Gazi Sakir Hossain

Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing l-amino acid deaminase from Proteus mirabilis

The goal of this work was to develop an immobilized whole-cell biocatalytic process for the environment-friendly synthesis of α-ketoglutaric acid (α-KG) from l-glutamic acid. We compared the suitability of Escherichia coli and Bacillus subtilis strains overexpressing Proteus mirabilisl-amino acid deaminase (l-AAD) as potential biocatalysts. Although both recombinant strains were biocatalytically active, the performance of B. subtilis was superior to that of E. coli. With l-glutamic acid as the substrate, α-KG production levels by membranes isolated from B. subtilis and E. coli were 55.3±1.73 and 21.7±0.39μg/mg protein/min, respectively. The maximal conversion ratio of l-glutamic acid to α-KG was 31% (w/w) under the following optimal conditions: 15g/L l-glutamic acid, 20g/L whole-cell biocatalyst, 5mM MgCl2, 40°C, pH 8.0, and 24-h incubation. Immobilization of whole cells with alginate increased the recyclability by an average of 23.33% per cycle. This work established an efficient one-step biotransformation process for the production of α-KG using immobilized whole B. subtilis overexpressing P. mirabilisl-AAD. Compared with traditional multistep chemical synthesis, the biocatalytic process described here has the advantage of reducing environmental pollution and thus has great potential for the large-scale production of α-KG.

One-step production of α-ketoglutaric acid from glutamic acid with an engineered l-amino acid deaminase from Proteus mirabilis

Currently, α-ketoglutaric acid (α-KG) is industrially produced by multi-step chemical synthesis, which can cause heavy environmental pollution. Here we reported a simple one-step approach for the production of α-KG by transforming l-glutamic acid with an engineered l-amino acid deaminase (l-AAD) from Proteus mirabilis. First, to facilitate the purification of membrane-bound l-AAD, one N-terminal transmembrane region (from 21 to 87th nucleotide) was removed from l-AAD to block the binding of l-AAD with membrane, and the relatively low-usage codons were replaced by high-usage codons in Escherichia coli to improve the expression level. However, inclusion bodies formed when expressing the ΔN-LAAD in E. coli BL 21, and then the soluble and active ΔN-LAAD was obtained by the solubilization and renaturation of ΔN-LAAD. Furthermore, the biochemical properties of the refolded ΔN-LAAD were characterized and compared with those of full-length l-AAD. Finally, the ΔN-LAAD was used to synthesize α-KG and the maximal formation rate of α-KG reached 12.6% (w/w) at 6h under the following conditions: 12g/L l-glutamic acid, 0.1g/L ΔN-LAAD, 5mM MgCl2, temperature 45°C and pH 8.0. Compared with the multi-step chemical synthesis, the transformation approach has less environmental pollution and has a great potential for α-KG production.

Improved production of α-ketoglutaric acid (α-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of l-amino acid deaminase and deletion of the α-KG utilization pathway

We previously developed a novel one-step biotransformation process for the production of α-ketoglutarate (α-KG) from L-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an L-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high α-KG degradation. In this study, we further improved α-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis α-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of L-glutamic acid from 31% to 83.25% and the α-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for L-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of α-KG production increased from 22.82 μM min(-1) to 56.7 μM min(-1). Finally, the sucA gene, encoding α-ketodehydrogenase, was deleted to reduce α-KG degradation, increasing the α-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the α-KG degradation pathway in B. subtilis improved α-KG production over that of previously developed processes.

One-step biosynthesis of α-keto-γ-methylthiobutyric acid from L-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered L-amino acid deaminase from Proteus vulgaris.

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of l-methionine, has great potential for use as an alternative to l-methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from l-methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered l-amino acid deaminase (l-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris l-AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of l-methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L l-methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris l-AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production.

Transporter engineering and enzyme evolution for pyruvate production from D/L-alanine with a whole-cell biocatalyst expressing L-amino acid deaminase from Proteus mirabilis

Pyruvate is an essential metabolite in the central metabolism of microbes, and it has been widely used in the food, pharmaceutical, and agrochemical industries. Both chemical and biological processes have been used for industrial pyruvate production. In this study, one-step pyruvate production from D/L-alanine with a whole-cell E. coli biocatalyst expressing L-amino acid deaminase (pm1) from Proteus mirabilis was investigated. Alanine uptake transporters (cycA, amaP) and a pyruvate uptake transporter (lldP) were knocked out to prevent substrate and product utilization by the biocatalyst. The pyruvate production titer from D/L-alanine increased from 1.14 g L−1 under control conditions to 5.38 g L−1 with the mutant whole-cell biocatalyst. Directed evolution was used to engineer pm1 and improve the catalytic activity with D/L-alanine. Three rounds of error-prone polymerase chain reaction generated the mutant pm1ep3, which showed improved affinity (6.76 mM for L-alanine) and catalytic efficiency (0.085 s−1 mM−1 and 0.027 s−1 mM−1 for L- and D-alanine, respectively). The final pyruvate titer was increased to 14.57 g L−1 and the conversion ratio was increased to 29.14% by using the engineered whole-cell biocatalyst containing the evolved pm1ep3.

