The BioC O-Methyltransferase Catalyzes Methyl Esterification of Malonyl-Acyl Carrier Protein,an Essential Step in Biotin Synthesis

Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682–688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-l-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-l-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis.     

Isolation and characterization of the Erwinia herbicola bio operon and the sequences of the bioA and bioB genes

The biotin operon of Erwinia herbicola was cloned and characterized. The operon consists of five genes arranged in the order, bioABFCD. The operon is negatively regulated via the interaction of a proposed biotin repressor with an operator sequence that lies between the bioA and bioB genes. The nucleotide sequences of bioA (7,8-diaminopelargonic acid transferase), bioB (biotin synthetase) and the regulatory region were determined and analyzed. The deduced amino acid sequences of bioA and bioB are also aligned with currently available homologs to obtain the UPGMA (unweighted pair group method with arithmetic mean) evolutionary tree.     

The ISC [corrected] proteins Isa1 and Isa2 are required for the function but not for the de novo synthesis of the Fe/S clusters of biotin synthase in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.     

Remarkable Diversity in the Enzymes Catalyzing the Last Step in Synthesis of the Pimelate Moiety of Biotin

Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG proteins have esterase activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli. Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.     

Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system

Background  

Escherichia coli strains lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) are capable of coutilizing glucose and other carbon sources due to the absence of catabolite repression by glucose. In these strains, the lack of this important regulatory and transport system allows the coexistence of glycolytic and gluconeogenic pathways. Strains lacking PTS have been constructed with the goal of canalizing part of the phosphoenolpyruvate (PEP) not consumed in glucose transport to the aromatic pathway. The deletion of the ptsHIcrr operon inactivates PTS causing poor growth on this sugar; nonetheless, fast growing mutants on glucose have been isolated (PB12 strain). However, there are no reported studies concerning the growth potential of a PTS^- strain in mixtures of different carbon sources to enhance the production of aromatics compounds.     

The SOS induction of umu'-'lacZ fusion gene by oxidative damage is influenced by polyamines in Escherichia coli

We report that polyamines have an effect on the SOS response of the umu operon in polyamine-deficient mutant and wild-type Escherichia coli strains carrying the umu'-'lacZ fusion. H₂O₂ effectively induces umu'-'lacZ in the wild type, but not significantly in the mutant strain. Exogenous polyamines did not restore the umu induction in the mutant to the wild-type level. In logarithmically growing cells, the basal expression of umu gene in the mutant is about five times higher than that of the wild type. The addition of polyamines to the growth medium markedly reduces the basal expression to the wild-type level. This reduction is due not to growth rate but to the polyamine itself. Our results suggest that polyamines are essentially involved in the SOS induction of the umu operon in E. coli.     

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments

Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD₆₀₀ 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017     

Biochemical and genetic characterization of dehydrobiotin resistant mutants of Escherichia coli

Summary Dehydrobiotin (DHB) resistant mutants were isolated from strains of Escherichia coli K-12 and were classified into two groups; dhbA and dhbB.In dhbB mutants the structural genes for enzymes of the biotin pathway are expressed constitutively at a high rate. The dhbB gene is co-transducible with argE at a frequency of about 50% by P1 transduction and maps on the chromosome between arg EC BH and rif. The dhbB ⁺ gene is trans-dominant over the mutant allele indicating that the dhbB ⁺ gene controls the production of a diffusible substance such as a repressor molecule.The dhbA mutants show biotin biosynthetic activity comparable to the wild type and are as sensitive to repression by biotin as the parent strain. The mutants appear to be deficient in DHB transport as suggested by the findings that the ability of the mutants to take up biotin is reduced significantly and that DHB, a competitive inhibitor of biotin uptake, is much less inhibitory to biotin uptake in the mutants than in the wild type.     

Metabolic engineering of<Emphasis Type="Italic"> Escherichia coli</Emphasis> for improved 6-deoxyerythronolide B production

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit (2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase (matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of ¹³C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.S. Murli and J. Kennedy contributed equally to this work     

Improvement of Escherichia coli strains overproducing lysine using recombinant DNA techniques

Summary Several genes of the lysine biosynthetic pathway were cloned separately on the high copy number plasmid pBR322 (Richaud et al. 1981). These hybrid plasmids were used to transform an Escherichia coli strain TOC R 21 that overproduces lysine due to mutations altering the aspartokinase reaction. The synthesis of lysine was studied in these different strains. It appears that only plasmids containing the dapA gene (encoding dihydrodipicolinate synthetase) lead to an increase in lysine production. This result allows us to identify this reaction as the limiting biosynthetic step in strain TOC R 21 and indicates that such a method of gene amplification can be used to improve strains overproducing metabolites.     

Studies on Production of Biotin by Microorganisms

The utilization of hydrocarbons by microorganisms was studied in many fields, but the production of biotin vitamers by hydrocarbon-utilizing bacteria has never been reported.

We have screened many hydrocarbon-utilizing bacteria which produce biotin vitamers in the culture broth. The effects of cultural conditions on biotin vitamers production by strain 5–2, tentatively assigned to the genus Pseudomonas, were studied.

