phosphoribosyl-

typhimurium
subtilis






(strain




LT2







AB0057)








CgHISMN_
9.3
B71459
Histidine
Acinetobacter
Saccharo-
P00499
ATP

Salmonella
Saccharo-


81


decarboxylase
baumannii
myces

phosphoribosyl-

typhimurium
myces






(strain
cerevisiae

transferase

LT2
cerevisiae






AB0057)








CgHISMN_
15529.4
B71459
Histidine
Acinetobacter
Yarrowia
P00499
ATP

Salmonella
Yarrowia


82


decarboxylase
baumannii
lipolytica

phosphoribosyl-

typhimurium
lipolytica






(strain


transferase

LT2







AB0057)








CgHISMN_

P00862
Histidine
Lactobacillus
Bacillus
Q9Z472
ATP
N215K,
Corynebacterium
Bacillus


83


decarboxylase
sp.
subtilis

phosphoribosyl-
L231F,
glutamicum
subtilis






(strain 30a)


transferase
T235A
ATCC 13032



CgHISMN_
2.6
P00862
Histidine
Lactobacillus
Saccharo-
Q9Z472
ATP
N215K,
Corynebacterium
Saccharo-


84


decarboxylase
sp.
myces

phosphoribosyl-
L231F,
glutamicum
myces






(strain 30a)
cerevisiae

transferase
T235A
ATCC 13032
cerevisiae


CgHISMN_
6134
P00862
Histidine
Lactobacillus
Yarrowia
Q9Z472
ATP
N215K,
Corynebacterium
Yarrowia


85


decarboxylase
sp.
lipolytica

phosphoribosyl-
L231F,
glutamicum
lipolytica






(strain 30a)


transferase
T235A
ATCC 13032



CgHISMN_
197
A0A0J6
Histidine
Chromo-
Bacillus
P00498
ATP

Saccharomyces
Bacillus


86

KM89
decarboxylase
bacterium
subtilis

phosphoribosyl-

cerevisiae
subtilis






sp. LK1


transferase

S288c
















TABLE 8







SEQ ID NO Cross-Reference Table











SEQ
















ID
Sequence Type with
Uniprot


Codon


NO
Modifications
ID
Activity name
Source organism
Optimization Abbrev.





 1
AA seq for
B71459
histidine decarboxylase
Acinetobacter baumannii




enzyme B71459


(strain AB0057)



 2
AA seq for
Q9KJU3
Imidazoleglycerol-
Corynebacterium glutamicum




enzyme Q9KJU3

phosphate dehydratase




 3
AA seq for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae




enzyme P00498






 4
AA seq for
P00862
histidine decarboxylase
Lactobacillus sp. (strain 30a)




enzyme P00862






 5
AA seq for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium




enzyme P00499 with


(strain LT2/SGSC1412/




deletion of Q207-E208


ATCC 700720)



 6
AA seq for
J6KM89
histidine decarboxylase
Chromobacterium sp. LK1




enzyme J6KM89






 7
AA seq for
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum




enzyme Q9Z472


(strain ATCC 13032/




with substitution


DSM 20300/JCM 1318/




N215K, L231F, T235A


LMG 3730/NCIMB 10025)



 8
AA seq for
E3QMN8
histidine decarboxylase
Methanosarcina barkeri




enzyme E3QMN8


str. Wiesmoor



 9
AA seq for
Q467R8
histidine decarboxylase
Methanosarcina barkeri




enzyme Q467R8


(strain Fusaro/DSM 804)



 10
AA seq for
Q05733
histidine decarboxylase
Drosophila melanogaster




enzyme Q05733






 11
AA seq for
P54772
histidine decarboxylase
Solanum lycopersicum




enzyme P54772






 12
AA seq for
P23738
histidine decarboxylase
Mus musculus




enzyme P23738






 13
AA seq for 
O68602
1-(5-phosphoribosyl)5[(5-
Corynebacterium glutamicum




enzyme O68602

phosphoribosylamino)







methylideneamino]







imidazole-4-







carboxamide isomerase




 14
AA seq for
Q9KJU4
Histidinol-phosphate
Corynebacterium glutamicum




enzyme Q9KJU4

aminotransferase




 15
AA seq for
Q8NNT5
Histidinol dehydrogenase
Corynebacterium glutamicum




enzyme Q8NNT5






 16
AA seq for
Q9Z471
Phosphoribosyl-
Corynebacterium glutamicum




enzyme Q9Z471

ATP pyrophosphatase




 17
AA seq for
O31139
Imidazole glycerol phosphate
Corynebacterium glutamicum




enzyme O31139

synthase subunit




 18
AA seq for
O69043
Imidazole glycerol phosphate
Corynebacterium glutamicum




enzyme O69043

synthase subunit




 19
AA seq for
Q8NNT9
phosphoribosyl-
Corynebacterium glutamicum




enzyme Q8NNT9

AMP cyclohydrolase




 22
AA seq for
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum




enzyme Q9Z472


(strain ATCC 13032/







DSM 20300/JCM 1318/







LMG 3730/NCIMB 10025)



