Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions.

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m(2)/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16-C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.

Construction of target-specific virus-like particles for the delivery of algicidal compounds to harmful algae.

Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPs in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems.

Effects of the algicide, thiazolidinedione derivative TD49, on microbial communities in a mesocosm experiment.

We investigated the effects of the algicide thiazolidinedione derivative TD49 on microbial community in mesocosm experiments. The TD49 concentration exponentially decreased over time, with half-life of 3.5 h, following addition in the seawater (R2=0.98, P<0.001). Among microbial communities, heterotrophic bacteria and heterotrophic nanoflagellates (HNFs) grew well in all treatments following the addition of TD49. The abundance of HNFs lagged behind the increase in heterotrophic bacteria by 24 h in the 0.2 and 0.4 µM TD49 concentrations (R2=0.28, P<0.05), and by 48 h in the 0.6 and 1.0 µM TD49 concentrations (R2=0.30, P<0.05). This implies a strong concentration-dependent top-down effect of TD49 on microbial communities, with indications that the degradation of planktonic organisms, including the target alga, led to high heterotrophic bacteria concentrations, which in turn stimulated the population growth of predatory HNF. However, total ciliate numbers remained relatively low in the TD49 treatments relative to the control and blank groups, suggesting limited carbon flow from bacteria to these grazers even though the abundance of aloricate ciliates gradually increased toward the end of the experimental period, particularly at the high TD49 concentrations. TD49 appears to provide an environmentally safe approach to the control of harmful algal blooms (HABs) in aquatic ecosystems.

Expression of each cistron in the gal operon can be regulated by transcription termination and generation of a galk-specific mRNA, mK2.

The gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378: 318-327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3' ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5' ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5' ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3' ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5' portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity.

Chromatographic methods for characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers.

The objective of this study was to develop chromatographic methods for the determination of the modification degree and the characterization of poly(ethylene glycol)-modified polyamidoamine dendrimers (PEG-PAMAMs). The PEG-PAMAMs were prepared by reacting PAMAM generation 4 with monomethoxy PEG-nitrophenyl carbonate (mPEG-NPC). The modification degrees of PEG-PAMAMs were determined by quantifying 4-nitrophenol released from mPEG-NPC after PEGylation reaction using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The PEG-PAMAMs, which have poor UV absorbances, were characterized by HPLC with charged aerosol detection. This study demonstrates that the combination of these two detectors is a powerful tool for the preparation and characterization of PEG-PAMAMs.

Biodegradation of Methyl Orange by alginate-immobilized Aeromonas sp. in a packed bed reactor: external mass transfer modeling.

Azo dyes are recalcitrant and xenobiotic nature makes these compounds a challenging task for continuous biodegradation up to satisfactorily levels in large-scale. In the present report, the biodegradation efficiency of alginate immobilized indigenous Aeromonas sp. MNK1 on Methyl Orange (MO) in a packed bed reactor was explored. The experimental results were used to determine the external mass transfer model. Complete MO degradation and COD removal were observed at 0.20 cm bead size and 120 ml/h flow rate at 300 mg/l of initial dye concentration. The degradation of MO decreased with increasing bead sizes and flow rates, which may be attributed to the decrease in surface of the beads and higher flux of MO, respectively. The experimental rate constants (k ps) for various beads sizes and flow rates were calculated and compared with theoretically obtained rate constants using external film diffusion models. From the experimental data, the external mass transfer effect was correlated with a model J D = K Re (-(1 - n)). The model was tested with K value (5.7) and the Colburn factor correlation model for 0.20, 0.40 and 0.60 bead sizes were J D = 5.7 Re (-0.15), J D = 5.7 Re (-0.36) and J D = 5.7 Re (-0.48), respectively. Based on the results, the Colburn factor correlation models were found to predict the experimental data accurately. The proposed model was constructive to design and direct industrial applications in packed bed reactors within acceptable limits.

