Recent advances in the production of malic acid by native fungi and engineered microbes.

Malic acid is mainly produced by chemical methods which lead to various environmental sustainability concerns associated with CO2 emissions and resulting global warming. Since malic acid is naturally synthesized, microorganisms offer an eco-friendly and cost-effective alternative for its production. An additional advantage of microbial production is the synthesis of pure L-form of malic acid. Due to its numerous applications, biotechnologically- produced L-malic acid is a much sought-after platform chemical. Malic acid can be produced by microbial fermentation via oxidative/reductive TCA and glyoxylate pathways. This article elaborates the potential and limitations of high malic acid producing native fungi belonging to Aspergillus, Penicillium, Ustilago and Aureobasidium spp. The utilization of industrial side streams and low value renewable substrates such as crude glycerol and lignocellulosic biomass is also discussed with a view to develop a competitive bio-based production process. The major impediments present in the form of toxic compounds from lignocellulosic residues or synthesized during fermentation along with their remedial measures are also described. The article also focuses on production of polymalic acid from renewable substrates which opens up a cost-cutting dimension in production of this biodegradable polymer. Finally, the recent strategies being employed for its production in recombinant organisms have also been covered.

Expression of Escherichia coli malic enzyme gene in Zymomonas mobilis for production of malic acid.

Malic acid is one of the organic acids which is used in various industries including food and pharmaceuticals. Biotechnological production of malic acid by an efficient microorganism is highly desirable as the process will be eco-friendly and cost-effective. In this study, malic acid synthesis by Zymomonas mobilis was studied by expressing Escherichia coli malic enzyme gene under Pchap, Ptac and Ppdc promoters. The mae+ recombinants were obtained by recombineering-based genomic integration of Pchap-mae, Ptac-mae and Ppdc-mae sequences. The Ppdc promoter showed the highest expression of malic enzyme and the Pchap the lowest. However, cell growth was limited in mae+ recombinant containing Ppdc promoter. The metabolic analysis showed the highest level of malic acid in Ppdc-mae recombinant (2.84 g/L), which was about eight times higher than that in the wild type strain. The study showed that these three promoters can be used to produce organic acids in Z. mobilis.

Characterization of Zymomonas mobilis promoters that are functional in Escherichia coli.

Zymomonas mobilis ZM4 is a gram-negative, facultative anaerobic, natural ethanologenic bacterium used in industrial production of bio-products. For expression of genes, promoters are required. However, most of the promoters reported from Z. mobilis poorly function in Escherichia coli. This makes the process of expression and screening labor-intensive. In the present study, we compared the strengths of two Z. mobilis promoters, Pchap and Ppap, which drive the expression of chaperonin and phosphatase PAP2 family protein, respectively, with Ptac promoter. In E. coli, the Ptac promoter was found to be the strongest followed by Ppap and Ppdc, while in Z. mobilis, Ppdc was found to be the strongest and Pchap the weakest promoter. Further characterization of the promoters was done by cloning the gfpuv gene which expresses the green fluorescent protein, under their control and measuring the fluorescence of the E. coli transformants. The activity of these promoters was also studied at different pH (pH 5, 7 and 9) and different temperatures (30°C, 37°C and 42°C) in exponential and stationary phases. Both Pchap and Ppap promoters showed higher activity in stationary phase than in exponential phase. Since the promoters were active at all temperatures and pH studied, they can be used for gene expression in E. coli under desired environmental conditions.

Deletion of pyruvate decarboxylase gene in Zymomonas mobilis by recombineering through bacteriophage lambda red genes.

Zymomonas mobilis ZM4 is a gram negative ethanologenic bacterium used in several biotechnological applications. Metabolic engineering in this bacterium is limited because of the available genome engineering tools. In the present study, we report genome engineering in this bacterium using bacteriophage lambda Red genes. Stability of plasmid replicons RK2 (pSIM9) and pBBR1 (pSIM7) containing the lambda Red genes was found to be 78% and 74%, respectively. We demonstrate successful deletion of pyruvate decarboxylase gene by recombineering in Z. mobilis. The deletion was confirmed by PCR and by estimating the metabolites formed. Ethanol, which was the main product in wild type cells, was formed in almost negligible amount in the pdc-deleted mutant. The developed Δpdc Z. mobilis cells can be exploited for production of desired bioproducts by expression of suitable enzymes that can regenerate NAD+.