 20
AA seq for
P00815
histidinol dehydrogenase,
Saccharomyces cerevisiae




enzyme P00815

phosphoribosyl-







AMP cyclohydrolase,







phosphoribosyl-







ATP diphosphatase




 21
AA seq for
P33734
Imidazole glycerol phosphate
Saccharomyces cerevisiae




enzyme P33734

synthase subunit HisF




 23
AA seq for
P07172
histidinol-
Saccharomyces cerevisiae




enzyme P07172

phosphate transaminase




 24
AA seq for
P06633
Imidazoleglycerol-
Saccharomyces cerevisiae




enzyme P06633

phosphate dehydratase




 25
AA seq for
P38635
histidinol-phosphatase
Saccharomyces cerevisiae




enzyme P38635






 26
AA seq for
A4QEF2
Glucose-6-phosphate 
Corynebacterium glutamicum




enzyme A4QEF2 with

1-dehydrogenase





substitution A243T

(G6PD) (EC 1.1.1.49)
(strain R)



 27
AA seq for
P23254
Transketolase
Saccharomyces cerevisiae




enzyme P23254


(strain ATCC 204508/S288c)







(Baker's yeast)



 28
AA seq for
Q12265
Ribose-phosphate 
Saccharomyces cerevisiae




enzyme Q12265

pyrophosphokinase 5
(strain ATCC 204508/S288c)






(EC 2.7.6.1)
(Baker's yeast)






(Phosphoribosyl







pyrophosphate synthase 5)




 30
DNA seq1 for
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
native



enzyme Q9Z472






 29
DNA seq1 for
P00815
histidinol dehydrogenase,
Saccharomyces cerevisiae
native



enzyme P00815

phosphoribosyl-







AMP cyclohydrolase,







phosphoribosyl-







ATP diphosphatase




 31
AA seq for
P40545
1-(5-phosphoribosyl)-5-[(5-
Saccharomyces cerevisiae




enzyme P40545

phosphoribosylamino)
(strain ATCC 204508/S288c)






methylideneamino]
(Baker's yeast)






imidazole-4-carboxamide







isomerase (EC 5.3.1.16)







(5-proFAR isomerase)







(Phosphoribosylformimino-5-







aminoimidazole carboxamide







ribotide isomerase)




 32
AA seq for
P00942
Triosephosphate isomerase
Saccharomyces cerevisiae




enzyme P00942 with

(TIM) (EC 5.3.1.1)
(strain ATCC 204508/S288c)




substitution 1170V

(Triose-phosphate isomerase)
(Baker's yeast)



 33
AA seq for enzyme
A0A089
Histidine decarboxylase
Pseudomonas rhizosphaerae




A0A089YPE5
YPE5
(HDC) (EC 4.1.1.22)




 34
AA seq for enzyme
A0A126
Histidine decarboxylase
Pseudomonas putida




AOA12656G9
56G9
(HDC) (EC 4.1.1.22)
(Arthrobacter siderocapsulatus)



 35
AA seq for enzyme
A0A0A1
Histidine decarboxylase
Citrobacter pasteurii




A0A0A1R6V3
R6V3
(HDC) (EC 4.1.1.22)




 36
AA seq for enzyme
A0A1W0
Histidine decarboxylase
Chromobacterium haemolyticum




A0A1W0CM88
CM88
(HDC) (EC 4.1.1.22)




 37
AA seq for enzyme
A0A0K6
Histidine decarboxylase
Lactobacillus reuteri




A0A0K6GJ74
GJ74
proenzyme




 38
AA seq for
T0QL99
Histidine decarboxylase
Aeromonas salmonicida




enzyme T0QL99

(EC 4.1.1.22) (Fragment)
subsp. pectinolytica 34mel



 39
AA seq for enzyme
A0A1B8
Histidine decarboxylase
Morganella psychrotolerans




A0A1B8HLR1
HLR1
(HDC) (EC 4.1.1.22)




 40
AA seq for enzyme
A0A0C1
Histidine decarboxylase
Lactobacillus fructivorans




A0A0C1PR48
PR48
proenzyme




 41
AA seq for
P0A717
Ribose-phosphate
Escherichia coli (strain K12)




enzyme P0A717

pyrophosphokinase







(RPPK) (EC 2.7.6.1)







(5-phospho-D-ribosyl







alpha-1-diphosphate)







(Phosphoribosyl







diphosphate synthase)







(Phosphoribosyl







pyrophosphate synthase) 







(P-Rib-PP synthase) 







(PRPP synthase) (PRPPase)




