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Butyl butyrate is a short-chain fatty acid ester (C8) with a fruity aroma. It has broad prospects in the fields of foods, cosmetics and biofuels. At present, butyl butyrate is produced by chemical synthesis in the industry, but it is highly dependent on petroleum-based products. The growing concerns regarding the future scarcity of fossil fuels have been strongly promoted the transition from traditional fossil fuels and products to renewable bioenergy and biochemicals. Therefore, it is necessary to develop a green biochemical technology to replace traditional petroleum-based materials. In recent years, microorganisms such as Escherichia coli and Clostridium have been engineered to serve as cell factories for the sustainable one-pot production of short-chain fatty acid esters, including butyl butyrate. This opinion highlights the recent development in the use of lipases and alcohol acyltransferases (AATs) for butyl butyrate production in microbial fermentation, as well as future perspectives., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Resistance to furan derivatives and phenolic compounds plays an important role in the use of lignocellulosic biomass for biological production of chemicals and fuels. This study confirmed that expression of short-chain dehydrogenase/reductase (SDR) from Clostridium beijerinckii NCIMB 8052 significantly improved the tolerance of C. tyrobutyricum to furfural due to the enhanced activity for furfural reduction. And on this basis, co-expression of SDR and heat shock chaperones GroESL could simultaneously enhance the tolerance of C. tyrobutyricum to furan derivatives and phenolic compounds, which were the main inhibitors presented in dilute-acid lignocellulosic hydrolysates. Consequently, the recombinant strain ATCC 25755/sdr+groESL exhibited good performance in butyric acid production with corncob acid hydrolysate as the substrate. Batch fermentation in bioreactor showed that the butyrate produced by ATCC 25755/sdr+groESL was 32.8 g/L, increased by 28.1% as compared with the wild-type strain. Meanwhile, the butyrate productivity increased from 0.19 g/L·h to 0.29 g/L·h., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Butyric acid is an important C4 organic acid with broad applications. It is currently produced by chemosynthesis from petroleum-based feedstocks. However, the fermentative production of butyric acid from renewable feedstocks has received growing attention because of consumer demand for green products and natural ingredients in foods, pharmaceuticals, animal feed supplements, and cosmetics. In this review, strategies for improving microbial butyric acid production, including strain engineering and novel fermentation process development are discussed and compared regarding product yield, titer, purity and productivity. Future perspectives on strain and process improvements for butyric acid production are also discussed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Butyric acid fermentation by Clostridium couples with the synthesis of acetic acid. But the presence of acetic acid reduces butyric acid yield and increases separation and purification costs of butyric acid. Hence, enhancing the butyrate/acetate ratio is important for economical butyric acid production. This study indicated that enhancing the acetyl-CoA to butyrate flux by overexpression of both the butyryl-CoA/acetate CoA transferase (cat1) and crotonase (crt) genes in C. tyrobutyricum could significantly reduce acetic acid concentration. Fed-batch fermentation of ATCC 25755/cat1 + crt resulted in increased butyrate/acetate ratio of 15.76 g/g, which was 2.24-fold higher than that of the wild-type strain. Furthermore, in order to simultaneously increase the butyrate/acetate ratio, butyric acid concentration and productivity, the recombinant strain ATCC 25755/ppcc (co-expression of 6-phosphofructokinase (pfkA) gene, pyruvate kinase (pykA) gene, cat1, and crt) was constructed. Consequently, ATCC 25755/ppcc produced more butyric acid (46.8 vs. 35.0 g/L) with a higher productivity (0.83 vs. 0.49 g/L·h) and butyrate/acetate ratio (13.22 vs. 7.22 g/g) as compared with the wild-type strain in batch fermentation using high glucose concentration (120 g/L). This study demonstrates that enhancing the acetyl-CoA to butyrate flux is an effective way to reduce acetic acid production and increase butyrate/acetate ratio.

