Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris.

BACKGROUND: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. RESULTS: Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. CONCLUSIONS: As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

Over 2000-Fold Increased Production of the Leaderless Bacteriocin Garvicin KS by Increasing Gene Dose and Optimization of Culture Conditions.

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 is, however, relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30°C). To improve the production, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes, a new medium formulation was made that increased GarKS production about 60-fold compared to that achieved in GM17. The new medium was composed of pasteurized milk and tryptone (PM-T). GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing the gene dose of the bacteriocin gene cluster (gak) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and cultivation at a constant pH 6 and a 50-60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto.

Production, Characterization, and Application of an Alginate Lyase, AMOR_PL7A, from Hot Vents in the Arctic Mid-Ocean Ridge.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. We describe a thermostable alginate lyase belonging to Polysaccharide Lyase family 7 (PL7), which can be used to degrade brown seaweed, Saccharina latissima, at conditions also suitable for a commercial cellulase cocktail (Cellic CTec2). This enzyme, AMOR_PL7A, is a ß-d-mannuronate specific (EC 4.2.2.3) endoacting alginate lyase, which degrades alginate and poly mannuronate within a broad range of pH, temperature and salinity. At 65 °C and pH 6.0, its Km and kcat values for sodium alginate are 0.51 ± 0.09 mg/mL and 7.8 ± 0.3 s-1 respectively. Degradation of seaweed with blends of Cellic CTec2 and AMOR_PL7A at 55 °C in seawater showed that the lyase efficiently reduces viscosity and increases glucose solublization. Thus, AMOR_PL7A may be useful in development of efficient protocols for enzymatic seaweed processing.

Comparative Assessment of Enzymatic Hydrolysis for Valorization of Different Protein-Rich Industrial Byproducts.

Hydrolyzed protein-rich byproducts from food production may find a variety of applications, for example, as rich ingredients of fermentation media. We have conducted a study of the enzymatic hydrolysis of three byproducts from Norwegian food industries: chicken byproducts, mixed pork and beef byproducts, and salmon viscera. The efficiency and optimization of the enzymatic hydrolysis were evaluated using endogenous enzymes alone and in combination with commercial proteases. Hydrolysis reactions were conducted with freshly thawed raw materials using short incubation times and including an initial temperature gradient from 4 to 60 °C to both harness the power of endogenous enzymes and minimize microbial contamination. Subsequently, hydrolysates were characterized by analyzing the total recovery of protein, the peptide molecular-weight distribution, and the composition of total and free amino acids. The action of endogenous enzymes played an important role in raw-material hydrolysis, particularly when hydrolyzing salmon viscera but less so when hydrolyzing chicken byproducts. For pork-beef and chicken byproducts, the addition of Alcalase or Papain improved protein recovery, reaching levels up to 90%. Next to showing efficient hydrolysis protocols, the present data also provide a comparison of the amino acid compositions of hydrolysates derived from these three different protein-rich byproducts. Growth studies showed that the obtained protein-rich hydrolysates from meat and fish industries are a promising alternative for expensive nitrogen sources that are commonly used for fermenting yeasts.

Microbial Protein Produced from Brown Seaweed and Spruce Wood as a Feed Ingredient.

The conversion of nonedible biomass to protein for use in feed is an attractive strategy toward improved sustainability in aquaculture. We have studied the possibility to produce protein-rich yeast Candida utilis on a medium consisting of enzymatically hydrolyzed sulphite-pulped spruce wood, mainly providing glucose, and enzymatically hydrolyzed brown seaweed, supplemented with ammonium sulfate. The results show that this blend constitutes a complete fermentation medium that enables good growth rates and cell yields. Results from a salmon feeding trial showed that the yeast can replace parts of a traditional fishmeal diet without harmful effects, although the apparent protein digestibility coefficient for the yeast was suboptimal. While further optimization of both the fermentation process and downstream processing is needed, the present proof-of-concept study shows a path to the production of microbial protein based on a simple, local and sustainable fermentation medium.

Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms.