 42
AA seq for
Q680A5
Ribose-phosphate
Arabidopsis thaliana




enzyme Q680A5

pyrophosphokinase 4
(Mouse-ear cress)






(EC 2.7.6.1)







(Phosphoribosyl







pyrophosphate synthase 4)




 43
AA seq for
P38620
Ribose-phosphate
Saccharomyces cerevisiae 




enzyme P38620

pyrophosphokinase 2
(strain ATCC 204508/S288c)






(EC 2.7.6.1)
(Baker's yeast)






(Phosphoribosyl







pyrophosphate synthase 2)




 44
AA seq for
P32895
Ribose-phosphate
Saccharomyces cerevisiae 




enzyme P32895

pyrophosphokinase 1
(strain ATCC 204508/S288c)






(EC 2.7.6.1)
(Baker's yeast)






(Phosphoribosyl







pyrophosphate synthase 1)




 45
AA seq for
P38689
Ribose-phosphate
Saccharomyces cerevisiae 




enzyme P38689

pyrophosphokinase 3
(strain ATCC 204508/S288c)






(EC 2.7.6.1)
(Baker's yeast)






(Phosphoribosyl







pyrophosphate synthase 3)




 46
AA seq for
P15019
Transaldolase (EC 2.2.1.2)
Saccharomyces cerevisiae 




enzyme P15019


(strain ATCC 204508/S288c)







(Baker's yeast)



 47
AA seq for
P06775
Histidine permease
Saccharomyces cerevisiae 




enzyme P06775


(strain ATCC 204508/S288c)







(Baker's yeast)



 48
AA seq for
O59667
Histidine biosynthesis
Schizosaccharomyces pombe




enzyme O59667

bifunctional protein
(strain 972/ATCC 24843)






his7 [Includes:
(Fission yeast)






Phosphoribosyl-







AMP cyclohydrolase







(EC 3.5.4.19);







Phosphoribosyl-







ATP pyrophosphatase







(EC 3.6.1.31)]




 49
AA seq for
O66000
Histidine decarboxylase
Oenococcus oeni




enzyme O66000

proenzyme
(Leuconostoc oenos)



 50
AA seq for enzyme
A0A0R1
Pyruvoyl family
Lactobacillus aviarius subsp.




A0A0R1Y874
Y874
histidine decarboxylase
aviarius DSM 20655



 51
AA seq for enzyme
A0A0J6K
Histidine decarboxylase 
Chromobacterium sp. LK1




A0A0J6KM89
M89
(HDC) (EC 4.1.1.22)




 52
DNA seq1 for
B71459
Histidine decarboxylase
Acinetobacter baumannii 
Yarrowia lipolytica



enzyme B71459


(strain AB0057)



 53
DNA seq1 for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae 
Yarrowia lipolytica



enzyme P00498


(strain ATCC 204508/S288c)







(Baker's yeast)



 54
DNA seq2 for
B71459
Histidine decarboxylase
Acinetobacter baumannii
Bacillus subtillus



enzyme B71459


(strain AB0057)



 55
DNA seq2 for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae 
Bacillus subtillus



enzyme P00498


(strain ATCC 204508/S288c)







(Baker's yeast)



 56
DNA seq3 for
B71459
Histidine decarboxylase
Acinetobacter baumannii
Saccharomyces cerevisiae



enzyme B71459


(strain AB0057)



 57
DNA seq3 for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae 
Saccharomyces cerevisiae



enzyme P00498


(strain ATCC 204508/S288c)







(Baker's yeast)



 58
DNA seq1 for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium 
Yarrowia lipolytica



enzyme P00499 with


(strain LT2/SGSC1412/




deletion of Q207-E208


ATCC 700720)



 59
DNA seq2 for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium 
Bacillus subtillus



enzyme P00499 with


(strain LT2/SGSC1412/




deletion of Q207-E208


ATCC 700720)



 60
DNA seq3 for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium 
Saccharomyces cerevisiae



enzyme P00499 with


(strain LT2/SGSC1412/




deletion of Q207-E208


ATCC 700720)



 61
DNA seq4 for
B71459
Histidine decarboxylase
Acinetobacter baumannii
modified codon usage for



enzyme B71459

(HDC) (EC 4.1.1.22)
(strain AB0057)
Corynebacterium glutamicum and







Saccharomyces cerevisiae


 62
DNA seq4 for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium
modified codon usage for



enzyme P00499 with


(strain LT2/SGSC1412/
Corynebacterium glutamicum and



deletion of Q207-E208


ATCC 700720)
Saccharomyces cerevisiae


 63
DNA seq1 for enzyme
A0A0J6K
Histidine decarboxylase
Chromobacterium sp. LK1
Yarrowia lipolytica



A0A0J6KM89
M89





 64
DNA seq1
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
Yarrowia lipolytica



for enzyme Q9Z472


(strain ATCC 13032/




with substitution


DSM 20300/JCM 1318/




N215K, L231F, T235A


LMG 3730/NCIMB 10025)