More than 98% of biotin vitamers produced from hydrocarbons by strain 5–2 was chromatographically determined as desthiobiotin. As nitrogen source, natural nutrients were more effective than inorganic nitrogen sources. The production of biotin vitamers was increased under the condition of good aeration. Exogenous pimelic or azelaic acid enhanced biotin vitamers production by strain 5–2.

The production of biotin vitamers from n-alkanes, n-alkenes or glucose by an isolated bacterium, strain 5-2, tentatively assigned to the genus Pseudomonas, was investigated. Among these carbon sources, n-undecane was the most excellent for biotin vitamers production.

The biosynthetic pathway of biotin vitamers, especially desthiobiotin, from n-undecane was also studied. It was found by thin-layer and gas-liquid chromatographical methods that pimelic and azelaic acids were the main acid components in n-undecane culture.

This result, together with previously reported enhancement of biotin vitamers production by these acids, suggests that pimelic and azelaic acids may be the intermediates of biotin vitamers biosynthesis from n-undecane.     

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

3‐amino‐benzoic acid (3AB) is an important building block molecule for production of a wide range of important compounds such as natural products with various biological activities. In the present study, we established a microbial biosynthetic system for de novo 3AB production from the simple substrate glucose. First, the active 3AB biosynthetic pathway was reconstituted in the bacterium Escherichia coli, which resulted in the production of 1.5 mg/L 3AB. In an effort to improve the production, an E. coli‐E. coli co‐culture system was engineered to modularize the biosynthetic pathway between an upstream strain and an downstream strain. Specifically, the upstream biosynthetic module was contained in a fixed E. coli strain, whereas a series of E. coli strains were engineered to accommodate the downstream biosynthetic module and screened for optimal production performance. The best co‐culture system was found to improve 3AB production by 15 fold, compared to the mono‐culture approach. Further engineering of the co‐culture system resulted in biosynthesis of 48 mg/L 3AB. Our results demonstrate co‐culture engineering can be a powerful new approach in the broad field of metabolic engineering.     

Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.     

The Arabidopsis Bio2 protein requires mitochondrial targeting for activity

Mitochondria are involved in the production of various vitamins, such as biotin, in plants. It is unclear why these biosynthetic pathways have been maintained partly or entirely within the mitochondria throughout evolution. The last step in biotin biosynthesis occurs within the mitochondria and is catalyzed by the biotin synthase complex containing the BIO2 gene product. We investigated whether the Arabidopsis Bio2 enzyme could function outside mitochondria, by trying to complement a bio2 mutant with a truncated version of BIO2 lacking the region encoding the mitochondrial targeting sequence. We describe the characterization of a new T-DNA allele of bio2, with the sole phenotype of an absence of biotin production, in contrast to the previously characterized EMS bio2 allele (Patton et al. 1998, Plant Physiol 116(3):935–946). We found that a cytosolic version of the Bio2 protein could not complement this mutant. Supplementation with the substrate dethiobiotin (DTB) also failed to rescue the mutant phenotype. Thus, the lack of availability of DTB in the cytosol is not the only factor preventing this reaction from occurring outside mitochondria. Bio2 requires mitochondrial targeting for activity, enabling it to fulfill its role in biotin synthesis. The reaction catalyzed by Bio2 may be subject to biochemical constraints, and the apparent close connection with the mitochondrial Fe-S machinery may account for the reaction being retained within the organelle.     

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new Y ehD f imbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Acetate scavenging activity in <Emphasis Type="Italic">Escherichia coli</Emphasis>: interplay of acetyl–CoA synthetase and the PEP–glyoxylate cycle in chemostat cultures

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl−CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl−CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl−CoA synthetase are upregulated. A mutant in acs (encoding acetyl−CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl−CoA synthetase by cAMP−CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl−CoA and acetyl−phosphate pools, although establishing an energy-dissipating substrate cycle.     

Expression of dha Operon Required for 1,3-PD Formation in Escherichia coli and Saccharomyces cerevisiae

The 1,3-propanediol (1,3-PD) synthesis operon (dha operon) was mainly composed of four genes: dhaB, dhaT, gdrA, and gdrB, which encoded glycerol dehydratase, 1,3-PD oxidoreductase and reactivating factor for glycerol dehydratase, respectively. In the present study, dha operon was cloned from 1,3-PD producing strain Klebsiella pneumoniae. Heterologous expression of cloned dha operon was carried out in Escherichia coli and Saccharomyces cerevisiae W303-1A, respectively. The results indicated that recombinant E. coli harboring the dha operon can produce 8–9 g/l 1,3-PD from glycerol while the 1,3-PD yield of recombinant strain W303-1A-dha could not be detected. In order to complete the 1,3-PD production from glucose, further, we also constructed the recombinant S. cerevisiae W303-1A-BT harboring plasmid pZ-BT. The 1,3-PD production and enzymatic activities of DhaB and DhaT were found in the engineered strain W303-1A-BT. Our results demonstrated that the recombinant S. cerevisiae strain W303-1A-BT that can produce 1,3-PD at low cost was constructed. This study might open a novel way to a safe and cost-efficient method for microbial production of 1,3-PD.