Recent advances, challenges and metabolic engineering strategies in the biosynthesis of 3-hydroxypropionic acid.

As an attractive and valuable platform chemical, 3-hydroxypropionic acid (3-HP) can be used to produce a variety of industrially important commodity chemicals and biodegradable polymers. Moreover, the biosynthesis of 3-HP has drawn much attention in recent years due to its sustainability and environmental friendliness. Here, we focus on recent advances, challenges, and metabolic engineering strategies in the biosynthesis of 3-HP. While glucose and glycerol are major carbon sources for its production of 3-HP via microbial fermentation, other carbon sources have also been explored. To increase yield and titer, synthetic biology and metabolic engineering strategies have been explored, including modifying pathway enzymes, eliminating flux blockages due to byproduct synthesis, eliminating toxic byproducts, and optimizing via genome-scale models. This review also provides insights on future directions for 3-HP biosynthesis.

Production of isoprene, one of the high-density fuel precursors, from peanut hull using the high-efficient lignin-removal pretreatment method.

BACKGROUND: Isoprene as the feedstock can be used to produce renewable energy fuels, providing an alternative to replace the rapidly depleting fossil fuels. However, traditional method for isoprene production could not meet the demands for low-energy consumption and environment-friendliness. Moreover, most of the previous studies focused on biofuel production out of lignocellulosic materials such as wood, rice straw, corn cob, while few studies concentrated on biofuel production using peanut hull (PH). As is known, China is the largest peanut producer in the globe with an extremely considerable amount of PH to be produced each year. Therefore, a novel, renewable, and environment-friendly pretreatment strategy to increase the enzymatic hydrolysis efficiency of cellulose and reduce the inhibitors generation was developed to convert PH into isoprene. RESULTS: The optimal pretreatment conditions were 100 °C, 60 min, 10% (w/v) solid loading with a 2:8 volume ratio of phosphoric acid and of hydrogen peroxide. In comparison with the raw PH, the hemicellulose and lignin were reduced to 85.0 and 98.0%, respectively. The cellulose-glucose conversion of pretreated PH reached up to 95.0% in contrast to that of the raw PH (19.1%). Only three kinds of inhibitors including formic acid, levulinic acid, and a little furfural were formed during the pretreatment process, whose concentrations were too low to inhibit the isoprene yield for Escherichia coli fermentation. Moreover, compared with the isoprene yield of pure glucose fermentation (298 ± 9 mg/L), 249 ± 6.7 and 294 ± 8.3 mg/L of isoprene were produced using the pretreated PH as the carbon source by the engineered strain via separate hydrolysis and fermentation and simultaneous saccharification and fermentation (SSF) methods, respectively. The isoprene production via SSF had a 9.8% glucose-isoprene conversion which was equivalent to 98.8% of isoprene production via the pure glucose fermentation. CONCLUSIONS: The optimized phosphoric acid/hydrogen peroxide combination pretreatment approach was proved effective to remove lignin and hemicellulose from lignocellulosic materials. Meanwhile, the pretreated PH could be converted into isoprene efficiently in the engineered Escherichia coli. It is concluded that this novel strategy of isoprene production using lignocellulosic materials pretreated by phosphoric acid/hydrogen peroxide is a promising alternative to isoprene production using traditional way which can fully utilize non-renewable fossil sources.

Computational study of the binding mechanism of medium chain acyl-CoA synthetase with substrate in Methanosarcina acetivorans.

The acyl-AMP forming family of adenylating enzymes catalyzes the formation of acyl-CoA from an acyl substrate, ATP, and CoA, which is a metabolite of many catabolic and anabolic processes. The medium-chain acyl-CoA synthetase from Methanosarcina acetivorans, designated MacsMa, uses 2-methylbutyrate as its preferred substrate. It is reported that the interaction between the sidechain of Cys298 and Lys256 of this enzyme is important for the catalytic activity. The mutation of these residues resulted in the changes of the structure stability and the reduced or absence catalytic activity. In the present study, the binding mechanism between the substrate 2-methylbutyrate- AMP (2MeBA) and MacsMa were explored by integrating multiple computational methods including molecular docking, molecular dynamics simulations, binding free energy calculation, active site access channel analysis and principal component analysis. The binding free energy between WT, mutated Macs and substrate was calculated by MM-GBSA method, which indicated that the binding affinity between this enzyme and substrate was stronger in the WT than that in the mutated forms (K256L, K256T and C298Y). Per-residue binding free energy decomposition identified some residues, such as Gly327, Phe350, Gly351, Gln352 and Lys461, which are important for the enzyme and substrate binding affinity. The access channels of the mutant system (MacsK256L, MacsK256T and MacsC298Y) were found to be different from those in the wild-type systems. It suggested that K256L and C298Y induced larger flexibility to the overall protein compared with the WT, whereas K256T induced larger flexibility to the partial protein compared with the WT by PCA vector porcupines. This study provides novel insight to understand the substrate binding mechanism of Macs and useful information for the rational enzyme design.

Engineering Escherichia coli to convert acetic acid to ß-caryophyllene.

BACKGROUND: Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce ß-caryophyllene by the engineered E. coli using acetic acid as the only carbon source. RESULTS: Firstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to ß-caryophyllene, acetyl-CoA synthase (ACS), ß-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance ß-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What's more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of ß-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and ß-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h(-1) g(-1) dry cells, and the conversion efficiency of acetic acid to ß-caryophyllene (gram to gram) reached 2.1%. The yield of ß-caryophyllene on acetic acid of this strain also reached approximately 5.6% of the theoretical yield. CONCLUSIONS: In the present study, a novel biosynthetic pathway for ß-caryophyllene has been investigated by means of conversion of acetic acid to ß-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in ß-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for ß-caryophyllene synthesis.