In vitro synthesis of 9,10-dihydroxyhexadecanoic acid using recombinant Escherichia coli.

BACKGROUND: Hydroxy fatty acids are widely used in food, chemical and cosmetic industries. A variety of dihydroxy fatty acids have been synthesized so far; however, no studies have been done on the synthesis of 9,10-dihydroxyhexadecanoic acid. In the present study recombinant E. coli has been used for the heterologous expression of fatty acid hydroxylating enzymes and the whole cell lysate of the induced culture was used for in vitro production of 9,10-dihydroxyhexadecanoic acid. RESULTS: A first of its kind proof of principle has been successfully demonstrated for the production of 9,10-dihydroxyhexadecanoic acid using three different enzymes viz. fatty acid desaturase (FAD) from Saccharomyces cerevisiae, epoxide hydrolase (EH) from Caenorhabditis elegance and epoxygenase (EPOX) from Stokasia laevis. The genes for these proteins were codon-optimised, synthesised and cloned in pET 28a (+) vector. The culture conditions for induction of these three proteins in E. coli were optimised in shake flask. The induced cell lysates were used both singly and in combination along with the trans-supply of hexadecanoic acid and 9-hexadecenoic acid, followed by product profiling by GC-MS. Formation of 9,10-dihydroxyhexadecanoic acid was successfully achieved when combination of induced cell lysates of recombinant E. coli containing FAD, EH, and EPOX were incubated with 9-hexadecenoic acid. CONCLUSIONS: The in vitro production of 9,10-dihydroxyhexadecanoic acid synthesis using three fatty acid modification genes from different sources has been successfully demonstrated. The strategy adopted can be used for the production of similar compounds.

Bioprocess strategies for enhanced production of xylanase by Melanocarpus albomyces IITD3A on agro-residual extract.

The production of high titer xylanase without cellulase is required for prebleaching of pulps in pulp and paper industry. The mutant IITD3A of Melanocarpus albomyces developed from the spores of the wild type organism was used in this study. The statistical optimization of the process parameters by response surface methodology revealed that the production of xylanase was most affected by changes in the pH of the production medium which contained a soluble extract of wheat straw as the sole carbon source. When the pH of the production medium in a 14 L bioreactor was controlled on-line at 7.8, xylanase activity of 415 IU mL⁻¹ was obtained after 36 h fermentation. On cycling the pH between 7.8 and 8.2, the same activity could be attained in 24 h with an overall productivity of 16,670 IU L⁻¹ h⁻¹. The production of xylanase was also influenced by the fungus morphology; the activity being maximum when it exhibited pellet form at an agitation speed of 600 rpm. On optimization of aeration rate to 0.25 vvm, the xylanase activity further increased to 550 IU mL⁻¹ with a very high overall volumetric productivity of 22,000 IU L⁻¹ h⁻¹. Thus, a 5.2-fold enhancement in overall volumetric productivity of xylanase could be obtained by the mutant in comparison to that obtained on insoluble wheat straw.

Exogenous hormones affecting morphology and biosynthetic potential of hairy root line (LYR2i) of Linum album.

A hairy root line of Linum album LYR2i was obtained via the genetic transformation of a cotyledon segment of the plant by cocultivating it with Agrobacterium rhizogenes and culturing it in hormone-free B5 Gamborg liquid medium. Characteristic changes in root morphology associated with variations in biosynthetic capabilities were achieved by adding different combinations of phytohormones to the basal medium. A reversible system between hairy roots and its cultures with a diversified morphology was established by including and excluding phytohormones in the basal medium. The medium containing indole 3 acetic acid (IAA) at a concentration of 3 mg/l (MI3) induced thicker root tips and that containing 2,4-dichlorophenoxy acetic acid (2,4-D) at a concentration of 1 mg/l (MD1) induced compact green callus. Podophyllotoxin and 6-methoxypodophyllotoxin content increased by 1.86-fold and 1.45-fold as a result of adding IAA (MI3) and 2,4-D (MD1) to the basal medium, respectively. After regeneration, the hairy roots regained their original morphology and biosynthetic capability on hormone-free basal medium. The transformation status of the regenerated hairy roots was confirmed by PCR analysis.