Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.     

Whole‐cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae

Rapid global industrialization in the past decades has led to extensive utilization of fossil fuels, which resulted in pressing environmental problems due to excessive carbon emission. This prompted increasing interest in developing advanced biofuels with higher energy density to substitute fossil fuels and bio‐alkane has gained attention as an ideal drop‐in fuel candidate. Production of alkanes in bacteria has been widely studied but studies on the utilization of the robust yeast host, Saccharomyces cerevisiae, for alkane biosynthesis have been lacking. In this proof‐of‐principle study, we present the unprecedented engineering of S. cerevisiae for conversion of free fatty acids to alkanes. A fatty acid α‐dioxygenase from Oryza sativa (rice) was expressed in S. cerevisiae to transform C_12–18 free fatty acids to C_11–17 aldehydes. Co‐expression of a cyanobacterial aldehyde deformylating oxygenase converted the aldehydes to the desired alkanes. We demonstrated the versatility of the pathway by performing whole‐cell biocatalytic conversion of exogenous free fatty acid feedstocks into alkanes as well as introducing the pathway into a free fatty acid overproducer for de novo production of alkanes from simple sugar. The results from this work are anticipated to advance the development of yeast hosts for alkane production. Biotechnol. Bioeng. 2017;114: 232–237. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O₂/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.     

Heme Iron Uptake by Caco-2 Cells is a Saturable,Temperature Sensitive and Modulated by Extracellular pH and Potassium

It is known that heme iron and inorganic iron are absorbed differently. Heme iron is found in the diet mainly in the form of hemoglobin and myoglobin. The mechanism of iron absorption remains uncertain. This study focused on the heme iron uptake by Caco-2 cells from a hemoglobin digest and its response to different iron concentrations. We studied the intracellular Fe concentration and the effect of time, K⁺ depletion, and cytosol acidification on apical uptake and transepithelial transport in cells incubated with different heme Fe concentrations. Cells incubated with hemoglobin-digest showed a lower intracellular Fe concentration than cells grown with inorganic Fe. However, uptake and transepithelial transport of Fe was higher in cells incubated with heme Fe. Heme Fe uptake had a low V _max and K _m as compared to inorganic Fe uptake and did not compete with non-heme Fe uptake. Heme Fe uptake was inhibited in cells exposed to K⁺ depletion or cytosol acidification. Heme oxygenase 1 expression increased and DMT1 expression decreased with higher heme Fe concentrations in the media. The uptake of heme iron is a saturable and temperature-dependent process and, therefore, could occur through a mechanism involving both a receptor and the endocytic pathway.     

Microbial synthesis of wax esters

Microbial synthesis of wax esters (WE) from low-cost renewable and sustainable feedstocks is a promising path to achieve cost-effectiveness in biomanufacturing. WE are industrially high-value molecules, which are widely used for applications in chemical, pharmaceutical, and food industries. Since the natural WE resources are limited, the WE production mostly rely on chemical synthesis from rather expensive starting materials, and therefore solution are sought from development of efficient microbial cell factories. Here we report to engineer the yeast Yarrowia lipolytica and bacterium Escherichia coli to produce WE at the highest level up to date. First, the key genes encoding fatty acyl-CoA reductases and wax ester synthase from different sources were investigated, and the expression system for two different Y. lipolytica hosts were compared and optimized for enhanced WE production and the strain stability. To improve the metabolic pathway efficiency, different carbon sources including glucose, free fatty acid, soybean oil, and waste cooking oil (WCO) were compared, and the corresponding pathway engineering strategies were optimized. It was found that using a lipid substrate such as WCO to replace glucose led to a 60-fold increase in WE production. The engineered yeast was able to produce 7.6 g/L WE with a yield of 0.31 (g/g) from WCO within 120 h and the produced WE contributed to 57% of the yeast DCW. After that, E. coli BL21(DE3), with a faster growth rate than the yeast, was engineered to significantly improve the WE production rate. Optimization of the expression system and the substrate feeding strategies led to production of 3.7–4.0 g/L WE within 40 h in a 1-L bioreactor. The predominant intracellular WE produced by both Y. lipolytica and E. coli in the presence of hydrophobic substrates as sole carbon sources were C₃₆, C₃₄ and C₃₂, in an order of decreasing abundance and with a large proportion being unsaturated. This work paved the way for the biomanufacturing of WE at a large scale.     

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.     

Production of artemisinin by genetically-modified microbes

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.     

Aromatic l‐amino acid decarboxylase phosphorylation and activation by PKGIαin vitro

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α₁/β₁ and α₂/β₁ heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.     

