Malate:quinone oxidoreductase knockout makes Mycobacterium tuberculosis susceptible to stress and affects its in vivo survival.

Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_2875, annotated as malate:quinone oxidoreductase (mqo), is thought to have a role in TCA cycle in converting malate to oxaloacetate. To study its physiological relevance, we developed mqo knockout (KO) in Mtb-Ra. A KO complemented (KOC) strain was also developed by complementing the KO with mqo over-expressing construct. Under normal in vitro conditions, KO does not show any growth defect but showed reduced CFU burden in macrophages and in mice lungs. In vitro studies with KO showed reduced fitness under oxidative and low pH stress, and also increased susceptibility to levofloxacin and D-cycloserine. Transcript analysis of mqo showed increased expression levels under oxidative and low pH stress. This is the first study to show physiological relevance of mqo encoded by MRA_2875 in Mtb-Ra under oxidative and low pH stress. In summary, the present study shows that MRA_2875 encoded malate:quinone oxidoreductase is a functional enzyme which contributes to oxidative stress and low pH tolerance, and survival in macrophages and in mice.

GlfT1 down-regulation affects Mycobacterium tuberculosis biofilm formation and its in-vitro and in-vivo survival.

Mycobacterial galactan biosynthesis is critical for cell viability and growth, therefore an effort was made to study galactofuranosyl transferase 1, encoded by MRA_3822 in Mycobacterium tuberculosis H37Ra (Mtb-Ra). Galactofuranosyl transferases are involved in the biosynthesis of mycobacterial cell wall galactan chain and have been shown to be essential for in-vitro growth of Mycobacterium tuberculosis. In Mtb-Ra and Mycobacterium tuberculosis H37Rv (Mtb-Rv), two galactofuranosyl transferases are present, GlfT1 acts as initiator of galactan biosynthesis and GlfT2 continues with the subsequent polymerization events. GlfT2 has been well studied however GlfT1 inhibition/down-regulation and its effect on mycobacterial survival fitness has not been evaluated. To study the Mtb-Ra survival after GlfT1 silencing, Mtb-Ra knockdown and complemented strains were developed. In this study we show that GlfT1 down-regulation leads to increased susceptibility to ethambutol. Expression of glfT1 was up-regulated in the presence of ethambutol, and also in the presence of oxidative and nitrosative stress and upon exposure to low pH. Also, reduced biofilm formation, increased accumulation of ethidium bromide, and reduced tolerance to peroxide, nitric oxide and acid stress, were observed. The present study also demonstrates that GlfT1 down-regulation leads to reduced survival of Mtb-Ra in macrophages and in mice.

Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist, and its survival in macrophages and in mice.

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.

Mycobacterium tuberculosis survival and biofilm formation studies: effect of D-amino acids, D-cycloserine and its components.

D-amino acids play an important role in cell wall peptidoglycan biosynthesis. Mycobacterium tuberculosis D-amino acid oxidase deletion led to reduced biofilm-forming ability. Other recent studies also suggest that the accumulation of D-amino acids blocks biofilm formation and could also disperse pre-formed biofilm. Biofilms are communities of bacterial cells protected by extracellular matrix and harbor drug-tolerant as well as persistent bacteria. In Mycobacterium tuberculosis, biofilm formation or its inhibition by D-amino acids is yet to be tested. In the present study, we used selected D-amino acids to study their role in the prevention of biofilm formation and also if D-cycloserine's activity was due to presence of D-Serine as a metabolite. It was observed that D-serine limits biofilm formation in Mycobacterium tuberculosis H37Ra (Mtb-Ra), but it shows no effect on pre-formed biofilm. Also, D-cycloserine and its metabolic product, hydroxylamine, individually and in combination, with D-Serine, limit biofilm formation in Mtb-Ra and also disrupts existing biofilm. In summary, we demonstrated that D-alanine, D-valine, D-phenylalanine, D-serine, and D-threonine had no disruptive effect on pre-formed biofilm of Mtb-Ra, either individually or in combination, and D-cycloserine and its metabolite hydroxylamine have potent anti-biofilm activity.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase.

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli. Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Knockout of MRA_1916 in Mycobacterium tuberculosis H37Ra affects its growth, biofilm formation, survival in macrophages and in mice.

