Frontiers in Bioengineering and Biotechnology, Apr 1, 2022
Corynebacterium glutamicum has been successfully employed for the industrial production of amino ... more Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate ligninderived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13 C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum. 13 C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13 C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.
In the last 20 years, biodiesel consumption in the United States has rapidly increased to ~2B gal... more In the last 20 years, biodiesel consumption in the United States has rapidly increased to ~2B gallons per year as a renewable supplement to fossil fuel. However, further expansion of biodiesel use is currently limited in part by poor cold weather performance which prevents year-round blending and necessitates blend walls ≤5% v/v. In order to provide a diesel fuel blendstock with improved cold weather performance (cloud point, pour point, cold filter plug point), while at the same time maintaining other required fuel performance specifications, several biodiesel redox analogues were synthesized and tested. The best performing candidate fuels from this class showed improvement in derived cetane number (29.3% shorter ignition delay), lower heating value (+4.7 MJ/kg), relative sooting tendency (-7.4 YSI/MJ), and cloud point (15°C lower) when compared to a B100 biodiesel composed of the identical fatty acid profile. It was observed as a general trend that the reduced form of biodiesel, Fatty Alkyl Ethers (FAEs), show performance improvements in all fuel property metrics. The suite of improved properties provided by FAEs gives biodiesel producers the opportunity to diversify their portfolio of products derived from lipid and alcohol feedstocks to include long-chain alkyl ethers, a biodiesel alternative with particular applicability for winter weather conditions across the US.
Sequential organosolv fragmentation approach (SOFA) enhances the self-assembling process of high-... more Sequential organosolv fragmentation approach (SOFA) enhances the self-assembling process of high-quality lignin nanoparticles (LNPs) by tailoring the lignin chemistry in biorefineries.
Microbial production of esters has recently garnered wide attention, but the current production m... more Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their 'direct esterification' using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production by engineering 'esterases' in E. coli. With increasing global efforts towards sustainability, esters, being biodegradable, are gaining significant recognition for an array of industrial applications, with a major market share in the cosmetics industry, followed by food, lubricants, pharmaceutical, and coating industries 1,2. The US esters market, which was 3.8 billion USD in 2019, is estimated to reach 5 billion USD by 2025, primarily driven by increasing demand for emulsifiers and stabilizers in personal care and detergent sectors 3. Predominantly, short-chain esters such as ethyl lactate and ethyl acetate occupy the commercial market owing to their biodegradable and environment-friendly characteristics 1. Esters are traditionally synthesized from carboxylic acids and alcohols in the presence of an acid catalyst, typically concentrated sulfuric acid 4. However, due to the use of excess alcohol and corrosive chemicals, advanced eco-friendly approaches, like microbial production route, are required to avoid further environmental damage. Microbial cell factories offer a sustainable platform for producing industrial solvents from renewable resources 5-9. Biocatalytic esterification can provide an eco-friendly alternative, as it occurs at ambient temperature and does not involve any corrosive waste products. Hence, biomanufacturing of green solvents, like esters, could be an effective substitute 10. Although microbes have long been exploited as cell factories for the production of various chemicals, the catalytic activity of the enzyme/s involved with the engineered pathway/s remains as one of the major bottlenecks. Hence, mining for new enzymes with better activity becomes essential. Under the current investigation, rigorous bioprospecting was performed to find potent esterifying enzymes from various natural producers 1,11,12 , and Escherichia coli was engineered for the ester production. Naturally, ester biosynthesis can be accomplished through either of the four categories of enzymes: acyltransferases; esterases; hemiacetal dehydrogenases; and monoxygenases 13. All of the earlier reports on microbial ester production have predominantly