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8g/L vs. 19.4g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28g/L·h vs. 0.16g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53g/L·h vs. 0.26g/L·h) and yield (0.32g/g vs. 0.28g/g). When the initial total sugar concentration was ~120g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4g/L, yield of 0.43g/g sugar consumed, productivity of 0.87g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Butyric acid fermentation by Clostridium couples with the synthesis of acetic acid. But the presence of acetic acid reduces butyric acid yield and increases separation and purification costs of butyric acid. Hence, enhancing the butyrate/acetate ratio is important for economical butyric acid production. This study indicated that enhancing the acetyl-CoA to butyrate flux by overexpression of both the butyryl-CoA/acetate CoA transferase (cat1) and crotonase (crt) genes in C. tyrobutyricum could significantly reduce acetic acid concentration. Fed-batch fermentation of ATCC 25755/cat1 + crt resulted in increased butyrate/acetate ratio of 15.76 g/g, which was 2.24-fold higher than that of the wild-type strain. Furthermore, in order to simultaneously increase the butyrate/acetate ratio, butyric acid concentration and productivity, the recombinant strain ATCC 25755/ppcc (co-expression of 6-phosphofructokinase (pfkA) gene, pyruvate kinase (pykA) gene, cat1, and crt) was constructed. Consequently, ATCC 25755/ppcc produced more butyric acid (46.8 vs. 35.0 g/L) with a higher productivity (0.83 vs. 0.49 g/L·h) and butyrate/acetate ratio (13.22 vs. 7.22 g/g) as compared with the wild-type strain in batch fermentation using high glucose concentration (120 g/L). This study demonstrates that enhancing the acetyl-CoA to butyrate flux is an effective way to reduce acetic acid production and increase butyrate/acetate ratio. [ABSTRACT FROM AUTHOR]

[Display omitted] • H 2 fermentation process of C. tyrobutyricum was optimized systematically. • The effect of endogenous hydrogenases overexpression on H 2 production was studied. • Introducing sucrose metabolic pathway realized H 2 production from sugarcane molasses. • The highest H 2 production achieved 1098.4 mL with a yield of 248.2 mL/g sugar. Clostridia is the dominant microorganism in dark-fermentative biohydrogen production, but there have been relatively few studies on hydrogen production by Clostridium tyrobutyricum using cheap biomass. In this study, the important process parameters for hydrogen fermentation, such as substrate concentration, pH and temperature, were optimized systematically. Then, the effect of endogenous hydrogenases overexpression on hydrogen production was studied. To achieve economical hydrogen production from cheap substrate sugarcane molasses, the sucrose metabolic pathway was further introduced into C. tyrobutyricum. As a result, the engineered C. tyrobutyricum produced 1098.4 mL hydrogen with a yield of 248.2 mL/g sugar and productivity of 199.6 mL/L/h under the optimum fermentation conditions, which is the highest hydrogen production (910.37 mmol/L) reported so far using molasses. This study demonstrates that C. tyrobutyricum is a promising cell factory for hydrogen production from cheap and renewable biomass resources. [ABSTRACT FROM AUTHOR]

• Clostridium tyrobutyricum is an excellent microorganism for bio-based butyric acid production. However, the main obstacles for its industrialization are low butyric acid concentration and productivity. This study indicated that overexpression of rate-limiting enzymes in the Embden-Meyerhof-Parnas pathway, including 6-phosphofructokinase ( pfkA ) and pyruvate kinase ( pykA ), in C. tyrobutyricum ATCC 25755 could enhance its intracellular NADH and ATP levels and resistance to butyric acid and glucose. The results of fed-batch fermentation by ATCC 25755/ pfkA  +  pykA showed that the butyrate concentration and productivity reached 48.2 g/L and 0.50 g/L h, which increased 38.1% and 38.9% as compared with the wild-type, respectively. The increased butyric acid tolerance and production could be due to the higher intracellular NADH and ATP levels by overexpression of pfkA and pykA in C. tyrobutyricum ATCC 25755. Furthermore, ATCC 25755/ pfkA + pykA exhibited higher glucose tolerance. Consequently, the productivity of butyrate was further increased by 64.0% in batch fermentation using high concentration of glucose (120 g/L). [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass is the most abundant and renewable substrate for biological fermentation, but the inhibitors present in the lignocellulosic hydrolysates could severely inhibit the cell growth and productivity of industrial strains. This study confirmed that overexpressing of native groESL in Clostridium tyrobutyricum could significantly improve its tolerance to lignocellulosic hydrolysate-derived inhibitors, especially for phenolic compounds. Consequently, ATCC 25755/ groESL showed a better performance in butyric acid fermentation with hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw, respectively. When corn straw and rice straw hydrolysates, which showed strong toxicity to C. tyrobutyricum , were used as the substrates, 29.6 g/L and 30.1 g/L butyric acid were obtained in batch fermentation, increased by 26.5% and 19.4% as compared with the wild-type strain, respectively. And more importantly, the butyric acid productivity reached 0.31 g/L·h (vs. 0.20–0.21 g/L·h for the wild-type strain) due to the shortened lag phase. [ABSTRACT FROM AUTHOR]