Recent developments in molecular biology and metabolic engineering have resulted in a large increase in the number of strains that need to be tested, positioning high-throughput screening of microorganisms as an important step in bioprocess development. Scalability is crucial for performing reliable screening of microorganisms. Most of the scalability studies from microplate screening systems to controlled stirred-tank bioreactors have been performed so far with unicellular microorganisms. We have compared cultivation of industrially relevant oleaginous filamentous fungi and microalga in a Duetz-microtiter plate system to benchtop and pre-pilot bioreactors. Maximal glucose consumption rate, biomass concentration, lipid content of the biomass, biomass, and lipid yield values showed good scalability for Mucor circinelloides (less than 20% differences) and Mortierella alpina (less than 30% differences) filamentous fungi. Maximal glucose consumption and biomass production rates were identical for Crypthecodinium cohnii in microtiter plate and benchtop bioreactor. Most likely due to shear stress sensitivity of this microalga in stirred bioreactor, biomass concentration and lipid content of biomass were significantly higher in the microtiter plate system than in the benchtop bioreactor. Still, fermentation results obtained in the Duetz-microtiter plate system for Crypthecodinium cohnii are encouraging compared to what has been reported in literature. Good reproducibility (coefficient of variation less than 15% for biomass growth, glucose consumption, lipid content, and pH) were achieved in the Duetz-microtiter plate system for Mucor circinelloides and Crypthecodinium cohnii. Mortierella alpina cultivation reproducibility might be improved with inoculation optimization. In conclusion, we have presented suitability of the Duetz-microtiter plate system for the reproducible, scalable, and cost-efficient high-throughput screening of oleaginous microorganisms.

Microtiter plate cultivation of oleaginous fungi and monitoring of lipogenesis by high-throughput FTIR spectroscopy.

BACKGROUND: Oleaginous fungi can accumulate lipids by utilizing a wide range of waste substrates. They are an important source for the industrial production of omega-6 polyunsaturated fatty acids (gamma-linolenic and arachidonic acid) and have been suggested as an alternative route for biodiesel production. Initial research steps for various applications include the screening of fungi in order to find efficient fungal producers with desired fatty acid composition. Traditional cultivation methods (shake flask) and lipid analysis (extraction-gas chromatography) are not applicable for large-scale screening due to their low throughput and time-consuming analysis. Here we present a microcultivation system combined with high-throughput Fourier transform infrared (FTIR) spectroscopy for efficient screening of oleaginous fungi. RESULTS: The microcultivation system enables highly reproducible fungal fermentations throughout 12 days of cultivation. Reproducibility was validated by FTIR and HPLC data. Analysis of FTIR spectral ester carbonyl peaks of fungal biomass offered a reliable high-throughput at-line method to monitor lipid accumulation. Partial least square regression between gas chromatography fatty acid data and corresponding FTIR spectral data was used to set up calibration models for the prediction of saturated fatty acids, monounsaturated fatty acids, polyunsaturated fatty acids, unsaturation index, total lipid content and main individual fatty acids. High coefficients of determination (R2 = 0.86-0.96) and satisfactory residual predictive deviation of cross-validation (RPDCV = 2.6-5.1) values demonstrated the goodness of these models. CONCLUSIONS: We have demonstrated in this study, that the presented microcultivation system combined with rapid, high-throughput FTIR spectroscopy is a suitable screening platform for oleaginous fungi. Sample preparation for FTIR measurements can be automated to further increase throughput of the system.

Metabolic Engineering of TCA Cycle for Production of Chemicals.

The tricarboxylic acid (TCA) cycle has been used for decades in the microbial production of chemicals such as citrate, L-glutamate, and succinate. Maximizing yield is key for cost-competitive production. However, for most TCA cycle products, the maximum pathway yield is lower than the theoretical maximum yield (Y(E)). For succinate, this was solved by creating two pathways to the product, using both branches of the TCA cycle, connected by the glyoxylate shunt (GS). A similar solution cannot be applied directly for production of compounds from the oxidative branch of the TCA cycle because irreversible reactions are involved. Here, we describe how this can be overcome and what the impact is on the yield.

Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production.

Itaconic acid, a C5-dicarboxylic acid, is a potential biobased building block for the polymer industry. It is obtained from the citric acid cycle by decarboxylation of cis-aconitic acid. This reaction is catalyzed by CadA in the native itaconic acid producer Aspergillus terreus. Recently, another enzyme encoded by the mammalian immunoresponsive gene 1 (irg1), was found to decarboxylate cis-aconitate to itaconate in vitro. We show that heterologous expression of irg1 enabled itaconate production in Escherichia coli with production titres up to 560 mg/L.

Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate.

Itaconic acid, an unsaturated C5-dicarboxylic acid, is a biobased building block for the polymer industry. The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway of E. coli. E. coli BW25113 (DE3) and the phosphate acetyltransferase (pta) and lactate dehydrogenase (ldhA) deficient strain E. coli BW25113 (DE3) Δpta-ΔldhA were used to study anaerobic itaconate production in E. coli. Heterologous expression of the gene encoding cis-aconitate decarboxylase (cadA) from A. terreus in E. coli BW25113 (DE3) did not result in itaconate production under anaerobic conditions, but 0.08 mM of itaconate was formed when the genes encoding citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum were also expressed. The same amount was produced when cadA was expressed in E. coli BW25113 (DE3) Δpta-ΔldhA. The titre increased 8 times to 0.66 mM (1.2 % Cmol) when E. coli BW25113 (DE3) Δpta-ΔldhA also expressed gltA and acnA. In addition, this strain produced 8.5 mM (13 % Cmol) of glutamate. The use of a nitrogen-limited growth medium reduced the accumulation of glutamate by nearly 50 % compared to the normal medium, and also resulted in a more than 3-fold increase of the itaconate titre to 2.9 mM. These results demonstrated that E. coli has potential to produce itaconate and glutamate under anaerobic conditions, closing the redox balance by co-production of succinate or ethanol with H2 and CO2.

Metabolic engineering of itaconate production in Escherichia coli.

Interest in sustainable development has led to efforts to replace petrochemical-based monomers with biomass-based ones. Itaconic acid, a C5-dicarboxylic acid, is a potential monomer for the chemical industry with many prospective applications. cis-aconitate decarboxylase (CadA) is the key enzyme of itaconate production, converting the citric acid cycle intermediate cis-aconitate into itaconate. Heterologous expression of cadA from Aspergillus terreus in Escherichia coli resulted in low CadA activities and production of trace amounts of itaconate on Luria-Bertani (LB) medium (<10 mg/L). CadA was primarily present as inclusion bodies, explaining the low activity. The activity was significantly improved by using lower cultivation temperatures and mineral medium, and this resulted in enhanced itaconate titres (240 mg/L). The itaconate titre was further increased by introducing citrate synthase and aconitase from Corynebacterium glutamicum and by deleting the genes encoding phosphate acetyltransferase and lactate dehydrogenase. These deletions in E. coli's central metabolism resulted in the accumulation of pyruvate, which is a precursor for itaconate biosynthesis. As a result, itaconate production in aerobic bioreactor cultures was increased up to 690 mg/L. The maximum yield obtained was 0.09 mol itaconate/mol glucose. Strategies for a further improvement of itaconate production are discussed.

The 'true' L-xylulose reductase of filamentous fungi identified in Aspergillus niger.

L-Xylulose reductase is part of the eukaryotic pathway for l-arabinose catabolism. A previously identified L-xylulose reductase in Hypocrea jecorina turned out to be not the 'true' one since it was not upregulated during growth on L-arabinose and the deletion strain showed no reduced L-xylulose reductase activity but instead lost the D-mannitol dehydrogenase activity. In this communication we identified the 'TRUE'L-xylulose reductase in Aspergillus niger. The gene, lxrA (JGI177736), is upregulated on L-arabinose and the deletion results in a strain lacking the NADPH-specific L-xylulose reductase activity and having reduced growth on l-arabinose. The purified enzyme had a K(m) for L-xylulose of 25 mM and a nu(max) of 650 U/mg.