 65
DNA seq2 for enzyme
A0A0J6K
Histidine decarboxylase
Chromobacterium sp. LK1
Saccharomyces cerevisiae



A0A0J6KM89
M89





 66
DNA seq2
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
Saccharomyces cerevisiae



for enzyme Q9Z472


(strain ATCC 13032/




with substitution


DSM 20300/JCM 1318/




N215K, L231F, T235A


LMG 3730/NCIMB 10025)



 67
DNA seq3
A0A0J6K
Histidine decarboxylase
Chromobacterium sp. LK1
Bacillus subtillus



for enzyme
M89






A0A0J6KM89






 68
DNA seq3
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
Bacillus subtillus



for enzyme Q9Z472


(strain ATCC 13032/




with substitution


DSM 20300/JCM 1318/




N215K, L231F, T235A


LMG 3730/NCIMB 10025)



 69
DNA seq1 for
P00862
Histidine decarboxylase
Lactobacillus sp. (strain 30a)
Bacillus subtillus



enzyme P00862

proenzyme




 70
DNA seq2 for
P00862
Histidine decarboxylase
Lactobacillus sp. (strain 30a)
Saccharomyces cerevisiae



enzyme P00862

proenzyme




 71
DNA seq3 for
P00862
Histidine decarboxylase
Lactobacillus sp. (strain 30a)
modified codon usage for



enzyme P00862

proenzyme

Corynebacterium glutamicum and







Saccharomyces cerevisiae


 72
DNA seq5 for
B71459
Histidine decarboxylase
Acinetobacter baumannii
modified codon usage for



enzyme B71459


(strain AB0057)
Corynebacterium glutamicum and







Saccharomyces cerevisiae


 73
DNA seq4 for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae 
modified codon usage for



enzyme P00498


(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


 74
AA seq for enzyme
A0A1H1
Histidine decarboxylase
Pseudomonas sp. bs2935
modified codon usage for



A0A1H1TEB8
TEB8
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and







Saccharomyces cerevisiae


 75
DNA seq1 for
E3QMN8
histidine decarboxylase
Methanosarcina barkeri
Corynebacterium glutamicum



enzyme E3QMN8


str. Wiesmoor



 76
DNA seq1 for
Q467R8
histidine decarboxylase
Methanosarcina barkeri
Corynebacterium glutamicum



enzyme Q467R8


(strain Fusaro/DSM 804)



 77
DNA seq4 for
P00862
histidine decarboxylase
Lactobacillus sp. (strain 30a)
Corynebacterium glutamicum



enzyme P00862






 78
DNA seq6 for
B71459
histidine decarboxylase
Acinetobacter baumannii
Corynebacterium glutamicum



enzyme B71459


(strain AB0057)



 79
DNA seq1 for
Q05733
histidine decarboxylase
Drosophila melanogaster
Corynebacterium glutamicum



enzyme Q05733






 80
DNA seq1 for
J6KM89
histidine decarboxylase
Chromobacterium sp. LK1
Corynebacterium glutamicum



enzyme J6KM89






 81
DNA seq5 for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium 
Corynebacterium glutamicum



enzyme P00499 with


(strain LT2/SGSC1412/




deletion of Q207-E208


ATCC 700720)



 82
DNA seq5 for
P00862
histidine decarboxylase
Lactobacillus sp. (strain 30a)
Saccharomyces cerevisiae



enzyme P00862






 83
DNA seq for
P54772
histidine decarboxylase
Solanum lycopersicum
Saccharomyces cerevisiae



enzyme P54772






 84
DNA seq for
P23738
histidine decarboxylase
Mus musculus
Saccharomyces cerevisiae



enzyme P23738






 85
DNA seq2 for
Q05733
histidine decarboxylase
Drosophila melanogaster
Saccharomyces cerevisiae



enzyme Q05733






 86
DNA seq2 for
J6KM89
histidine decarboxylase
Chromobacterium sp. LK1
Saccharomyces cerevisiae



enzyme J6KM89






 87
DNA seq2 for
E3QMN8
histidine decarboxylase
Methanosarcina barkeri
Saccharomyces cerevisiae



enzyme E3QMN8


str. Wiesmoor



 88
DNA seq2 for
Q467R8
histidine decarboxylase
Methanosarcina barkeri
Saccharomyces cerevisiae



enzyme Q467R8


(strain Fusaro/DSM 804)



 89
DNA seq4
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
Saccharomyces cerevisiae



for enzyme Q9Z472


(strain ATCC 13032/




with substitution


DSM 20300/JCM 1318/




N215K, L231F, T235A


LMG 3730/NCIMB 10025)



 90
DNA seq6 for
P00499
ATP phosphoribosyltransferase
Salmonella typhimurium 
Saccharomyces cerevisiae



enzyme P00499 with


(strain LT2/SGSC1412/




deletion of Q207-E208


ATCC 700720)