Abstract: A strictly anaerobic, thermoacidophilic, H2-producing bacterium was isolated and designated as Thermoanaerobacterium aotearoense. The optimized cultivation conditions for H2 production are 55°C, pH 6.5 and 10gl−1 of glucose or xylose. A metabolic pathway analysis showed that lactate occupied most of the liquid metabolites and consumed a large amount of NADH. To increase the efficiency of hydrogen production, the gene encoding the l-lactate dehydrogenase was knocked out to redirect the NADH flow. Genetic manipulation resulted in the 2 and 2.5 folds increase of the H2 yield and production rate, respectively. The maximum H2 yields using the Δldh mutant were 2.71, 1.45 and 2.28molH2 mol−1 sugar under glucose, xylose and glucose/xylose mixture tests, respectively. The recombinant Δldh strain could ferment the mixture of glucose and xylose to produce H2 effectively, indicating that the performance of Thermoanaerobacterium in H2 production can be significantly improved by metabolic engineering technique. [ABSTRACT FROM AUTHOR]

• C. tyrobutyricum was engineered to overexpress galactose metabolism-related genes. • Removing lipids could significantly enhance the hydrolysis efficiency of SCGs. • The engineered strain utilized galactose and glucose simultaneously. • 34.3 g/L butyrate at a productivity of 0.36 g/L·h and yield of 0.37 g/g was obtained. • This provides an economically viable approach for butyric acid production. Clostridium tyrobutyricum cannot utilize galactose, which is abundant in lignocellulose and red algae, as a carbon source for butyric acid production. Hence, when using galactose-rich coffee ground hydrolysate as the substrate, the fermentation performance of C. tyrobutyricum is poor. In this work, a recombinant strain, C. tyrobutyricum ATCC 25755/ ketp , overexpressing galactose catabolism genes (galK , galE , galT , and galP) from Clostridium acetobutylicum ATCC 824 was constructed for the co-utilization of glucose and galactose. Batch fermentation in the bioreactor showed that ATCC 25755/ ketp could efficiently utilize galactose without glucose-induced carbon catabolite repression and consume nearly 100% of the galactose present in the spent coffee ground hydrolysate. Correspondingly, the butyric acid concentration and productivity of ATCC 25755/ ketp reached 34.3 g/L and 0.36 g/L·h, respectively, an increase of 78.6% and 56.5% compared with the wild-type strain, indicating its potential for butyric acid production from hydrolysates of inexpensive and galactose-rich biomass. [ABSTRACT FROM AUTHOR]

• scrA , scrB and scrK were successfully overexpressed in C. tyrobutyricum ATCC 25755. • The cane molasses could be utilized efficiently by C. tyrobutyricum ATCC 25755/ scrBAK. • The medium composition and initial sugar concentration were optimized. • 45.71 g/L butyrate at a yield of 0.39 g/g was obtained in fed-batch fermentation. The sucrose metabolic genes (scrA, scrB and scrK) from C. acetobutylicum ATCC 824 were successfully overexpressed in C. tyrobutyricum ATCC 25755, endowing it with the ability to co-utilize sucrose, fructose and glucose in the cane molasses. As a result, the engineering strain C. tyrobutyricum ATCC 25755/ scrBAK produced 18.07 g/L and 18.98 g/L butyric acid when sucrose and cane molasses were used as the carbon source, respectively. Furthermore, the medium composition and initial cane molasses concentration were optimized to make full use of the untreated cane molasses. Based on these results, 45.71 g/L butyric acid with a yield of 0.39 g/g was obtained in fed-batch fermentation, and the feedstock cost of using untreated cane molasses was decreased by ~47% when compared with the conventional glucose fermentation. This study demonstrated the potential application of C. tyrobutyricum ATCC 25755/ scrBAK for economic butyric acid production from untreated cane molasses. [ABSTRACT FROM AUTHOR]