overexpressed 'acyltransferase' enzymes such as alcohol O-acetyltransferase 1 (Atf1) from yeasts, vesca alcohol acyltransferase (VAAT) from the wild strawberry plant (Fig. 1a) 1,2,14,15. They require acyl-CoA and alcohol as their substrates, wherein relatively less substrate (acyl-CoA) availability could be one of the primary limitations in acyltransferase catalysis. On the other hand, substrates for 'esterase' , organic acids and alcohols, are usually accumulated by the microbial hosts like E. coli at relatively higher titers (Fig. 1a) 16. Hence, their direct esterification will be favorable. In addition, a further increase in the precursor titers through metabolic engineering can improve the carbon flux towards the ester production 17,18. Despite the above advantages, esterases have never been exploited as candidates for ester production via metabolic engineering. A positive
Microwave assisted biocatalytic transformation is a green process for chiral drug resolution. Dif... more Microwave assisted biocatalytic transformation is a green process for chiral drug resolution. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Lipase Amano AS and Lipase AYS amino were screened for the kinetic resolution of RS-(±)-ketorolac under microwave irradiation. To study kinetics and mechanism, the effect of different parameters on the conversion, initial rates and enantiomeric excess of substrate were studied. Under microwave synergism, an increase in initial rates up to 1.5-fold was observed. Novozym 435 effectively catalyzed the enantioselective esterification of RS-(±)-ketorolac, showing excellent conversion (50%) and enantiomeric excess (>99%) in 3 h at 50 • C and 300 rpm. Based on initial rate and progress curve data, the reaction was found to follow the Ping Pong bi-bi mechanism with inhibition by n-octanol.
Combinatorial pretreatments with a low holding temperature were developed in an effort to synergi... more Combinatorial pretreatments with a low holding temperature were developed in an effort to synergistically improve the carbohydrate output and lignin processability from corn stover.
Microbial production of esters has recently garnered wide attention, but the current production m... more Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their ‘direct esterification’ using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production b...
Frontiers in Bioengineering and Biotechnology, Apr 1, 2022
Corynebacterium glutamicum has been successfully employed for the industrial production of amino ... more Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate ligninderived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13 C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of β-ketoadipate pathway in C. glutamicum. 13 C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13 C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.
In the last 20 years, biodiesel consumption in the United States has rapidly increased to ~2B gal... more In the last 20 years, biodiesel consumption in the United States has rapidly increased to ~2B gallons per year as a renewable supplement to fossil fuel. However, further expansion of biodiesel use is currently limited in part by poor cold weather performance which prevents year-round blending and necessitates blend walls ≤5% v/v. In order to provide a diesel fuel blendstock with improved cold weather performance (cloud point, pour point, cold filter plug point), while at the same time maintaining other required fuel performance specifications, several biodiesel redox analogues were synthesized and tested. The best performing candidate fuels from this class showed improvement in derived cetane number (29.3% shorter ignition delay), lower heating value (+4.7 MJ/kg), relative sooting tendency (-7.4 YSI/MJ), and cloud point (15°C lower) when compared to a B100 biodiesel composed of the identical fatty acid profile. It was observed as a general trend that the reduced form of biodiesel, Fatty Alkyl Ethers (FAEs), show performance improvements in all fuel property metrics. The suite of improved properties provided by FAEs gives biodiesel producers the opportunity to diversify their portfolio of products derived from lipid and alcohol feedstocks to include long-chain alkyl ethers, a biodiesel alternative with particular applicability for winter weather conditions across the US.
Sequential organosolv fragmentation approach (SOFA) enhances the self-assembling process of high-... more Sequential organosolv fragmentation approach (SOFA) enhances the self-assembling process of high-quality lignin nanoparticles (LNPs) by tailoring the lignin chemistry in biorefineries.