 91
DNA seq for
O68602
1-(5-phosphoribosyl)5[(5-
Corynebacterium glutamicum
native



enzyme O68602

phosphoribosylamino)







methylideneamino]imidazole-







4-carboxamide isomerase




 92
DNA seq for
Q9KJU3
Imidazoleglycerol-
Corynebacterium glutamicum
native



enzyme Q9KJU3

phosphate dehydratase




 93
DNA seq for
Q9KJU4
Histidinol-phosphate 
Corynebacterium glutamicum
native



enzyme Q9KJU4

aminotransferase




 94
DNA seq for
Q8NNT5
Histidinol dehydrogenase
Corynebacterium glutamicum
native



enzyme Q8NNT5






 95
DNA seq for
Q9Z471
Phosphoribosyl-ATP
Corynebacterium glutamicum
native



enzyme Q9Z471

pyrophosphatase




 96
DNA seq for
O31139
Imidazole glycerol phosphate
Corynebacterium glutamicum
native



enzyme O31139

synthase subunit




 97
DNA seq for
O69043
Imidazole glycerol phosphate
Corynebacterium glutamicum
native



enzyme O69043

synthase subunit




 98
DNA seq for
Q8NNT9
phosphoribosyl-
Corynebacterium glutamicum
native



enzyme Q8NNT9

AMP cyclohydrolase




 99
DNA seq5 for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae
native



enzyme P00498

Imidazole glycerol phosphate




100
DNA seq1 for
P33734
synthase subunit HisF
Saccharomyces cerevisiae
native



enzyme P33734






101
DNA seq1 for
P07172
histidinol-phosphate 
Saccharomyces cerevisiae
native



enzyme P07172

transaminase




102
DNA seq for
P06633
Imidazoleglycerol-
Saccharomyces cerevisiae
native



enzyme P06633

phosphate dehydratase




103
DNA seq1 for
P38635
histidinol-phosphatase
Saccharomyces cerevisiae
native



enzyme P38635






104
DNA seq
A0A0C1
Histidine decarboxylase
Lactobacillus fructivorans
modified codon usage for



for enzyme
PR48
proenzyme

Corynebacterium glutamicum and



A0A0C1PR48



Saccharomyces cerevisiae


105
DNA seq for
T0QL99
Histidine decarboxylase
Aeromonas salmonicida
modified codon usage for



enzyme T0QL99

(EC 4.1.1.22) (Fragment)
subsp. pectinolytica 34mel
Corynebacterium glutamicum and







Saccharomyces cerevisiae


106
DNA seq
A0A1B8
Histidine decarboxylase
Morganella psychrotolerans
modified codon usage for



for enzyme
HLR1
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and



A0A1B8HLR1



Saccharomyces cerevisiae


107
DNA seq2 for
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
modified codon usage for



enzyme Q9Z472

(ATP-PRT) (ATP-PRTase)
(strain ATCC 13032/
Corynebacterium glutamicum and






DSM 20300/JCM 1318/
Saccharomyces cerevisiae






LMG 3730/NCIMB 10025)



108
DNA seq for
P0A717
Ribose-phosphate
Escherichia coli (strain K12)
modified codon usage for



enzyme P0A717

pyrophosphokinase

Corynebacterium glutamicum and





(RPPK) (EC 2.7.6.1)

Saccharomyces cerevisiae





(5-phospho-D-ribosyl







alpha-1-diphosphate)







(Phosphoribosyl







diphosphate synthase)







(Phosphoribosyl







pyrophosphate synthase)







(P-Rib-PP synthase) 







(PRPP synthase) (PRPPase)




109
DNA seq for
Q12265
Ribose-phosphate
Saccharomyces cerevisiae
modified codon usage for



enzyme Q12265

pyrophosphokinase 5
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





(EC 2.7.6.1) (Phosphoribosyl
(Baker's yeast)
Saccharomyces cerevisiae





pyrophosphate synthase 5)




110
DNA seq for
P32895
Ribose-phosphate
Saccharomyces cerevisiae
modified codon usage for



enzyme P32895

pyrophosphokinase 1
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





(EC 2.7.6.1) (Phosphoribosyl
(Baker's yeast)
Saccharomyces cerevisiae





pyrophosphate synthase 1)