Mycobacterium tuberculosis H37Ra (Mtb-Ra) ORF MRA_1916 is annotated as a D-amino acid oxidase (DAO). These enzymes perform conversion of d-amino acids to corresponding imino acids followed by conversion into α-keto-acids. In the present study Mtb-Ra recombinants with DAO knockout (KO) and knockout complemented with DAO over-expressing plasmid (KOC) were constructed. The growth studies showed loss of growth of KO in medium containing glycerol as a primary carbon source. Substituting glycerol with acetate or with FBS addition, restored the growth. Growth was also restored in complemented strain (KOC). KO showed increased permeability to hydrophilic dye EtBr and reduced biofilm formation. Also, its survival in macrophages was low. Phagosome maturation studies suggested enhanced colocalization of KO, compared to WT, with lysosomal marker cathepsin D. Also, an increased intensity of Rab5 and iNOS was observed in macrophages infected with KO, compared to WT and KOC. The in vivo survival studies showed no increase in CFU of KO. This is the first study to show functional relevance of DAO encoded by MRA_1916 for Mtb-Ra growth on glycerol, its permeability and biofilm formation. Also, this study clearly demonstrates that DAO deletion leads to Mtb-Ra failing to grow in macrophages and in mice.

Down-regulation of malate synthase in Mycobacterium tuberculosis H37Ra leads to reduced stress tolerance, persistence and survival in macrophages.

Malate synthase is a condensing enzyme responsible for conversion of glyoxylate to malate in the presence of acetyl-CoA. This reaction helps in bypassing the TCA cycle reactions involving carbon loss and leads to diverting some of the carbon skeletons to gluconeogenic events while rest can continue to provide TCA cycle intermediates. Malate synthase (GlcB) is encoded by MRA_1848 of Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a knockdown (KD) Mtb-Ra strain by down-regulating GlcB. The survival studies suggested increased susceptibility to oxidative and nitrosative stress as well as to rifampicin. The susceptibility profile was reversed in the presence of free radical scavengers. Also, KD showed reduced biofilm maturation, failed to enter persistent state, and showed reduced growth inside macrophages. The study of post-endocytosis events showed differences in late stage endosomal maturation behavior in macrophages infected with KD compared to WT. Increased iNOS, LAMP1 and cathepsin D expression was observed in macrophages infected with KD compared to WT.

Biochemical and functional characterization of MRA_1571 of Mycobacterium tuberculosis H37Ra and effect of its down-regulation on survival in macrophages.

Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA_1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties. Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ΔilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ΔilvA + pET32a-ilvA). The E. coli-ΔilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ΔilvA + pET32a-ilvA. E. coli-ΔilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Maximum IlvA expression was observed at 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC).

Purification, characterization and thermostability improvement of xylanase from Bacillus amyloliquefaciens and its application in pre-bleaching of kraft pulp.

Xylanases have important industrial applications but are most extensively utilized in the pulp and paper industry as a pre-bleaching agent. We characterized a xylanase from Bacillus amyloliquefaciens strain SK-3 and studied it for kraft pulp bleaching. The purified enzyme had a molecular weight of ~50 kDa with optimal activity at pH 9.0 and 50 °C. The enzyme showed good activity retention (85%) after 2 h incubation at 50 °C and pH 9.0. This enzyme obeyed Michaelis-Menten kinetics with regard to beechwood xylan with K m and V max values of 5.6 mg/ml, 433 µM/min/mg proteins, respectively. The enzyme activity was stimulated by Mn2+, Ca2+ and Fe2+ metal ions. Further, it also showed good tolerance to phenolics (2 mM) in the presence of syringic acid (no loss), cinnamic acid (97%), benzoic acid (94%) and phenol (97%) activity retention. The thermostability of xylanase was increased by 6.5-fold in presence of sorbitol (0.75 M). Further, pulp treated with 20U/g of xylanase (20IU/g) alone and with sorbitol (0.75M) reduced kappa number by 18.3 and 23.8%, respectively after 3 h reaction. In summary, presence of xylanase shows good pulp-bleaching activity, good tolerance to phenolics, lignin and metal ions and is amenable to thermostability improvement by addition of polyols. The SEM image showed significant changes on the surface of xylanase-treated pulp fiber as a result of xylan hydrolysis.

MSMEG_5684 down-regulation in Mycobacterium smegmatis affects its permeability, survival under stress and persistence.