Comparison of biomass production and total lipid content of freshwater green microalgae cultivated under various culture conditions.

The growth and total lipid content of four green microalgae (Chlorella sp., Chlorella vulgaris CCAP211/11B, Botryococcus braunii FC124 and Scenedesmus obliquus R8) were investigated under different culture conditions. Among the various carbon sources tested, glucose produced the largest biomass or microalgae grown heterotrophically. It was found that 1% (w/v) glucose was actively utilized by Chlorella sp., C. vulgaris CCAP211/11B and B. braunii FC124, whereas S. obliquus R8 preferred 2% (w/v) glucose. No significant difference in biomass production was noted between heterotrophic and mixotrophic (heterotrophic with light illumination/exposure) growth conditions, however, less production was observed for autotrophic cultivation. Total lipid content in cells increased by approximately two-fold under mixotrophic cultivation with respect to heterotrophic and autotrophic cultivation. In addition, light intensity had an impact on microalgal growth and total lipid content. The highest total lipid content was observed at 100 µmol m(-2)s(-1) for Chlorella sp. (22.5%) and S. obliquus R8 (23.7%) and 80 µmol m(-2)s(-1) for C. vulgaris CCAP211/11B (20.1%) and B. braunii FC124 (34.9%).

A novel thiazolidinedione derivative TD118 showing selective algicidal effects for red tide control.

Thiazolidinedione (TD) derivatives have been found to have an algicidal effect on harmful algal bloom microalgae. In this study, 75 TD derivatives were synthesized and analyzed for algicidal activity. Among these synthetic TDs, 18 TD derivatives showed specific algicidal activity on two strains belonging to the classes Raphidophyceae (Chattonella marina and Heterosigma akashiwo) and Dinophyceae (Cochlodinium polykrikoides). Two strains belonging to Bacillariophyceae (Navicula pelliculosa and Phaeodactylum EPV), one strain belonging to Dinophyceae (Amphidinium sp.), and a Eustigmatophycean microalga (Nannochloropsis oculata) showed less sensitivity to the TD derivatives than the other two phyla. The most reactive TD derivative, compound 2 (TD118), was selected and tested for morphological and physiological changes. TD118 effectively damaged the cell membrane of C. marina, H. akashiwo and C. polykrikoides. The O2 evolution and photosystem II efficiency (F(v)/F(m)) of C. marina, H. akashiwo and C. polykrikoides were also severely reduced by TD118 treatment. Amphidinium sp., N. pelliculosa, Phaeodactylum EPV and N. oculata showed less reduction of O2 evolution and the F(v)/F(m) by TD118. These results imply that the species-specific TD structure relationship may be due to structural and/or physiological differences among microalgal species.

Regulation of the ald gene encoding alanine dehydrogenase by AldR in Mycobacterium smegmatis.

The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding L-alanine dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of L-alanine. The purified AldR protein exists as a homodimer in the absence of L-alanine, while it adopts the quaternary structure of a homohexamer in the presence of L-alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by L-alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N2-ATC-N2-TC and one putative AldR binding site with the sequence GA-N2-GTT-N2-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of L-alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of alanine.

Crystallization and preliminary X-ray crystallographic analysis of MxaJ, a component of the methanol-oxidizing system operon from the marine bacterium Methylophaga aminisulfidivorans MPT.

The methanol-oxidizing system (mox) is essential for methylotrophic bacteria to extract energy during the oxidoreduction reaction and consists of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes is the α2ß2 methanol dehydrogenase complex (type I MDH), which converts methanol to formaldehyde during the 2e⁻ transfer through the prosthetic group pyrroloquinoline quinone. MxaJ, a product of mxaJ of the mox operon, is a component of the MDH complex and enhances the methanol-converting activity of the MDH complex. However, the exact functional mechanism of MxaJ in the complex is not clearly known. To investigate the functional role of MxaJ in MDH activity, an attempt was made to determine its crystal structure. Diffraction data were collected from a selenomethionine-substituted crystal to 1.92 Å resolution at the peak wavelength. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 37.127, b = 63.761, c = 99.246 Å. The asymmetric unit contained one MxaJ molecule with a calculated Matthews coefficient of 2.11 Ų Da⁻¹ and a solvent content of 41.7%. Three-dimensional structure determination of the MxaJ protein is currently in progress by the single-wavelength anomalous dispersion technique and model building.

