Glucose In Batch Fermentation Research Articles

Pathway Engineering for Phenethylamine Production in Escherichia coli.

In this study, the metabolic pathway of phenethylamine synthesis was reconstructed by chromosomal integration and overexpression of the Enterococcus faecium pdc gene encoding phenylalanine decarboxylase in Escherichia coli. The genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG), shikimate kinase II (aroL), chorismate mutase/prephenate dehydratase (pheA), and tyrosine aminotransferase (tyrB) in the phenethylamine synthetic pathway were sequentially chromosomally overexpressed. The phosphotransferase system was replaced by deleting the ptsH-ptsI-crr genes and chromosomally overexpressing the genes encoding galactose permease (galP) and glucokinase (glk). In addition, the zwf gene encoding glucose-6-phosphate dehydrogenase in the pentose phosphate pathway was chromosomally overexpressed, generating the final engineered E. coli strain AUD9. The AUD9 strain produced 2.65 g L-1 phenethylamine with a yield of 0.27 g of phenethylamine g-1 glucose in batch fermentation; fed-batch fermentation of AUD9 produced 38.82 g L-1 phenethylamine with a productivity of 1.08 g L-1 h-1 phenethylamine, demonstrating its potential for industrial fermentative production of phenethylamine.

Development of a shuttle plasmid without host restriction sites for efficient transformation and heterologous gene expression in Clostridium cellulovorans.

Clostridium cellulovorans capable of producing large amounts of acetate and butyrate from cellulose is a promising candidate for biofuels and biochemicals production from lignocellulosic biomass. However, the restriction modification (RM) systems of C. cellulovorans hindered the application of existing shuttle plasmids for metabolic engineering of this organism. To overcome the hurdle of plasmid digestion by host, a new shuttle plasmid (pYL001) was developed to remove all restriction sites of two major RM systems of C. cellulovorans, Cce743I and Cce743II. The pYL001 plasmid remained intact after challenge by C. cellulovorans cell extract. Post-electroporation treatments and culturing conditions were also modified to improve cell growth and colony formation on agar plates. With the improvements, the pYL001 plasmid, without in vivo methylation, was readily transformed into C. cellulovorans with colonies of recombinant cells formed on agar plates within 24h. Three pYL001-derived recombinant plasmids free of Cce743I/Cce743II restriction sites, after synonymous mutation of the heterologous genes, were constructed and transformed into C. cellulovorans. Functional expression of these genes was confirmed with butanol and ethanol production from glucose in batch fermentations by the transformants. The pYL001 plasmid and improved transformation method can facilitate further metabolic engineering of C. cellulovorans for cellulosic butanol production.

Production of 5-aminovaleric acid in recombinant Corynebacterium glutamicum strains from a Miscanthus hydrolysate solution prepared by a newly developed Miscanthus hydrolysis process

This study examined nine expired industrial Corynebacterium glutamicum strains with high lysine producing capability for enhanced production of 5-AVA. C. glutamicum KCTC 1857 exhibiting the highest lysine production was transformed with either original Pseudomonas putida davBA genes, encoding the 5-AVA biosynthesis pathway, or C. glutamicum codon-optimized davBA genes. C. glutamicum KCTC 1857 expressing the original genes had superior cell viability and 5-AVA production capability compared to the other strain. This strain produced 39.93g/L of 5-AVA, which is the highest titer reported to date in fed-batch fermentation from glucose. Indeed, Miscanthus hydrolysate solution prepared from a novel process, comprising pretreatment, hydrolysis, purification, and concentration, was used as feedstock for 5-AVA production. A total of 12.51g/L 5-AVA was produced from the Miscanthus hydrolysate; this value is 34.7% higher than that obtained from glucose in batch fermentation.

Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase.

The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (Δack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH5.0 and 6.0. Compared to C. tyrobutyricum (Δack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22 - 0.26g/g (vs. 0.10 - 0.13g/g) and productivity to 0.35g/lh (vs. 0.13g/lh) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation.

Engineering Clostridium acetobutylicum with a histidine kinase knockout for enhanced n-butanol tolerance and production.

Clostridium acetobutylicum JB200, a mutant strain of C. acetobutylicum ATCC 55025 obtained through strain evolution in a fibrous bed bioreactor, had high butanol tolerance and produced up to ~21 g/L butanol from glucose in batch fermentation, an improvement of ~67 % over the parental strain (~12.6 g/L). Comparative genomic analysis revealed a single-base deletion in the cac3319 gene leading to C-terminal truncation in its encoding histidine kinase (HK) in JB200. To study the effects of cac3319 mutation on cell growth and fermentation, the cac3319 gene in ATCC 55025 was disrupted using the ClosTron group II intron-based gene inactivation system. Compared to ATCC 55025, the cac3319 HK knockout mutant, HKKO, produced 44.4 % more butanol (18.2 ± 1.3 vs. 12.6 ± 0.2 g/L) with a 90 % higher productivity (0.38 ± 0.03 vs. 0.20 ± 0.02 g/L h) due to increased butanol tolerance, confirming, for the first time, that cac3319 plays an important role in regulating solvent production and tolerance in C. acetobutylicum. This work also provides a novel metabolic engineering strategy for generating high-butanol-tolerant and high-butanol-producing strains for industrial applications.

Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Conventional acetone-butanol-ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L(-1) of ABE (17.6 g L(-1) butanol, 10.5 g L(-1) ethanol, and 4.4 g L(-1) acetone) from 85.2 g L(-1) glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell-recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L(-1) h(-1) , respectively, could be achieved at the dilution rate of 0.85 h(-1) . Further cell recycling experiments were carried out with controlled cell-bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h(-1) with the bleeding rate of 0.04 h(-1) . Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L(-1) h(-1) , and the yields of 0.17 and 0.34 g g(-1) , respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known-processes.

Gluconic acid production by Aspergillus terreus

Aspergillus terreus produces itaconic acid at low pH but lovastatin and other secondary metabolites at higher pH in the fermentation. The utilization of glucose as a carbon substrate was investigated for secondary metabolite production by A. terreus. With a starting pH of 6.5, glucose was rapidly metabolized to gluconic acid by the wild-type strain and by transformants harbouring Aspergillus niger genes encoding 6-phosphofructo-1-kinases with superior kinetic and regulatory properties for bioproduction of metabolites from glucose. On exhaustion of the glucose in batch fermentations, the accumulated gluconic acid was utilized as a carbon source. A novel pathway of glucose catabolism was demonstrated in A. terreus, a species whose wild type is, without any strain development, capable of producing gluconic acid at high molar conversion efficiency (up to 0.7 mol mol(-1) glucose consumed). Aspergillus terreus is a potential novel producer organism for gluconic acid, a compound with many uses as a bulk chemical. With a new knowledge of glucose catabolism by A. terreus, fermentation strategies for secondary metabolite production can be devised with glucose feeding using feedback regulation by pH.

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.