111
DNA seq for
P23254
Transketolase
Saccharomyces cerevisiae
modified codon usage for



enzyme P23254


(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


112
DNA seq for
P06775
Histidine permease
Saccharomyces cerevisiae
modified codon usage for



enzyme P06775


(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


113
DNA seq2 for
P00815
trifunctional histidinol
Saccharomyces cerevisiae
modified codon usage for



enzyme P00815

dehydrogenase/
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





phosphoribosyl-AMP
(Baker's yeast)
Saccharomyces cerevisiae





cyclohydrolase/







phosphoribosyl-ATP







diphosphatase




114
DNA seq2 fo 
P07172
Histidinol-phosphate
Saccharomyces cerevisiae
modified codon usage for



enzyme P07172

aminotransferase
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


115
DNA seq6 for
P00498
ATP phosphoribosyltransferase
Saccharomyces cerevisiae
modified codon usage for



enzyme P00498

(ATP-PRT) (ATP-PRTase)
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


116
DNA seq for
P40545
1-(5-phosphoribosyl)-5-[(5-
Saccharomyces cerevisiae
modified codon usage for



enzyme P40545

phosphoribosylamino)
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





methylideneamino]
(Baker's yeast)
Saccharomyces cerevisiae





imidazole-4-carboxamide 







isomerase (EC 5.3.1.16)







(5-proFAR isomerase)







(Phosphoribosylformimino-







5-aminoimidazole







carboxamide







ribotide isomerase)




117
DNA seq2 for
P33734
Imidazole
Saccharomyces cerevisiae
modified codon usage for



enzyme P33734

glycerol phosphate
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





synthase hisHF
(Baker's yeast)
Saccharomyces cerevisiae


118
DNA seq for
P00942
Triosephosphate isomerase
Saccharomyces cerevisiae
modified codon usage for



enzyme P00942 with

(TIM) (EC 5.3.1.1)
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and



substitution 1170V

(Triose-phosphate isomerase)
(Baker's yeast)
Saccharomyces cerevisiae


119
DNA seq3 for
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
modified codon usage for



enzyme Q9Z472

(ATP-PRT) (ATP-PRTase)
(strain ATCC 13032/
Corynebacterium glutamicum and





(EC 2.4.2.17)
DSM 20300/JCM 1318/
Saccharomyces cerevisiae


120
DNA seq5
Q9Z472
ATP phosphoribosyltransferase
LMG 3730/NCIMB 10025)
modified codon usage for



for enzyme Q9Z472

(ATP-PRT) (ATP-PRTase)
(strain ATCC 13032/
Corynebacterium glutamicum and



with substitution

(EC 2.4.2.17)
DSM 20300/JCM 1318/
Saccharomyces cerevisiae



N215K, L231F, T235A


LMG 3730/NCIMB 10025)



121
DNA seq for
O66000
Histidine decarboxylase
Oenococcus oeni
modified codon usage for



enzyme O66000

proenzyme
(Leuconostoc oenos)
Corynebacterium glutamicum and







Saccharomyces cerevisiae


122
DNA seq for enzyme
A0A0R1
Pyruvoyl family
Lactobacillus aviarius
modified codon usage for



A0A0R1Y874
Y874
histidine decarboxylase
subsp. aviarius DSM 20655
Corynebacterium glutamicum and







Saccharomyces cerevisiae


123
DNA seq for enzyme
A0A1H1
Histidine decarboxylase
Pseudomonas sp. bs2935
modified codon usage for



A0A1H1TEB8 with
TEB8
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and



substitution S9R



Saccharomyces cerevisiae


124
DNA seq for enzyme
A0A089
Histidine decarboxylase
Pseudomonas rhizosphaerae
modified codon usage for



A0A089YPE5
YPE5
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and







Saccharomyces cerevisiae


125
DNA seq for enzyme
A0A126
Histidine decarboxylase
Pseudomonas putida
modified codon usage for



A0A12656G9
56G9
(HDC) (EC 4.1.1.22)
(Arthrobacter siderocapsulatus)
Corynebacterium glutamicum and







Saccharomyces cerevisiae


126
DNA seq4 for enzyme
A0A0J6K
Histidine decarboxylase
Chromobacterium sp. LK1
modified codon usage for



A0A0J6KM89
M89
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and







Saccharomyces cerevisiae


127
DNA seq for enzyme
A0A0A1
Histidine decarboxylase
Citrobacter pasteurii
modified codon usage for



A0A0A1R6V3
R6V3
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and







Saccharomyces cerevisiae


128
DNA seq for enzyme
A0A1W0
Histidine decarboxylase
Chromobacterium haemolyticum
modified codon usage for



A0A1W0CM88
CM88
(HDC) (EC 4.1.1.22)

Corynebacterium glutamicum and





Histidine decarboxylase

Saccharomyces cerevisiae


129
DNA seq6 for
P00862
proenzyme
Lactobacillus sp. (strain 30a)
modified codon usage for



enzyme P00862

(EC 4.1.1.22) (Pi chain)

Corynebacterium glutamicum and





[Cleaved into: Histidine

Saccharomyces cerevisiae





decarboxylase beta chain;







Histidine decarboxylase







alpha chain]