Expression and bioconversion of recombinant m- and p-hydroxybenzoate hydroxylases from a novel moderate halophile, Chromohalobacter sp.

p-Hydroxybenzoate hydroxylase (pobA) and m-hydroxybenzoate hydroxylase (mobA) genes, from the moderate halophile Chromohalobacter sp. HS-2, were expressed and characterized. Solubilities of overexpressed recombinant MobA and PobA were enhanced by the induction of the heat-shock proteins DnaJ and DnaK. Each MobA and PobA maintained stable activity under high NaCl concentrations. V (max) and K (m) values for MobA with m-hydroxybenzoate were 70 µmol min(-1) mg(-1) protein and 81 µM, respectively. Similarly, those of PobA with p-hydroxybenzoate as substrate were 5 µmol min(-1) mg(-1) protein and 129 µM, respectively. The Escherichia coli expression system, including induction of heat shock proteins, was used to convert hydroxybenzoates into protocatechuate (3,4-dihydroxybenzoate) and revealed that resting cells harboring mobA converted 15 mM m-hydroxybenzoate to 15 mM protocatechuate while those harboring pobA converted 50 mM p-hydroxybenzoate to 35 mM protocatechuate at 30 °C, respectively.

Optimization of culture conditions and comparison of biomass productivity of three green algae.

Culture conditions for the mass production of three green algae, Chlorella sp., Dunaliella salina DCCBC2 and Dunaliella sp., were optimized using a response surface methodology (RSM). A central composite design was applied to investigate the effects of initial pH, nitrogen and phosphate concentrations on the cultivation of microalgae. The optimal growth conditions estimated from the design are as follows: Chlorella sp. (initial pH 7.2, ammonium 17 mM, phosphate 1.2 mM), D. salina DCCBC2 (initial pH 8.0, nitrate 3.3 mM, phosphate 0.0375 mM) and Dunaliella sp. (initial pH 8.0, nitrate 3.7 mM, phosphate 0.17 mM). Culturing the microalgae with the optimized conditions confirmed that the maximum growth rates were attained for these parameters. The optimum CO(2) concentrations of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 1.0, 3.0 and 1.0% (v/v), respectively. The specific growth rates (µ) of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 0.58, 0.78 and 0.56 day(-1), respectively, and the biomass productivities were 0.28, 0.54 and 0.30 g dry cell wt l(-1) day(-1), respectively. The CO(2) fixation rates of Chlorella sp., D. salina DCCBC2 and Dunaliella sp. were 42.8, 90.9 and 45.5 mg l(-1) day(-1), respectively. Mixotrophic cultivation of Chlorella sp. with glucose increased biomass productivity from 0.28 to 0.51 g dry cell wt l(-1) day(-1). However, D. salina DCCBC2 and Dunaliella sp. were not stimulated by several organic compounds tested.

Comparative analysis of two types of methanol dehydrogenase from Methylophaga aminisulfidivorans MPT grown on methanol.