Industrial Bioprocesses and the Biorefinery Concept

Abstract Both economic and environmental drivers have been major considerations in the development of sustainable chemical production using renewable crop-based feedstocks. Biomass is characterized by a plentiful carbon-neutral renewable feedstock for the production of fuels and chemicals, replacing fossil fuels and petrochemicals. First-generation biorefineries use corn, soybeans, and sugarcane for bioethanol and biodiesel production, which can benefit from integrated biorefining that extracts high-value nutritional products while using the main feedstock component for biofuel and chemical production and further converting low-value by-products to additional marketable products such as animal feed and energy. Second-generation biorefineries use lignocellulosic biomass, including agricultural and forestry residues, and provide the opportunity to meet a significant portion of fuel and chemical needs. Third-generation biorefineries use aquacultures of either microalgae or macroalgae, which use sunlight and CO2 for growth, which could deliver all upcoming fuel needs without disturbing current land use for agricultural purposes. With continuing progress and improvements in new energy crops, aquaculture, synthetic biology for cell engineering, and conversion technologies, biorefining will have an increasingly important role in supplying energy, fuel, and chemicals to sustain economic growth without a substantial negative effect on the environment. Nevertheless, there are also numerous challenges facing the biorefinery industry. Lignocellulosic refining is not yet rationally cost-effective because of the complex nature of the feedstock as well as high costs of pretreatment and enzymatic hydrolysis of cellulose. Although a number of new bioprocesses of cellulosic biomass have been industrialized, it is clear that economic and technical barriers exist that must be addressed before the full potential of this area can be recognized. In addition, processes using microalgae with photosynthesis for cell growth and oil production are not only difficult to scale up but also far from cost-effective. To achieve the sustainable and economical production of biofuels and bio-based chemicals, new advances in process engineering and metabolic engineering for biomass conversion will be required. Moreover, a biorefinery should consume all components of a given biomass feedstock to produce fuels, chemicals, and energy, to take full advantage of product values, decrease waste generation, and recover process economics, which necessitates a combination of technologies from various areas, including new energy crops with higher biomass yields, improved and inexpensive enzymes for hydrolysis, innovative and upgraded cells and catalysts for biomass conversion to chemicals, fuels, and other marketable products, and more efficient processes for the production of these bio-based products on an industrial scale.

Metabolic engineering of cofactor flavin adenine dinucleotide (FAD) synthesis and regeneration in Escherichia coli for production of α-keto acids†

Cofactor flavin adenine dinucleotide (FAD) plays a vital role in many FAD‐dependent enzymatic reactions; therefore, how to efficiently accelerate FAD synthesis and regeneration is an important topic in biocatalysis and metabolic engineering. In this study, a system involving the synthesis pathway and regeneration of FAD was engineered in Escherichia coli to improve α‐keto acid production—from the corresponding l‐amino acids—catalyzed by FAD‐dependent l‐amino acid deaminase (l‐AAD). First, key genes, ribH, ribC, and ribF, were overexpressed and fine‐tuned for FAD synthesis. In the resulting E. coli strain PHCF7, strong overexpression of pma, ribC, and ribF and moderate overexpression of ribH yielded a 90% increase in phenylpyruvic acid (PPA) titer: 19.4 ± 1.1 g · L−1. Next, formate dehydrogenase (FDH) and NADH oxidase (NOX) were overexpressed to strengthen the regeneration rate of cofactors FADH2/FAD using FDH for FADH2/FAD regeneration and NOX for NAD+/NADH regeneration. The resulting E. coli strain PHCF7‐FDH‐NOX yielded the highest PPA production: 31.4 ± 1.1 g · L−1. Finally, this whole‐cell system was adapted to production of other α‐keto acids including α‐ketoglutaric acid, α‐ketoisocaproate, and keto‐γ‐methylthiobutyric acid to demonstrate the broad utility of strengthening of FAD synthesis and FADH2/FAD regeneration for production of α‐keto acids. Notably, the strategy reported herein may be generally applicable to other flavin‐dependent biocatalysis reactions and metabolic pathway optimizations. Biotechnol. Bioeng. 2017;114: 1928–1936.

Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by L-amino acid deaminase

L-Amino acid deaminases (LAADs; EC 1.4.3.2) belong to a family of amino acid dehydrogenases that catalyze the formation of α-keto acids from L-amino acids. In a previous study, a whole cell biocatalyst with the L-amino acid deaminase (pm1) from Proteus mirabilis was developed for the one-step production of α-ketoglutarate (α-KG) from L-glutamic acid, and the α-KG titer reached 12.79 g L−1 in a 3 L batch bioreactor. However, the product α-KG strongly inhibited pm1 activity, and the titer of α-KG was comparatively lower than expected. Therefore, in this study, multiple rounds of error-prone polymerase chain reaction (PCR) and gene shuffling were integrated for the molecular engineering of pm1 to further improve the catalytic performance and α-KG titer. A variant (pm1338g4), which contained mutations in 34 amino acid residues, was found to have enhanced catalytic efficiency. In a batch system, the α-KG titer reached 53.74 g L−1 when 100 g of monosodium glutamate was used as a substrate. Additionally, in a fed-batch biotransformation system, the maximum α-KG titer reached 89.11 g L−1 when monosodium glutamate was continuously fed at a constant rate of 6 g L−1 h−1 (from 4 to 23 h) with an initial concentration of 50 g L−1. Analysis of the kinetics of the mutant variant showed that these improvements were achieved due to enhancement of the reaction velocity (from 56.7 μM min−1 to 241.8 μM min−1) and substrate affinity (the Km for glutamate decreased from 23.58 to 6.56 mM). A possible mechanism for the enhanced substrate affinity was also evaluated by structural modeling of the mutant. Our findings showed that the integration of error-prone PCR and gene shuffling was an effective method for improvement of the catalytic performance of industrial enzymes.

Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling

Phenylpyruvic acid (PPA) is widely used in the pharmaceutical, food, and chemical industries. Here, a two-step bioconversion process, involving growing and resting cells, was established to produce PPA from l-phenylalanine using the engineered Escherichia coli constructed previously. First, the biotransformation conditions for growing cells were optimized (l-phenylalanine concentration 20.0 g·L−1, temperature 35°C) and a two-stage temperature control strategy (keep 20°C for 12 h and increase the temperature to 35°C until the end of biotransformation) was performed. The biotransformation conditions for resting cells were then optimized in 3-L bioreactor and the optimized conditions were as follows: agitation speed 500 rpm, aeration rate 1.5 vvm, and l-phenylalanine concentration 30 g·L−1. The total maximal production (mass conversion rate) reached 29.8 ± 2.1 g·L−1 (99.3%) and 75.1 ± 2.5 g·L−1 (93.9%) in the flask and 3-L bioreactor, respectively. Finally, a kinetic model was established, and it was revealed that the substrate and product inhibition were the main limiting factors for resting cell biotransformation.

Metabolic engineering of Raoultella ornithinolytica BF60 for the production of 2, 5-furandicarboxylic acid from 5-hydroxymethylfurfural

ABSTRACT 2,5-Furandicarboxylic acid (FDCA) is an important renewable biotechnological building block because it serves as an environmentally friendly substitute for terephthalic acid in the production of polyesters. Currently, FDCA is produced mainly via chemical oxidation, which can cause severe environmental pollution. In this study, we developed an environmentally friendly process for the production of FDCA from 5-hydroxymethyl furfural (5-HMF) using a newly isolated strain, Raoultella ornithinolytica BF60. First, R. ornithinolytica BF60 was identified by screening and was isolated. Its maximal FDCA titer was 7.9 g/liter, and the maximal molar conversion ratio of 5-HMF to FDCA was 51.0% (mol/mol) under optimal conditions (100 mM 5-HMF, 45 g/liter whole-cell biocatalyst, 30°C, and 50 mM phosphate buffer [pH 8.0]). Next, dcaD, encoding dicarboxylic acid decarboxylase, was mutated to block FDCA degradation to furoic acid, thus increasing FDCA production to 9.2 g/liter. Subsequently, aldR, encoding aldehyde reductase, was mutated to prevent the catabolism of 5-HMF to HMF alcohol, further increasing the FDCA titer, to 11.3 g/liter. Finally, the gene encoding aldehyde dehydrogenase 1 was overexpressed. The FDCA titer increased to 13.9 g/liter, 1.7 times that of the wild-type strain, and the molar conversion ratio increased to 89.0%. IMPORTANCE In this work, we developed an ecofriendly bioprocess for green production of FDCA in engineered R. ornithinolytica. This report provides a starting point for further metabolic engineering aimed at a process for industrial production of FDCA using R. ornithinolytica.