130
DNA seq for enzyme
A0A0K6
Histidine decarboxylase
Lactobacillus reuteri
modified codon usage for



A0A0K6GJ74
GJ74
proenzyme

Corynebacterium glutamicum and







Saccharomyces cerevisiae


131
DNA seq for
P38620
Ribose-phosphate
Saccharomyces cerevisiae
modified codon usage for



enzyme P38620

pyrophosphokinase 2
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





(EC 2.7.6.1) (Phosphoribosyl
(Baker's yeast)
Saccharomyces cerevisiae





pyrophosphate synthase 2)




132
DNA seq for
P38689
Ribose-phosphate
Saccharomyces cerevisiae
modified codon usage for



enzyme P38689

pyrophosphokinase 3
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and





(EC 2.7.6.1) (Phosphoribosyl
(Baker's yeast)
Saccharomyces cerevisiae





pyrophosphate synthase 3)




133
DNA seq for
P15019
Transaldolase (EC 2.2.1.2)
Saccharomyces cerevisiae
modified codon usage for



enzyme P15019


(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


134
DNA seq for
Q680A5
Ribose-phosphate
Arabidopsis thaliana
modified codon usage for



enzyme Q680A5

pyrophosphokinase 4
(Mouse-ear cress)
Corynebacterium glutamicum and





(EC 2.7.6.1) (Phosphoribosyl

Saccharomyces cerevisiae





pyrophosphate synthase 4)




135
DNA seq for
O59667
Histidine biosynthesis
Schizosaccharomyces pombe
modified codon usage for



enzyme O59667

bifunctional 
(strain 972/ATCC 24843)
Corynebacterium glutamicum and





protein his7 [Includes:
(Fission yeast)
Saccharomyces cerevisiae





Phosphoribosyl-AMP







cyclohydrolase (EC 3.5.4.19);







Phosphoribosyl-ATP







pyrophosphatase







(EC 3.6.1.31)]




136
DNA seq2 for
P38635
Histidinol-phosphatase
Saccharomyces cerevisiae
modified codon usage for



enzyme P38635

(HolPase) (EC 3.1.3.15)
(strain ATCC 204508/S288c)
Corynebacterium glutamicum and






(Baker's yeast)
Saccharomyces cerevisiae


137
DNA seq for
A4QEF2
Glucose-6-phosphate
Corynebacterium glutamicum
modified codon usage for



enzyme

1-dehydrogenase
(strain R)
Corynebacterium glutamicum and



A4QEF2 with

(G6PD) (EC 1.1.1.49)

Saccharomyces cerevisiae



substitution A243T






138
DNA seq7 for
P00862
Histidine decarboxylase
Lactobacillus sp. (strain 30a)
Yarrowia lipolytica



enzyme P00862

proenzyme




139
DNA seq5
A0A0J6K
Histidine decarboxylase
Chromobacterium sp. LK1
modified codon usage for



for enzyme
M89


Corynebacterium glutamicum and



A0A0J6KM89



Saccharomyces cerevisiae


140
DNA seq6
Q9Z472
ATP phosphoribosyl transferase
Corynebacterium glutamicum
modified codon usage for



for enzyme Q9Z472


ATCC 13032
Corynebacterium glutamicum and



with substitution



Saccharomyces cerevisiae



N215K, L231F, T235A






141
DNA seq4 for
Q9Z472
ATP phosphoribosyltransferase
Corynebacterium glutamicum
Saccharomyces cerevisiae



enzyme Q9Z472


(strain ATCC 13032/







DSM 20300/JCM 1318/







LMG 3730/NCIMB 10025)



142
AA seq for







N-terminal







solubility tag






143
AA seq for
E7EAU9
Ribose-phosphate
Bacillus amyloliquefaciens
Yarrowia lypolytica



enzyme E7EAU9

pyrophosphokinase (RPPK)









REFERENCES


    1. Gezginc, Y., et al., Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt. Food Chem, 2013. 138(1): p. 655-62.
    2. Byun, B. Y. and J. H. Mah, Occurrence of biogenic amines in Miso, Japanese traditional fermented soybean paste. J Food Sci, 2012. 77(12): p. T216-23.
    3. Landete, J. M., et al., Molecular methods for the detection of biogenic amine-producing bacteria on foods. Int J Food Microbiol, 2007. 117(3): p. 258-69.
    4. Ferstl, R., et al., Histamine receptor 2 is a key influence in immune responses to intestinal histamine-secreting microbes. J Allergy Clin Immunol, 2014. 134(3): p. 744-746 e3.
    5. Tabanelli, G., et al., Effect of chemico-physical parameters on the histidine decarboxylase (HdcA) enzymatic activity in Streptococcus thermophilus PRI60. J Food Sci, 2012. 77(4): p. M231-7.
    6. Wauters, G., et al., Histidine decarboxylase in Enterobacteriaceae revisited. J Clin Microbiol, 2004. 42(12): p. 5923-4.
    7. Lee, M. E., et al., A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synth Biol, 2015. 4(9): p. 975-86.
    8. Roland, B. P., et al., Triosephosphate isomerase I170V alters catalytic site, enhances stability and induces pathology in a Drosophila model of TPI deficiency. Biochim Biophys Acta, 2015. 1852(1): p. 61-9.

