D‐Mannitol production by resting state whole cell biotransformation of D‐fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium

Abstract

An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.

A short story about a big magic bug

“…Under the strict control of the repressor protein XylR, the system was used for the controllable production of intracellular recombinant proteins like β-galactosidase, glucose dehydrogenase, formate dehydrogenase and toxin A. [12][13][14][15] This promoter provided the basis for the effective and removal without cell disruption. Next, a series of secretion vectors based on the xylose-inducible vector system ( Fig.…”

“…The biosynthesis of all tetrapyrroles including vitamin B 12 and heme uses the same precursor molecule, uroporphyrinogen III. After uroporphyrinogen III, the biosynthetic pathways of vitamin B 12 and heme diverge. To repress the flux of this metabolite to the heme biosynthetic path, an antisense RNA strategy to inactivate the hemZ mRNA involved in this competing pathway was established.…”

Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium

“…Based on the development of strong inducible promoters and optimal ribosome binding sites, the identification of highly effective export signals and the integration of sequences encoding affinity tags, a set of B. megaterium expression systems was constructed which allowed the production of recombinant proteins intracellularly and also facilitated the secretion of recombinant proteins in the gram-per-liter scale (Biedendieck et al 2007b, c;Gamer et al 2009;Korneli et al 2013a;Malten et al 2006;Meinhardt et al 1989;Rygus and Hillen 1991;Stammen et al 2010a;Wittchen and Meinhardt 1995). However, one major bottleneck in context of recombinant protein production was the low translation efficiency of mRNA derived from heterologous genes (Bäumchen et al 2007;Yang et al 2007). Strong Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6744-5) contains supplementary material, which is available to authorized users.…”

“…So far, one requirement to enable the production of a recombinant protein in B. megaterium is a CAI above 0.3 of its corresponding gene to the B. megaterium codon usage (Bäumchen et al 2007;Grote et al 2005;Yang et al 2007). However, no generally applicable B. megaterium production strain providing rare codon tRNA (rctRNA) genes in trans is available.…”

Experimental and Modeling Investigation of Supercritical Extraction of Mannitol from Olive Leaves

“…Therefore, research in developing a safer, inexpensive, and more effective method for the production of mannitol is needed. New processes, including chemical, enzymatic, and microbial processes, are currently being developed and evaluated against the existing hydrogenation method in order to improve the process of mannitol production in terms of safety, technological and economical aspects [5][6][7].…”