Microbial production of esters has recently garnered wide attention, but the current production m... more Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their 'direct esterification' using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production by engineering 'esterases' in E. coli. With increasing global efforts towards sustainability, esters, being biodegradable, are gaining significant recognition for an array of industrial applications, with a major market share in the cosmetics industry, followed by food, lubricants, pharmaceutical, and coating industries 1,2. The US esters market, which was 3.8 billion USD in 2019, is estimated to reach 5 billion USD by 2025, primarily driven by increasing demand for emulsifiers and stabilizers in personal care and detergent sectors 3. Predominantly, short-chain esters such as ethyl lactate and ethyl acetate occupy the commercial market owing to their biodegradable and environment-friendly characteristics 1. Esters are traditionally synthesized from carboxylic acids and alcohols in the presence of an acid catalyst, typically concentrated sulfuric acid 4. However, due to the use of excess alcohol and corrosive chemicals, advanced eco-friendly approaches, like microbial production route, are required to avoid further environmental damage. Microbial cell factories offer a sustainable platform for producing industrial solvents from renewable resources 5-9. Biocatalytic esterification can provide an eco-friendly alternative, as it occurs at ambient temperature and does not involve any corrosive waste products. Hence, biomanufacturing of green solvents, like esters, could be an effective substitute 10. Although microbes have long been exploited as cell factories for the production of various chemicals, the catalytic activity of the enzyme/s involved with the engineered pathway/s remains as one of the major bottlenecks. Hence, mining for new enzymes with better activity becomes essential. Under the current investigation, rigorous bioprospecting was performed to find potent esterifying enzymes from various natural producers 1,11,12 , and Escherichia coli was engineered for the ester production. Naturally, ester biosynthesis can be accomplished through either of the four categories of enzymes: acyltransferases; esterases; hemiacetal dehydrogenases; and monoxygenases 13. All of the earlier reports on microbial ester production have predominantly overexpressed 'acyltransferase' enzymes such as alcohol O-acetyltransferase 1 (Atf1) from yeasts, vesca alcohol acyltransferase (VAAT) from the wild strawberry plant (Fig. 1a) 1,2,14,15. They require acyl-CoA and alcohol as their substrates, wherein relatively less substrate (acyl-CoA) availability could be one of the primary limitations in acyltransferase catalysis. On the other hand, substrates for 'esterase' , organic acids and alcohols, are usually accumulated by the microbial hosts like E. coli at relatively higher titers (Fig. 1a) 16. Hence, their direct esterification will be favorable. In addition, a further increase in the precursor titers through metabolic engineering can improve the carbon flux towards the ester production 17,18. Despite the above advantages, esterases have never been exploited as candidates for ester production via metabolic engineering. A positive
Microwave assisted biocatalytic transformation is a green process for chiral drug resolution. Dif... more Microwave assisted biocatalytic transformation is a green process for chiral drug resolution. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM, Lipase Amano AS and Lipase AYS amino were screened for the kinetic resolution of RS-(±)-ketorolac under microwave irradiation. To study kinetics and mechanism, the effect of different parameters on the conversion, initial rates and enantiomeric excess of substrate were studied. Under microwave synergism, an increase in initial rates up to 1.5-fold was observed. Novozym 435 effectively catalyzed the enantioselective esterification of RS-(±)-ketorolac, showing excellent conversion (50%) and enantiomeric excess (>99%) in 3 h at 50 • C and 300 rpm. Based on initial rate and progress curve data, the reaction was found to follow the Ping Pong bi-bi mechanism with inhibition by n-octanol.
Combinatorial pretreatments with a low holding temperature were developed in an effort to synergi... more Combinatorial pretreatments with a low holding temperature were developed in an effort to synergistically improve the carbohydrate output and lignin processability from corn stover.
Microbial production of esters has recently garnered wide attention, but the current production m... more Microbial production of esters has recently garnered wide attention, but the current production metrics are low. Evidently, the ester precursors (organic acids and alcohols) can be accumulated at higher titers by microbes like Escherichia coli. Hence, we hypothesized that their ‘direct esterification’ using esterases will be efficient. We engineered esterases from various microorganisms into E. coli, along with overexpression of ethanol and lactate pathway genes. High cell density fermentation exhibited the strains possessing esterase-A (SSL76) and carbohydrate esterase (SSL74) as the potent candidates. Fed-batch fermentation at pH 7 resulted in 80 mg/L of ethyl acetate and 10 mg/L of ethyl lactate accumulation by SSL76. At pH 6, the total ester titer improved by 2.5-fold, with SSL76 producing 225 mg/L of ethyl acetate, and 18.2 mg/L of ethyl lactate, the highest reported titer in E. coli. To our knowledge, this is the first successful demonstration of short-chain ester production b...
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Papers by Somnath Shinde