Two types of methanol dehydrogenase (MDH) were obtained from a novel marine methylotrophic bacterium, Methylophaga aminisulfidivorans MP(T), grown on methanol. Type I MDH consisted of two identical dimers of α (65.98 kDa) and ß (7.58 kDa) subunits organized to form the α(2)ß(2) tetramer. Type II MDH contained an additional MxaJ protein (27.86 kDa) and had more specific activity than type I MDH. The K(m) values of type I and II MDH for methanol under cytochrome c(L) reduction assay system were estimated to be 50.3 and 13.0 µM, respectively, and the isoelectric points of type I and II MDH were determined to be 5.4 and 5.8, respectively. The average molar ratios of α:ß, α:MxaJ, and ß:MxaJ in type II MDH were approximately 1:0.99, 1:0.41 and 1:0.42, respectively. Based on these results, the original conformation of the MDH of M. aminisulfidivorans MP(T) is most likely the α(2)ß(2)-MxaJ complex. During purification, the lysozyme and freeze-thawing cell disruption method significantly increased the amount of type II MDH in the soluble fraction compared with strong physical disruption methods such as sonication and French Press.

Draft genome sequence of Methylophaga aminisulfidivorans MP T.

Methylophaga aminisulfidivorans MP(T) is a restricted facultatively marine methylotrophic bacterium that grows on methanol, methylated amines, dimethyl sulfide, and dimethyl sulfoxide. Here we present the high-quality draft genome sequence of M. aminisulfidivorans MP(T) (KCTC 12909(T) = JCM 14647(T)), consisting of a chromosome (3,092,085 bp) and a plasmid (16,875 bp).

Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism.

A bacterial flavin-containing monooxygenase (bFMO) catalyses the oxygenation of indole to produce indigoid compounds. In the reductive half of the indole oxygenation reaction, NADPH acts as a reducing agent, and NADP(+) remains at the active site, protecting bFMO from reoxidation. Here, the crystal structures of bFMO and bFMO in complex with NADP(+), and a mutant bFMO(Y207S), which lacks indole oxygenation activity, with and without indole are reported. The crystal structures revealed overlapping binding sites for NADP(+) and indole, suggestive of a double-displacement reaction mechanism for bFMO. In biochemical assays, indole inhibited NADPH oxidase activity, and NADPH in turn inhibited the binding of indole and decreased indoxyl production. Comparison of the structures of bFMO with and without bound NADP(+) revealed that NADPH induces conformational changes in two active site motifs. One of the motifs contained Arg-229, which participates in interactions with the phosphate group of NADPH and appears be a determinant of the preferential binding of bFMO to NADPH rather than NADH. The second motif contained Tyr-207. The mutant bFMO(Y207S) exhibited very little indoxyl producing activity; however, the NADPH oxidase activity of the mutant was higher than the wild-type enzyme. It suggests a role for Y207, in the protection of hydroperoxyFAD. We describe an indole oxygenation reaction mechanism for bFMO that involves a ping-pong-like interaction of NADPH and indole.

Escherichia coli ribonuclease III activity is downregulated by osmotic stress: consequences for the degradation of bdm mRNA in biofilm formation.

During the course of experiments aimed at identifying genes with ribonuclease III (RNase III)-dependent expression in Escherichia coli, we found that steady state levels of bdm mRNA were dependent on cellular concentrations of RNase III. The half-lives of adventitiously overexpressed bdm mRNA and the activities of a transcriptional bdm'-'cat fusion were observed to be dependent on cellular concentrations of RNase III, indicating the existence of cis-acting elements in bdm mRNA responsive to RNase III. In vitro and in vivo cleavage analyses of bdm mRNA identified two RNase III cleavage motifs, one in the 5'-untranslated region and the other in the coding region of bdm mRNA, and indicated that RNase III cleavages in the coding region constitute a rate-determining step for bdm mRNA degradation. We also discovered that downregulation of the ribonucleolytic activity of RNase III is required for the sustained elevation of RcsB-induced bdm mRNA levels during osmotic stress and that cells overexpressing bdm form biofilms more efficiently. These findings indicate that the Rcs signalling system has an additional regulatory pathway that functions to modulate bdm expression and consequently, adapt E. coli cells to osmotic stress.

Gold nanoparticle-assisted delivery of small, highly structured RNA into the nuclei of human cells.