The Mycobacterium tuberculosis (Mtb) genome sequence and annotation details have been available for a long time; however physiological relevance of many ORFs remains poorly described. Mtb is a pathogenic strain; hence, surrogate strains such as Mycobacterium bovis BCG and Mycobacterium smegmatis (Msmeg) have also been studied to gain an understanding of mycobacterial physiology and metabolism. The Mycobacterium smegmatis mc2 155 ORF MSMEG_5684 is annotated as a part of serine biosynthetic pathway, however, its physiological significance remains to be established experimentally. To understand the relevance of SerC for Msmeg physiology we developed a recombinant Mycobacterium smegmatis with SerC knockdown (KD) and also complemented it with serC over-expressing construct (KDC). The KD showed reduced growth compared to wild-type (WT) and complemented strain on glycerol as carbon source. The growth of KD was restored after supplementation of serine. The survival studies with WT and KD under oxidative, nitrosative and detergent stresses showed increased susceptibility of KD. The KD also showed increased susceptibility to antimycobacterial agents and poor ability for in vitro persistence. Also, the serC transcript profiling showed increased expression under stress. The complementation studies with Msmeg serC showed growth restoration of E. coli-ΔserC in minimal medium.

Genotoxicity evaluation of tannery effluent treated with newly isolated hexavalent chromium reducing Bacillus cereus.

In this study, the efficiency of free and immobilized cells of newly isolated hexavalent chromium [Cr(VI)] reducing Bacillus cereus strain Cr1 (accession no. KJ162160) was studied in the treatment of tannery effluent. The analysis of effluents revealed high chemical oxygen demand (COD-1260 mg/L), biological oxygen demand (BOD5-660 mg/L), total dissolved solids (TDS-14000 mg/L), electrical conductivity (EC-21.5 mS/cm) and total chromium (TC-2.4 mg/L). The effluents also showed genotoxic effects to Allium cepa. Treatment of tannery effluent with isolated B. cereus strain led to considerable reduction of pollutant load. The pollutant load reduction was studied with both immobilized and free cells and immobilized cells were more effective in reducing COD (65%), BOD (80%), TDS (67%), EC (65%) and TC (92%) after 48 h. GC-MS analysis of pre and post-treatment tannery effluent samples revealed reduction of organic load after treatment with free and immobilized cells. An improvement in mitotic index and reduction in chromosomal aberrations was also observed in A. cepa grown with post-treatement effluent samples compared to untreated sample. Results demonstrate that both methods of bacterial treatment (free and immobilized) were efficient in reducing the pollutant load of tannery effluent as well as in reducing genotoxic effects, however, treatment with immobilized cells was more effective.

MRA_1571 is required for isoleucine biosynthesis and improves Mycobacterium tuberculosis H37Ra survival under stress.

Threonine dehydratase is a pyridoxal 5-phosphate dependent enzyme required for isoleucine biosynthesis. Threonine dehydratase (IlvA) participates in conversion of threonine to 2-oxobutanoate and ammonia is released as a by-product. MRA_1571 is annotated to be coding for IlvA in Mycobacterium tuberculosis H37Ra (Mtb-Ra). We developed a recombinant (KD) Mtb-Ra strain by down-regulating IlvA. The growth studies on different carbon sources suggested reduced growth of KD compared to wild-type (WT), also, isoleucine concentration dependent KD growth restoration was observed. The expression profiling of IlvA suggested increased expression of IlvA during oxygen, acid and oxidative stress. In addition, KD showed reduced survival under pH, starvation, nitric oxide and peroxide stresses. KD was more susceptible to antimycobacterial agents such as streptomycin (STR), rifampicin (RIF) and levofloxacin (LVF), while, no such effect was noticeable when exposed to isoniazid. Also, an increase in expression of IlvA was observed when exposed to STR, RIF and LVF. The dye accumulation studies suggested increased permeability of KD to ethidium bromide and Nile Red as compared to WT. TLC and Mass studies confirmed altered lipid profile of KD. In summary down-regulation of IlvA affects Mtb growth, increases its susceptibility to stress and leads to altered cell wall lipid profile.

Evaluation of bioremediation potentiality of ligninolytic Serratia liquefaciens for detoxification of pulp and paper mill effluent.

