Acetoin and 2,3-butanediol (2,3-BD) have a large number of industrial applications. The production of acetoin and 2,3-BD has traditionally relied on oil supplies. Microbial production of acetoin and ...2,3-BD will alleviate the dependence on oil. Acetoin and 2,3-BD are neighboring metabolites in the 2,3-BD metabolic pathway of bacteria. This review summarizes metabolic engineering strategies for improvement of microbial acetoin and 2,3-BD production. We also propose enhancements to current acetoin and 2,3-BD production strategies, by offering a metabolic engineering approach that is guided by systems biology and synthetic biology.
Carbamoyl phosphate is an important precursor for L-arginine and pyrimidines biosynthesis. In view of this importance, the cell factory should enhance carbamoyl phosphate synthesis to improve related ...compound production. In this work, we verified that carbamoyl phosphate is essential for L-arginine production in
Corynebacterium
sp., followed by engineering of carbamoyl phosphate synthesis for further strain improvement. First,
carAB
encoding carbamoyl phosphate synthetase II was overexpressed to improve the synthesis of carbamoyl phosphate. Second, the regulation of glutamine synthetase increases the supply of L-glutamine, providing an effective substrate for carbamoyl phosphate synthetase II. Third, carbamate kinase, which catalyzes inorganic ammonia synthesis carbamoyl phosphate, was screened and selected to assist in carbamoyl phosphate supply. Finally, we disrupted
ldh
(encoding lactate dehydrogenase) to decrease by-production formation and save NADH to regenerate ATP through the electron transport chain. Subsequently, the resulting strain allowed a dramatically increased L-arginine production of 68.6 ± 1.2 g∙L
−1
, with an overall productivity of 0.71 ± 0.01 g∙L
−1
∙h
−1
in 5-L bioreactor. Stepwise rational metabolic engineering based on an increase in the supply of carbamoyl phosphate resulted in a gradual increase in L-arginine production. The strategy described here can also be implemented to improve L-arginine and pyrimidine derivatives.
Key points
•
The L-arginine production strongly depended on the supply of carbamoyl phosphate.
•
The novel carbamoyl phosphate synthesis pathway for C. crenatum based on carbamate kinase was first applied to L-arginine synthesis.
•
ATP was regenerated followed with the disruption of lactate formation.
NAD
-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw ...of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10C
) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10C
, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.
FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.
Acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), which catalyzes the interconversion between acetoin and 2,3-butanediol, plays an important role in distribution of the products pools. This ...work characterized the Bacillus subtilis AR/BDH for the first time. The enzyme showed very different pH preferences of pH 6.5 for reduction and pH 8.5 for oxidation. Based on these above results, a two-stage pH control strategy was optimized for acetoin production, in which the pH was controlled at 6.5 for quickly converting glucose to acetoin and 2,3-butanediol, and then 8.0 for reversely transforming 2,3-butanediol to acetoin. By over-expression of AR/BDH in the wild-type B. subtilis JNA 3-10 and applying fed-batch fermentation based on the two-stage pH control strategy, acetoin yield of B. subtilis was improved to a new record of 73.6 g/l, with the productivity of 0.77 g/(l · h). The molar yield of acetoin was improved from 57.5% to 83.5% and the ratio of acetoin/2,3-butanediol was switched from 2.7:1 to 18.0:1.
l-arginine, a semi essential amino acid, is an important amino acid in food flavoring and pharmaceutical industries. Its production by microbial fermentation is gaining more and more attention. In ...previous work, we obtained a new l-arginine producing Corynebacterium crenatum (subspecies of Corynebacterium glutamicum) through mutation breeding. In this work, we enhanced l-arginine production through improvement of the intracellular environment. First, two NAD(P)H-dependent H2O2-forming flavin reductases Frd181 (encoded by frd1 gene) and Frd188 (encoded by frd2) in C. glutamicum were identified for the first time. Next, the roles of Frd181 and Frd188 in C. glutamicum were studied by overexpression and deletion of the encoding genes, and the results showed that the inactivation of Frd181 and Frd188 was beneficial for cell growth and l-arginine production, owing to the decreased H2O2 synthesis and intracellular reactive oxygen species (ROS) level, and increased intracellular NADH and ATP levels. Then, the ATP level was further increased by deletion of noxA (encoding NADH oxidase) and amn (encoding AMP nucleosidase), and overexpression of pgk (encoding 3-phosphoglycerate kinase) and pyk (encoding pyruvate kinase), and the l-arginine production and yield from glucose were significantly increased. In fed-batch fermentation, the l-arginine production and yield from glucose of the final strain reached 57.3g/L and 0.326g/g, respectively, which were 49.2% and 34.2% higher than those of the parent strain, respectively. ROS and ATP are important elements of the intracellular environment, and l-arginine biosynthesis requires a large amount of ATP. For the first time, we enhanced l-arginine production and yield from glucose through reducing the H2O2 synthesis and increasing the ATP supply.