Previous studies have shown that functionalized gold nanoparticles (AuNPs) can be used as a general platform for loading and delivering DNA oligonucleotides and short hairpin RNA to living systems. Here, we report the ability of functionalized AuNP to deliver RNA aptamers into the nuclei of human cells. An in vitro-synthesized RNA aptamer specific to the ß-catenin protein was delivered into the HepG2 human cell line more efficiently via functionalized AuNP than liposome-based delivery, and resulted in nearly complete inhibition of ß-catenin binding to the p50 subunit of NF-κB in the nucleus. This inhibition led to repression of NF-κB p50-dependent transcription of CRP. Also, the ß-catenin aptamer in the nucleus led to down-regulation of ß-catenin-mediated transcriptional activity through the TCF complex and resulted in decrease in the levels of cyclin D, and c-myc mRNA by ~47% and ~57%, respectively. In addition, we used functionalized AuNP to deliver another RNA aptamer targeted to the p50 subunit of NF-κB into the A549 human cell line, and this was sufficient to induce apoptosis of the cells. Our findings demonstrate that AuNP GDS can be used to deliver small, highly structured RNA aptamers into the nucleus of human cells where they modulate the activity of transactivators by interacting with target proteins.

Purification, crystallization and preliminary X-ray crystallographic analysis of a methanol dehydrogenase from the marine bacterium Methylophaga aminisulfidivorans MP(T).

Methylophaga aminisulfidivorans MP(T) is a marine methylotrophic bacterium that utilizes C(1) compounds such as methanol as a carbon and energy source. The released electron from oxidation flows through a methanol-oxidizing system (MOX) consisting of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes in the pathway is methanol dehydrogenase (MDH), which contains the prosthetic group pyrroloquinoline quinone (PQQ) and converts methanol to formaldehyde in the periplasm by transferring two electrons from the oxidation of one methanol molecule to the electron acceptor cytochrome c(L). In order to obtain molecular insights into the oxidation mechanism, a native heterotetrameric α(2)ß(2) MDH complex was directly purified from M. aminisulfidivorans MP(T) grown in the presence of methanol and crystallized. The crystal diffracted to 1.7 Šresolution and belonged to the monoclinic space group P2(1) (unit-cell parameters a = 63.9, b = 109.5, c = 95.6 Å, ß = 100.5°). The asymmetric unit of the crystal contained one heterotetrameric complex, with a calculated Matthews coefficient of 2.24 Å(3) Da(-1) and a solvent content of 45.0%.

Achieving Maximal Production of Fusaricidins from Paenibacillus kribbensis CU01 via Continuous Fermentation.

In this study, we investigated the potential of Paenibacillus kribbensis CU01 in producing fusaricidin, a strong antifungal substance, via optimization of metal ions and carbon and nitrogen source, and continuous fermentation. In the cultivation of a 2-l batch, maximal production of fusaricidins (581 mg l-1) was achieved in a modified M9 medium containing metal ions, 10 g l-1 glucose, and 1 g l-1 ammonium chloride. Most of glucose was consumed at a rate of 0.74 g l-1 h-1 within 24 h and fusaricidin production began 15 h after batch cultivation. Continuous fermentation was performed using a 7-l fermenter with 2-l working volume of modified M9 medium containing 10 g l-1 glucose, 1 × 10-3 M FeSO4, and 1 × 10-6 M MnCl2. After 24 h of the start of cultivation, fresh M9 medium was continuously supplied at a flow rate of 2.5 ml min-1, and simultaneously, the same amount of cell culture broth was removed. In a continuous system, the highest fusaricidin concentration (579 mg l-1) was obtained using a dilution rate of 0.075 h-1 with an average productivity of 10.4 mg l-1 h-1 for 24 to 72 h of incubation. Based on these results, it was found that fusaricidin production using P. kribbens CU01 strain increased by at least 28 times the values reported in previous studies.

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.