Due to high pollution load and colour contributing substances, pulp and paper mill effluents cause serious aquatic and soil pollution. A lignin-degrading bacterial strain capable of decolourising Azure-B dye was identified as lignin peroxidase (LiP) producing strain LD-5. The strain was isolated from pulp and paper mill effluent contaminated site. Biochemical and 16S rDNA gene sequence analysis suggested that strain LD-5 belonged to the Serratia liquefaciens. The strain LD-5 effectively reduced pollution parameters (colour 72%, lignin 58%, COD 85% and phenol 95%) of real effluent after 144h of treatment at 30°C, pH 7.6 and 120rpm. Extracellular LiP produced by S. liquefaciens during effluent decolourisation was purified to homogeneity using ammonium sulfate (AMS) precipitation and DEAE cellulose column chromatography. The molecular weight of the purified lignin peroxidase was estimated to be ∼28kDa. Optimum pH and temperature for purified lignin peroxidase activity were determined as pH 6.0 and 40°C, respectively. Detoxified effluent was evaluated for residual toxicity by alkaline single cell (comet) gel electrophoresis (SCGE) assay using Saccharomyces cerevisiae MTCC 36 as model organism. The toxicity reduction to treated effluent was 49.4%. These findings suggest significant potential of S. liquefaciens for bioremediation of pulp and paper mill effluent.

The Mycobacterium tuberculosis H37Ra gene MRA_1916 causes growth defects upon down-regulation.

D-amino acid oxidases play an important role in converting D-amino acids to their corresponding α-keto acids. MRA_1916 of Mycobacterium tuberculosis H37Ra (Mtb-Ra) is annotated to be a D-amino acid oxidase (DAO). However, not much information is available about its physiological role during Mtb-Ra growth and survival. The present study was taken-up to understand the role of DAO during different stages of growth and effect of its down-regulation on growth. Recombinant Mtb-Ra strains with DAO and GlcB (malate synthase: MRA_1848) gene knockdown were developed and their growth was studied using Microtiter Alamar Blue Assay (MABA) with glycerol, acetate and glycine as a carbon source. Ethyl bromopyruvate (BrP) was used as an inhibitor of GlcB. MABA study showed inhibition of wild-type (WT) and knockdowns in the presence of BrP (2.5mM). However, growth inhibition of WT was less noticeable at lower concentrations of BrP. Mtb-Ra with DAO knockdown showed poor utilization of glycine in the presence of BrP. The DAO localization study showed its prominent distribution in cytosolic fraction and to some extent in cell wall and membrane fractions. Growth profile of WT under oxygen and nutritional stress showed changes in expression of DAO, GlcB, PckA (phosphoenolpyruvate carboxykinase: MRA_0219) and GlyA1 (serine hydroxymethyltransferase: MRA_1104).

Characterization of a new Providencia sp. strain X1 producing multiple xylanases on wheat bran.

Providencia sp. strain X1 showing the highest xylanase activity among six bacterial isolates was isolated from saw-dust decomposing site. Strain X1 produced cellulase-free extracellular xylanase, which was higher in wheat bran medium than in xylan medium, when cultivated at pH 8.0 and 35°C. Zymogram analysis of crude preparation of enzymes obtained while growing on wheat bran and birchwood xylan revealed the presence of seven and two distinct xylanases with estimated molecular weight of 33; 35; 40; 48; 60; 75; and 95 kDa and 33 and 44 kDa, respectively. The crude xylanases were produced on wheat bran medium and showed optimum activity at pH 9.0 and 60°C. The thermotolerance studies showed activity retention of 100% and 85% at 40°C and 60°C after 30 min preincubation at pH 9.0. It was tolerant to lignin, ferulic acid, syringic acid, and guaiacol and retained 90% activity after ethanol treatment. The enzyme preparation was also tolerant to methanol and acetone and showed good activity retention in the presence of metal ions such as Fe2+, Mg2+, Zn2+, and Ca2+. The crude enzyme preparation was classified as endoxylanase based on the product pattern of xylan hydrolysis. Pretreatment of kraft pulp with crude xylanases for 3 h at 60°C led to a decrease in kappa number by 28.5%. The properties of present xylanases make them potentially useful for industrial applications.

Emerging approaches in fermentative production of statins.