Physiological characterization of recombinant Saccharomyces cerevisiae expressing the Aspergillus nidulans phosphoketolase pathway: validation of activity through 13C-based metabolic flux analysis

Several bacterial species and filamentous fungi utilize the phosphoketolase pathway (PHK) for glucose dissimilation as an alternative to the Embden-Meyerhof-Parnas pathway. In Aspergillus nidulans, the utilization of this metabolic pathway leads to increased carbon flow towards acetate and acetyl CoA. In the first step of the PHK, the pentose phosphate pathway intermediate xylulose-5-phosphate is converted into acetylphosphate and glyceraldehyde-3-phosphate through the action of xylulose-5-phosphate phosphoketolase, and successively acetylphosphate is converted into acetate by the action of acetate kinase. In the present work, we describe a metabolic engineering strategy used to express the fungal genes of the phosphoketolase pathway in Saccharomyces cerevisiae and the effects of the expression of this recombinant route in yeast. The phenotype of the engineered yeast strain MP003 was studied during batch and chemostat cultivations, showing a reduced biomass yield and an increased acetate yield during batch cultures. To establish whether the observed effects in the recombinant strain MP003 were due directly or indirectly to the expression of the phosphoketolase pathway, we resolved the intracellular flux distribution based on (13)C labeling during chemostat cultivations. From flux analysis it is possible to conclude that yeast is able to use the recombinant pathway. Our work indicates that the utilization of the phosphoketolase pathway does not interfere with glucose assimilation through the Embden-Meyerhof-Parnas pathway and that the expression of this route can contribute to increase the acetyl CoA supply, therefore holding potential for future metabolic engineering strategies having acetyl CoA as precursor for the biosynthesis of industrially relevant compounds.     

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas’ disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.     

Defects in the biosynthesis of mitochondrial heme c and heme a in yeast and mammals

Defects in heme biosynthesis have been associated with a large number of diseases, but mostly recognized in porphyrias, which are neurovisceral or cutaneous disorders caused by the accumulation of biosynthetic intermediates. However, defects in the maturation of heme groups that are part of the oxidative phosphorylation system are now also recognized as important causes of disease. The electron transport chain contains heme groups of the types a, b and c, all of which are directly involved in electron transfer reactions. In this article, we review the effect of mutations in enzymes involved in the maturation of heme a (the prosthetic group of cytochrome c oxidase) and heme c (the prosthetic group of cytochrome c) both in yeast and in humans. COX10 and COX15 are two genes, initially identified in Saccharomyces cerevisiae that have been found to cause infantile cytochrome c oxidase deficiency in humans. They participate in the farnesylation and hydroxylation of heme b, steps that are necessary for the formation of heme a, the prosthetic group required for cytochrome oxidase assembly and activity. Deletion of the cytochrome c heme lyase gene in a single allele has also been associated with a human disease, known as Microphthalmia with Linear Skin defects (MLS) syndrome. The cytochrome c heme lyase is necessary to covalently attach the heme group to the apocytochrome c polypeptide. The production of mouse models recapitulating these diseases is providing novel information on the pathogenesis of clinical syndromes.     

Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear.

In this review, special emphasis is placed on the stability properties of the heme-bound O₂. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O₂ to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO₂ or HbO₂ are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia

Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.     

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3₁₀-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3₁₀-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.     

Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9

The yeast Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butanediol, isoprenoids, and other chemicals. The construction of a successful production strain usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect the metabolic fluxes for the conversion of sugars and other feed stocks into the desired product. RNA-guided Cas9 based genome editing has been demonstrated in many prokaryotic and eukaryotic hosts including S. cerevisiae, in which it has been additionally exploited as a tool for metabolic engineering. To extend the utilization of RNA-guided Cas9 as a metabolic pathway building tool, we demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the beta-carotene biosynthetic pathway. Furthermore, we generated a combinatorial strain library for the beta-carotene biosynthetic pathway, directly integrated into the yeast genome to create a diverse library of strains. This enabled the screening of combinatorial libraries in stable chromosomally integrated strains for rapid improvements of product titers. This combinatorial approach for pathway assembly will significantly accelerate the current speed of metabolic engineering for S. cerevisiae as an industrial platform, and increase the number of strains that can be simultaneously evaluated for enzyme screening, expression optimization and protein engineering to achieve the titer, rate and yield necessary for the commercialization of new industrial fermentation products.     

1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdB^N1). Herein, we report the near complete ¹H, ¹³C and ¹⁵N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.     

Characterization and engineering of the ethylmalonyl-CoA pathway towards the improved heterologous production of polyketides in Streptomyces venezuelae

Streptomyces venezuelae has an inherent advantage as a heterologous host for polyketide production due to its fast rate of growth that cannot be endowed easily through metabolic engineering. However, the utility of S. venezuelae as a host has been limited thus far due to its inadequate intracellular reserves of the (2S)-ethylmalonyl-CoA building block needed to support the biosynthesis of polyketides preventing the efficient production of the desired metabolite, such as tylactone. Here, via precursor supply engineering, we demonstrated that S. venezuelae can be developed into a more efficient general heterologous host for the quick production of polyketides. We first identified and functionally characterized the ethylmalonyl-CoA pathway which plays a major role in supplying the (2S)-ethylmalonyl-CoA extender unit in S. venezuelae. Next, S. venezuelae was successfully engineered to increase the intracellular ethylmalonyl-CoA concentration by the deletion of the meaA gene encoding coenzyme B₁₂-dependent ethylmalonyl-CoA mutase in combination with ethylmalonate supplementation and was engineered to upregulate the expression of the heterologous tylosin PKS by overexpression of the pathway specific regulatory gene pikD. Thus, a dramatic increase (～10-fold) in tylactone production was achieved. In addition, the detailed insights into the role of the ethylmalonyl-CoA pathway, which is present in most streptomycetes, provides a general strategy to increase the ethylmalonyl-CoA supply for polyketide biosynthesis in the most prolific family of polyketide-producing bacteria.     

Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.