Engineering of Bacillus subtilis for Enhanced Total Synthesis of Folic Acid (original) (raw)
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Applied and Environmental Microbiology, 2007
The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (pabC) was initially annotated, but detailed analysis revealed that pabC was fused with the 3 end of the gene coding for chorismate synthetase component II (pabB). Therefore, we hypothesize that all three enzyme activities needed for pABA production are present in L. lactis, allowing for the production of pABA. Indeed, the overexpression of the pABA gene cluster in L. lactis resulted in elevated pABA pools, demonstrating that the genes are involved in the biosynthesis of pABA. Moreover, a pABA knockout (KO) strain lacking pabA and pabBC was constructed and shown to be unable to produce folate when cultivated in the absence of pABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and pABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of L. lactis. It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway. L. lactis strain NZ7024, overexpressing both the folate and pABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without pABA. This is in sharp contrast to L. lactis strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the pABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and pABA biosynthesis pathway in the wild-type strain.
Increased Production of Folate by Metabolic Engineering of Lactococcus lactis
Applied and Environmental Microbiology, 2003
The dairy starter bacterium Lactococcus lactis is able to synthesize folate and accumulates large amounts of folate, predominantly in the polyglutamyl form. Only small amounts of the produced folate are released in the extracellular medium. Five genes involved in folate biosynthesis were identified in a folate gene cluster in L. lactis MG1363: folA, folB, folKE, folP, and folC. The gene folKE encodes the biprotein 2-amino-4-hydroxy-6hydroxymethyldihydropteridine pyrophosphokinase and GTP cyclohydrolase I. The overexpression of folKE in L. lactis was found to increase the extracellular folate production almost 10-fold, while the total folate production increased almost 3-fold. The controlled combined overexpression of folKE and folC, encoding polyglutamyl folate synthetase, increased the retention of folate in the cell. The cloning and overexpression of folA, encoding dihydrofolate reductase, decreased the folate production twofold, suggesting a feedback inhibition of reduced folates on folate biosynthesis. on October 29, 2015 by guest http://aem.asm.org/ Downloaded from on October 29, 2015 by guest http://aem.asm.org/ Downloaded from FIG. 4. Effect of nisin concentration on dihydrofolate reductase activity measured in cell extracts of L. lactis strains harboring pNZ8048 (empty vector) (white bars), pNZ7013 (overexpressing folA) (hatched bars), or pNZ7014 (overexpressing antisense RNA of folA) (black bars). Error bars indicate standard deviations.
Multivitamin production in Lactococcus lactis using metabolic engineering
Metabolic Engineering, 2004
The dairy starter bacterium Lactococcus lactis has the potential to synthesize both folate (vitamin B11) and riboflavin (vitamin B2). By directed mutagenesis followed by selection and metabolic engineering we have modified two complicated biosynthetic pathways in L. lactis resulting in simultaneous overproduction of both folate and riboflavin: Following exposure to the riboflavin analogue roseoflavin we have isolated a spontaneous mutant of L. lactis strain NZ9000 that was changed from a riboflavin consumer into a riboflavin producer. This mutant contained a single base change in the regulatory region upstream of the riboflavin biosynthetic genes. By the constitutive overproduction of GTP cyclohydrolase I in this riboflavin-producing strain, the production of folate was increased as well. Novel foods, enriched through fermentation using these multivitamin-producing starters, could compensate the B-vitamin-deficiencies that are common even in highly developed countries and could specifically be used in dietary foods for the large fraction of the Caucasian people (10-15%) with mutations in the methylene tetrahydrofolate reductase (MTHFR).
Metabolic engineering of cobalamin (vitamin B 12 ) production in Bacillus megaterium
Microbial Biotechnology, 2010
Cobalamin (vitamin B12) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B 12 biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG A cbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B 12 riboswitch upstream of the cbiXJCDETLFGAcysG A cbiYbtuR operon and the recombinant production of three different vitamin B 12 binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B 12-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B 12 concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.
Enhanced recombinant protein production in pyruvate kinase mutant of Bacillus subtilis
Applied Microbiology and Biotechnology, 2010
Previous work demonstrated that acetate production was substantially lower in pyruvate kinase (pyk) mutant of Bacillus subtilis. The significantly lower acetate production in the pyk mutant is hypothesized to have positive effect on recombinant protein production either by lifting the inhibitory effect of acetate accumulation in the medium or redirecting the metabolic fluxes beneficial to biomass/protein synthesis. In this study, the impact of the pyk mutation on recombinant protein production was investigated. Green fluorescent protein (GFP+) was selected as a model protein and constitutively expressed in both the wild-type strain and a pyk mutant. In batch cultures, the pyk mutant produced 3-fold higher levels of recombinant protein when grown on glucose as carbon source. Experimental measurements and theoretical analysis show that the higher protein yield of the mutant is not due to removal of an acetate-associated inhibition of expression or gene dosage or protein stability but a much lower acetate production in the mutant allows for a greater fraction of carbon intake to be directed to protein synthesis.
High-Level Folate Production in Fermented Foods by the B12 Producer Lactobacillus reuteri JCM1112
Applied and Environmental Microbiology, 2008
We observed that Lactobacillus reuteri JCM1112 produces B 12 and folate. However, the folate/B 12 mass ratio found was far below that desired for human consumption (ϳ170:1). We used metabolic engineering applying genetic and physiological approaches to improve this ratio and developed a generic and natural process that significantly increases folate production.
Transformation of Folate-Consuming Lactobacillus gasseri into a Folate Producer
Applied and Environmental Microbiology, 2004
Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri. As a result L. gasseri changed from a folate consumer to a folate producer. This principle can be used to increase folate levels in many fermented food products.
Folate production by probiotic bacteria
Nutrients, 2011
Probiotic bacteria, mostly belonging to the genera Lactobacillus and Bifidobacterium, confer a number of health benefits to the host, including vitamin production. With the aim to produce folate-enriched fermented products and/or develop probiotic supplements that accomplish folate biosynthesis in vivo within the colon, bifidobacteria and lactobacilli have been extensively studied for their capability to produce this vitamin. On the basis of physiological studies and genome analysis, wild-type lactobacilli cannot synthesize folate, generally require it for growth, and provide a negative contribution to folate levels in fermented dairy products. Lactobacillus plantarum constitutes an exception among lactobacilli, since it is capable of folate production in presence of para-aminobenzoic acid (pABA) and deserves to be used in animal trials to validate its ability to produce the vitamin in vivo. On the other hand, several folate-producing strains have been selected within the genus Bifi...