A novel MVA-mediated pathway for isoprene production in engineered E. coli.

BACKGROUND: To deal with the increasingly severe energy crisis and environmental consequences, biofuels and biochemicals generated from renewable resources could serve as a promising alternative for replacing petroleum as a source of fuel and chemicals, among which isoprene (2-methyl-1,3-butadiene) in particular is of great significance in that it is an important platform chemical, which has been used in industrial production of synthetic rubber for tires and coatings or aviation fuel. RESULTS: We firstly introduced fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species into E. coli to directly convert MVA(mevalonate) into 3-methy-3-buten-1-ol. And then to transform 3-methy-3-buten-1-ol to isoprene, oleate hydratase (OhyAEM) from Elizabethkingia meningoseptica was overexpressed in E. coli. A novel biosynthetic pathway of isoprene in E. coli was established by co-expressing the heterologous mvaE gene encoding acetyl-CoA acetyltransferase/HMG-CoA reductase and mvaS gene encoding HMG-CoA synthase from Enterococcus faecalis, fatty acid decarboxylase (OleTJE) and oleate hydratase (OhyAEM). Furthermore, to enhance isoprene production, a further optimization of expression level of OleTJE, OhyAEM was carried out by using different promoters and copy numbers of plasmids. Thereafter, the fermentation process was also optimized to improve the production of isoprene. The final engineered strain, YJM33, bearing the innovative biosynthetic pathway of isoprene, was found to produce isoprene up to 2.2 mg/L and 620 mg/L under flask and fed-batch fermentation conditions, respectively. CONCLUSIONS: In this study, by using metabolic engineering techniques, the novel MVA-mediated biosynthetic pathway of isoprene was successfully assembled in E. coli BL21(DE3) with the heterologous MVA upper pathway, OleTJE from Jeotgalicoccus species and OhyAEM from Elizabethkingia meningoseptica. Compared with traditional MVA pathway, the novel pathway is shortened by 3 steps. In addition, this is the first report on the reaction of converting MVA into 3-methy-3-buten-1-ol by fatty acid decarboxylase (OleTJE) from Jeotgalicoccus species. In brief, this study provided an alternative method for isoprene biosynthesis, which is largely different from the well-developed MEP pathway or MVA pathway.

Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene.

BACKGROUND: α-Pinene is an important natural product that is widely used in flavorings, fragrances, medicines, fine chemicals and high-density renewable fuels. Currently, α-Pinene used in industry is mainly produced either by tapping trees (gum turpentine) or as a byproduct of paper pulping (crude sulfate turpentine, CST). However, the extraction of it from trees is tedious and inefficient and requires substantial expenditure of natural resources. Therefore, it is necessary to seek sustainable technologies for α-pinene production. RESULTS: To construct the microbial synthetic pathway of α-pinene in E. coli, we co-expressed native geranyl diphosphate synthase (IspA) from E. coli and α-pinene synthase (Pt30) from Pinus taeda, and then to increase the geranyl diphosphate (GPP) content in the cells, a suitable geranyl diphosphate synthase (GPPS2) was selected from two different origins. Furthermore, to enhance α-pinene production, a novel biosynthetic pathway of α-pinene was assembled in E. coli BL21(DE3) with the heterologous hybrid mevalonate (MVA) pathway, GPPS2 and α-pinene synthase (Pt30). The final genetic strain, YJM28, harboring the above novel biosynthetic pathway of α-pinene, accumulated α-pinene up to 5.44 mg/L and 0.97 g/L under flask and fed-batch fermentation conditions, respectively. The conversion efficiency of glucose to α-pinene (gram to gram) in the metabolically engineered strain reached 2.61%. CONCLUSIONS: In this paper, by using metabolic engineering techniques, the more efficient biosynthetic pathway of α-pinene was successfully assembled in E. coli BL21(DE3) with the heterologous hybrid MVA pathway, GPPS2 and α-pinene synthase (Pt30). In addition, this is the first report on α-pinene fed-batch fermentation, and our results represent improvements over previous reports.

Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli.

The depleting petroleum reserve, increasingly severe energy crisis, and global climate change are reigniting enthusiasm for seeking sustainable technologies to replace petroleum as a source of fuel and chemicals. In this paper, the efficiency of the MVA pathway on isoprene production has been improved as follows: firstly, in order to increase MVA production, the source of the "upper pathway" which contains HMG-CoA synthase, acetyl-CoA acetyltransferase and HMG-CoA reductase to covert acetyl-CoA into MVA has been changed from Saccharomyces cerevisiae to Enterococcus faecalis; secondly, to further enhance the production of MVA and isoprene, a alanine 110 of the mvaS gene has been mutated to a glycine. The final genetic strain YJM25 containing the optimized MVA pathway and isoprene synthase from Populus alba can accumulate isoprene up to 6.3 g/L after 40 h of fed-batch cultivation.

[Progress of antisense technology applied in metabolic regulation of bacteria].

With the rapid development of genetic engineering and metabolic regulation, antisense technology displays its fascination to the world as a mild regulation genetic tool. Compared with other loss-of-function research methods (e.g. gene knockout), antisense technologies have advantages such as low cost, short period, and easy operation. It has been increasingly used in bacterial metabolic regulation as a powerful genetic tool. This review briefly summarized the latest progress and problems in antisense technologies that are recently used in metabolic engineering of bacteria, and compares the advantages and disadvantages of these technologies.