•Two new H2O2-forming flavin reductases from C. glutamicum are identified.•Inactivation of the flavin reductases facilitates L-arginine production.•Increasing the ATP supply facilitates L-arginine production.
L-valine is an essential amino acid that has wide and expanding applications with a suspected growing market demand. Its applicability ranges from animal feed additive, ingredient in cosmetic and ...special nutrients in pharmaceutical and agriculture fields. Currently, fermentation with the aid of model organisms, is a major method for the production of L-valine. However, achieving the optimal production has often been limited because of the metabolic imbalance in recombinant strains. In this review, the constrains in L-valine biosynthesis are discussed first. Then, we summarize the current advances in engineering of microbial cell factories that have been developed to address and overcome major challenges in the L-valine production process. Future prospects for enhancing the current L-valine production strategies are also discussed.
Prodigiosin (PG), a red linear tripyrrole pigment produced by
Serratia marcescens
, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. Although many ...studies have been used to dissect the biosynthetic pathways and regulatory network of prodigiosin production in
S. marcescens
, few studies have been focused on improving prodigiosin production through metabolic engineering in this strain. In this study, transcription factor engineering and promoter engineering was used to promote the production of prodigiosin in
S. marcescens
JNB5-1. Firstly, through construing of a Tn5G transposon insertion library of strain JNB5-1, it was found that the DNA-binding response regulator BVG89_19895 (OmpR) can promote prodigiosin synthesis in this strain. Then, using RNA-Seq analysis, reporter green fluorescent protein analysis and RT-qPCR analysis, the promoter P17 (P
RplJ
) was found to be a strong constitutive promoter in strain JNB5-1. Finally, the promoter P17 was used for overexpressing of prodigiosin synthesis activator OmpR and PsrA in strain JNB5-1 and a recombinant strain PG-6 was obtained. Shake flask analysis showed that the prodigiosin titer of this strain was increased to 10.25 g/L, which was 1.62-times that of the original strain JNB5-1 (6.33 g/L). Taken together, this is the first well-characterized constitutive promoter library from
S. marcescens
, and the transcription factor engineering and promoter engineering can be also useful strategies to improve the production of other high value-added products in
S. marcescens
.
The fat mass and obesity-associated protein (FTO) is an N6-Methyladenosine (m6A) demethylase, which has been revealed to play critical roles in tumorigenesis. However, its role in the development and ...progression of prostate cancer (PCa) remains poorly understood. Here, we aimed to investigate the function and clinical relevance of FTO in PCa. Our results demonstrated that FTO was notably downregulated in PCa tissues compared with the paired normal tissues. In addition, the decreased expression of FTO was correlated with poor prognosis of PCa. Functional experiments showed that depletion of FTO promoted the proliferation and metastasis of PCa both in vitro and in vivo. Conversely, ectopic expression of FTO exhibited the opposite effects. Combined with RNA-sequencing, MeRIP-RT-qPCR, and mRNA stability assays indicated chloride intracellular channel 4(CLIC4) was a functional target of FTO-mediated m6A modification. FTO depletion significantly increased the m6A level of CLIC4 mRNA and then reduced the mRNA stability. In conclusion, our findings suggest that FTO suppresses PCa proliferation and metastasis through reducing the degradation of CLIC4 mRNA in an m6A dependent manner. FTO may be used as a promising novel therapeutic target and prognostic evaluation biomarker for PCa.
Previously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. ...amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.
In the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.
Co-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.
As one of the most significant steroid hormone precursors, androst-1,4-diene-3,17-dione (ADD) could be used to synthesize many valuable hormone drugs. The microbial transformation of sterols to ADD ...has received extensive attention in recent years. In a previous study,
JC-12 was isolated and converted sterols to the major product, ADD. In this work, we enhanced ADD yield by improving the cell intracellular environment. First, we introduced a nicotinamide adenine dinucleotide (NADH) oxidase from
to balance the intracellular NAD
availability in order to strengthen the ADD yield. Then, the catalase gene from
was also over-expressed to simultaneously scavenge the generated H
O
and eliminate its toxic effects on cell growth and sterol transformation. Finally, using a 5 L fermentor, the recombinant strain JC-12
produced 9.66 g/L ADD, which increased by 80% when compared with the parent strain. This work shows a promising way to increase the sterol transformation efficiency by regulating the intracellular environment.
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