Microbial metabolites have many important applications in pharmaceutical and health-care industry. The products of microbial origin are usually produced by submerged fermentation. The solid-state fermentation represents an alternative mode of fermentation, which is increasingly being employed as an alternative to submerged fermentation for metabolite production. The prospect of producing high-value product using low-value raw material offers a substantial premium to switch to these technologies. The cost of statins being one major factor, solid-state fermentation with agro-industrial residues as carbon, nitrogen and support matrix, promises to substantially lower the cost of production. Hence, newer approaches are required to exploit the agro-industrial residues for statin production. The development of these technologies offers an opportunity to exploit low-cost substrates without substantial investment in newer production methodologies. The emerging evidence of beneficial effect of statins in applications other than lipid lowering such as in Alzheimer disease, HIV, age-related dementia, and cancer chemotherapy makes it very important to develop methods for economic production of statins.

A Highly Thermostable Xylanase from Stenotrophomonas maltophilia: Purification and Partial Characterization.

Seven xylanolytic bacterial strains were isolated from saw-dust dump soil. The bacterial strain X6 was selected on the basis of the highest xylanase activity with no cellulase contamination. It was identified as Stenotrophomonas maltophilia by biochemical tests and 16S rRNA gene sequencing approach. Xylanase production studies by S. maltophilia on different commercial xylans and agro-industrial residues suggested that wheat bran was the best carbon source for xylanase production (26.4 ± 0.6 IU/mL). The studies with inorganic and organic nitrogen sources suggested yeast extract as the best support for xylanase production (25 ± 0.6 IU/mL). Maximum xylanase production was observed at initial medium pH = 8.0 (23.8 ± 0.4 IU/mL) with production at pH = 7.0 and pH = 9.0 being almost comparable. Xylanase produced by S. maltophilia was purified to homogeneity using ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The final purification was 5.43-fold with recovery of 19.18%. The molecular weight of the purified xylanase protein was ~142 kDa. Both crude and purified xylanase had good stability at pH = 9.0 and 80°C with activity retention greater than 90% after 30 min incubation. The enzyme stability at high temperature and alkaline pH make it potentially effective for industrial applications.

Effect of precultural and nutritional parameters on compactin production by solid-state fermentation.

In the present study, production of compactin by Penicillium brevicompactum WA 2315 was studied. In the first step, various precultural parameters were studied by substituting one factor at a time. Subsequently, the effect of maltodextrin DE 18 on compactin production was studied. The optimized parameters gave maximum compactin production of 850 mug/gds as compared with 678 mug/gds before optimization. Statistical study was performed to further improve the production and develop a robust model. An improved yield of 950 mug/gds was obtained using the conditions proposed by the experimental model. The present study emphasizes the importance of precultural and nutritional parameters on the production of compactin, and further confirms the usefulness of solid-state fermentation for the production of industrially important secondary metabolites. It also confirms that complex nitrogen sources such as oil cakes can be used for the production of compactin.

Polyhydroxybutyrate production using agro-industrial residue as substrate by Bacillus sphaericus NCIM 5149

The aim of this work was to study the production of polyhydroxybutyrate (PHB) using agro- industrial residues as the carbon source. Seven substrates, viz., wheat bran, potato starch, sesame oil cake, groundnut oil cake, cassava powder, jackfruit seed powder and corn flour were hydrolyzed using commercial enzymes and the hydrolyzates assessed for selecting the best substrate for PHB production. Jackfruit seed powder gave the maximum production of PHB under submerged fermentation using Bacillus sphaericus (19 percent) at the initial pH of 7.5.

Application of response surface method for studying the role of dissolved oxygen and agitation speed on gamma-linolenic acid production.

To study the effect of agitation speed (rpm) and dissolved oxygen concentration (DO) on the production of gamma linolenic acid by Mucor sp. RRL001, a central composite design experiment was performed in a 5-L stirred tank bioreactor. The design consisted of a total of 10 runs consisting of runs at five levels for each factor and was divided in two blocks. The ANOVA analysis and Pareto chart of effects suggested agitation speed (p = 0.0142) linear effect and DO concentration (p = 0.0342) quadratic effects were significant factors with significant contribution to the response. The validation run based on the optimum production zone in response surface plot resulted in the maximum 350.3 mg l(-1) GLA yield as compared with model predicted value of 340.7 mg l(-1). The study suggests that agitation rate is having more pronounced effect on GLA yield than dissolved oxygen concentration by ensuring enhanced mass transfer and by preventing wall growth at elevated agitation speed. Also, it shows that higher GLA yields can be obtained in a simple medium at moderate oxygen saturation and that the Mucor sp. RRL001 is resistant to high agitation linked shear stress and suitable for GLA production at higher scale.