## Claims

1. An engineered fungal cell that expresses:
a non-native histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 having SEQ ID NO:6 or from Acinetobacter baumannii strain AB0057 having SEQ ID NO:1; and
at least one ATP phosphoribosyltransferase, wherein the ATP phosphoribosyltransferase comprises:
an ATP phosphoribosyltransferase having at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c having SEQ ID NO:3; and/or
a feedback-deregulated ATP phosphoribosyltransferase comprising a feedback-deregulated variant of a Corynebacterium glutamicum ATP phosphoribosyltransferase having at least 70% amino acid sequence identity with SEQ ID NO:7;

wherein the engineered fungal cell produces histamine at a level at least 75 mg/L of culture medium.

2. The engineered fungal cell of claim 1, wherein the engineered fungal cell comprises increased activity of one or more upstream histamine pathway enzyme(s) in addition to said ATP phosphoribosyltransferase(s), said increased activity being increased relative to a control cell.

3. The engineered fungal cell of claim 2, wherein the one or more upstream histamine pathway enzyme(s) are selected from the group consisting of a phosphoribosyl-ATP pyrophosphatase, a phosphoribosyl-AMP cyclohydrolase, a 5′ProFAR isomerase, an imidazole-glycerol phosphate synthase, an imidazole-glycerol phosphate dehydratase, a histidinol-phosphate aminotransferase, a histidinol-phosphate phosphatase, histidinol dehydrogenase, and a ribose phosphate pyrophosphokinase.

4. The engineered fungal cell of claim 1, wherein the engineered fungal cell comprises reduced activity of one or more enzyme(s) that consume one or more histamine pathway precursors, said reduced activity being reduced relative to a control cell.

5. The engineered fungal cell of claim 1, wherein the engineered fungal cell expresses a feedback-deregulated glucose-6-phosphate dehydrogenase or said feedback-deregulated ATP phosphoribosyltransferase.

6. A culture of engineered fungal cells according to claim 1.

7. The culture of claim 6, wherein the culture comprises histamine at a level at least 300 mg/L of culture medium.

8. A method of culturing engineered fungal cells according to claim 1, the method comprising culturing the cells under conditions suitable for producing histamine.

9. A method for preparing histamine using the engineered fungal cells of claim 1, the method comprising:
(a) cultivating the fungal cells in a suitable culture medium under conditions that permit the fungal cells to produce histamine, wherein the histamine is released into the culture medium; and
(b) isolating histamine from the culture medium.

10. The engineered fungal cell of claim 1, wherein the fungal cell is a cell of the genus Saccharomyces and of the species cerevisiae.

11. The engineered fungal cell of claim 10, wherein the fungal cell expresses:
said non-native histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 having SEQ ID NO:6 or from Acinetobacter baumannii strain AB0057 having SEQ ID NO:1;
said ATP phosphoribosyltransferase having at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c having SEQ ID NO:3; and
said feedback-deregulated ATP phosphoribosyltransferase comprising a feedback-deregulated variant of a Corynebacterium glutamicum ATP phosphoribosyltransferase having at least 70% amino acid sequence identity with SEQ ID NO:7.

12. The engineered fungal cell of claim 11, wherein the fungal cell expresses:
said non-native histidine decarboxylase, which has 100% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 having SEQ ID NO:6;
said ATP phosphoribosyltransferase, which has 100% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c having SEQ ID NO:3; and
said feedback-deregulated ATP phosphoribosyltransferase comprising a feedback-deregulated variant of a Corynebacterium glutamicum ATP phosphoribosyltransferase, which has 100% amino acid sequence identity with SEQ ID NO:7.

13. The engineered fungal cell of claim 1, wherein the fungal cell is a cell of the genus Yarrowia and of the species lipolytica.

14. The engineered fungal cell of claim 13, wherein the fungal cell expresses:
said non-native histidine decarboxylase having at least 70% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 having SEQ ID NO:6 or from Acinetobacter baumannii strain AB0057 having SEQ ID NO:1; and
said ATP phosphoribosyltransferase having at least 70% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c having SEQ ID NO:3.

15. The engineered fungal cell of claim 14, wherein the fungal cell expresses:
said non-native histidine decarboxylase, which has 100% amino acid sequence identity with a histidine decarboxylase from Chromobacterium sp. LK1 having SEQ ID NO:6 or from Acinetobacter baumannii strain AB0057 having SEQ ID NO:1; and
said ATP phosphoribosyltransferase, which has 100% amino acid sequence identity with an ATP phosphoribosyltransferase from S. cerevisiae S288c having SEQ ID NO:3.