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The combination of Escherichia coli BL21 (DE3) and the pET expression system is used extensively for the expression of various recombinant proteins (RPs). However, RP overexpression often introduces a growth burden for the host, especially in the case of toxic proteins. The key to solving this problem is to reduce the host burden associated with protein overproduction, which is often achieved by regulating the expression or activity of T7 RNAP or growth-decoupled systems. However, these strategies mainly relieve or interrupt the robbing of host resources, and do not eliminate other types of host burdens in the production process. In this study, we constructed a production system based on a dynamic equilibrium to precisely relieve the host burden and increase the RP production. The system is composed of three modules, including the overexpression of basic growth-related genes (rRNA, RNAP core enzyme, sigma factors), prediction and overexpression of key proteins using the enzyme-constrained model ec_iECBD_1354, and dynamic regulation of growth-related and key protein expression intensity based on a burden-driven promoter. Using this system, the production of many high-burden proteins, including autolysis protein and E. coli membrane proteins, was increased to varying degrees. Among them, the cytosine transporter protein (CodB) was most significantly improved, with a 4.02-fold higher production compared to the wild strain. This system can effectively reduce the optimizing costs, and is suitable for developing various types of RP expression hosts rapidly. Key points: • The basic growth-related resources can relieve the host burden from recombinant protein. • The enzyme-constrained model can accurately predict key genes to improve yield. • The expression intensity can be dynamically adjusted with changes in burden. [ABSTRACT FROM AUTHOR]

As natural metabolites, organic acids have been widely applied in food, pharmaceutical, and bio-based materials industries. Particularly, the short-chain organic acids, including C2, C3, C4, C5, and C6 organic acids, are necessary intermediate metabolites in cells and are also alternatives to some commercial chemical products. As the necessary metabolites in cells, most major short-chain organic acids can be produced through microbial fermentation. Specifically, with the development of synthetic biology, metabolic engineering could endow cells with the ability to produce more short-chain organic acid products including propionic acid, pyruvate, lactic acid, 3-hydroxypropionic acid, malic acid, succinic acid, fumaric acid, butyric acid, itaconic acid, α-ketoglutaric acid, glutaric acid, citric acid, gluconic acid, muconic acid, adipic acid, xylonic acid, and so on. The recent advances in the biological production of short-chain organic acids, as well as the challenges and perspectives, are summarized in this review to promote the generation of microbial cell factories for the production of short-chain organic acids. Key points • Outlines the production strategy of short-chain organic acids • Provide guidance for efficient synthesis of short-chain organic acids • Impacts the necessary factor of acid resistance on the successful production of host cells [ABSTRACT FROM AUTHOR]

Despite the long-term interest in solventogenic clostridia-based ABE (acetone-butanol-ethanol) fermentation, clostridial butanol tolerance and its underlying mechanism remain poorly understood, which is a major obstacle hindering further improvements of this important fermentative process. In this study, a two-component system (TCS), BtrK/BtrR, was identified and demonstrated to positively regulate butanol tolerance and ABE solvent formation in Clostridium acetobutylicum, a representative species of solventogenic clostridia. The transcriptomic analysis results showed that BtrK/BtrR has a pleiotropic regulatory function, affecting a large number of crucial genes and metabolic pathways. Of the differentially expressed genes, btrTM, encoding a putative ABC-type transporter (named BtrTM), was shown to be under the direct control of BtrR, the response regulator of the BtrK/BtrR TCS. Furthermore, BtrTM was shown to contribute to more butanol tolerance (46.5% increase) by overexpression, revealing a novel regulatory mechanism consisting of the BtrK/BtrR TCS and the BtrTM transporter in C. acetobutylicum. Based on these findings, we achieved faster growth and solvent production of C. acetobutylicum by overexpressing BtrK/BtrR or its direct target BtrTM, although no significant improvement in the final butanol titer and yield. These results further confirm the importance of BtrK/BtrR and BtrTM in this organism. Also, of significance, a specific number of btrR-btrT-btrM-btrK-like gene clusters were identified in other Clostridium species, including the pathogens Clostridium perfringens and Clostridium botulinum, indicating a broad role for this regulatory module in the class Clostridia. [ABSTRACT FROM AUTHOR]

Clostridium acetobutylicum is an important industrial microorganism for n-butanol bioproduction, and its transcription factor AbrB0310 regulates various important cellular processes. However, the roles of two abrB homologues, abrB1941 and abrB3647, have not been determined because they appear inactive during transcription. Here, we performed a detailed investigation into the function of abrB1941 and abrB3647 in C. acetobutylicum. Interestingly, we observed that AbrB3647 exerts an important influence on biphasic fermentation that opposes the influence of AbrB0310, while AbrB1941 might not be essential. When abrB3647 was disrupted using the Targetron system, a greatly improved cellular growth occurred. The following analysis shows that all three AbrBs participated in metabolically regulating acidogenesis, solventogenesis, and a two-phase transition in C. acetobutylicum, but the AbrB0310 and AbrB3647 functions were the most important. Moreover, the target genes subject to AbrB0310 and AbrB3647 regulation closely overlap. Based on these results, we will better understand the roles of the three AbrBs in regulating solventogenic clostridia cell physiology. [ABSTRACT FROM AUTHOR]