Absence of trehalose synthesis in Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus ferrivorans CF27 at low temperature

Trehalose is a type of carbohydrate that protects against different types of stress and is also used as a source of carbon storage in prokaryotes. There are four different ways of synthesizing trehalose in Acidithiobacillus ferrivorans and two in Acidithiobacillus ferrooxidans, but its purpose remains unknown. This study aimed to measure the production of trehalose under different conditions by quantifying it in three culture media at two different temperatures. The growth kinetics of both species were also assessed, and the trehalose concentration was analysed during the early stationary phase using an enzymatic method. The results showed that the modified 9K medium with ferrous iron at 28°C had the highest production of trehalose, with A. ferrivorans CF27 having a higher production of 0.34 µmol/mg protein compared to A. ferrooxidans ATCC 23270 at 0.31 µmol/mg protein. When using CuS, the production of trehalose was lower, with 0.02 and 0.03 µmol/mg protein for A. ferrivorans CF27 and A. ferrooxidans ATCC 23270, respectively, while no trehalose was detected in the presence of zinc. At 15°C, the enzymatic method did not detect any trehalose in all three culture media, this would indicate that this carbohydrate does not protect against low temperatures in either species.

La trehalosa es un tipo de carbohidrato, que en procariotas protege contra diferentes tipos de estrés y también se utiliza como fuente de almacenamiento de carbono. Hay cuatro formas diferentes de sintetizar trehalosa en Acidithiobacillus ferrivorans y dos en Acidithiobacillus ferrooxidans, pero su propósito sigue siendo desconocido. Este estudio tuvo como objetivo medir la producción de trehalosa en diferentes condiciones mediante su cuantificación en tres medios de cultivo a dos temperaturas diferentes. También se evaluó la cinética de crecimiento de ambas especies y se analizó la concentración de trehalosa durante la fase estacionaria temprana mediante un método enzimático. Los resultados mostraron que el medio 9K modificado con hierro ferroso a 28 °C tuvo la mayor producción de trehalosa, con A. ferrivorans CF27 con una mayor producción de 0.34 µmol/mg de proteína en comparación con A. ferrooxidans ATCC 23270 a 0.31 µmol/mg de proteína. Al utilizar CuS, la producción de trehalosa fue menor, con 0.02 y 0.03 µmol/mg de proteína para A. ferrivorans CF27 y A. ferrooxidans ATCC 23270, respectivamente, mientras que en presencia de zinc no se detectó trehalosa. A 15°C, el método enzimático no detectó trehalosa en los tres medios de cultivo, lo que indicaria que este carbohidrato no protege contra las bajas temperaturas en ninguna de las especies.

Research progress on chemical structures and pharmacological effects of natural cytisine and its derivatives / 中国中药杂志

Cytisine derivatives are a group of alkaloids containing the structural core of cytisine, which are mainly distributed in Fabaceae plants with a wide range of pharmacological activities, such as resisting inflammation, tumors, and viruses, and affecting the central nervous system. At present, a total of 193 natural cytisine and its derivatives have been reported, all of which are derived from L-lysine. In this study, natural cytisine derivatives were classified into eight types, namely cytisine type, sparteine type, albine type, angustifoline type, camoensidine type, cytisine-like type, tsukushinamine type, and lupanacosmine type. This study reviewed the research progress on the structures, plant sources, biosynthesis, and pharmacological activities of alkaloids of various types.

Advances in the production of chemicals by organelle compartmentalization in Saccharomyces cerevisiae / 生物工程学报

As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.

Advances on the production of organic acids by yeast / 生物工程学报

Organic acids are organic compounds that can be synthesized using biological systems. They often contain one or more low molecular weight acidic groups, such as carboxyl group and sulphonic group. Organic acids are widely used in food, agriculture, medicine, bio-based materials industry and other fields. Yeast has unique advantages of biosafety, strong stress resistance, wide substrate spectrum, convenient genetic transformation, and mature large-scale culture technology. Therefore, it is appealing to produce organic acids by yeast. However, challenges such as low concentration, many by-products and low fermentation efficiency still exist. With the development of yeast metabolic engineering and synthetic biology technology, rapid progress has been made in this field recently. Here we summarize the progress of biosynthesis of 11 organic acids by yeast. These organic acids include bulk carboxylic acids and high-value organic acids that can be produced naturally or heterologously. Finally, future prospects in this field were proposed.

Controlling antifungal activity with light: Optical regulation of fungal ergosterol biosynthetic pathway with photo-responsive CYP51 inhibitors

Invasive fungal infections (IFIs) have been associated with high mortality, highlighting the urgent need for developing novel antifungal strategies. Herein the first light-responsive antifungal agents were designed by optical control of fungal ergosterol biosynthesis pathway with photocaged triazole lanosterol 14α-demethylase (CYP51) inhibitors. The photocaged triazoles completely shielded the CYP51 inhibition. The content of ergosterol in fungi before photoactivation and after photoactivation was 4.4% and 83.7%, respectively. Importantly, the shielded antifungal activity (MIC80 ≥ 64 μg/mL) could be efficiently recovered (MIC80 = 0.5-8 μg/mL) by light irradiation. The new chemical tools enable optical control of fungal growth arrest, morphological conversion and biofilm formation. The ability for high-precision antifungal treatment was validated by in vivo models. The light-activated compound A1 was comparable to fluconazole in prolonging survival in Galleria mellonella larvae with a median survival of 14 days and reducing fungal burden in the mouse skin infection model. Overall, this study paves the way for precise regulation of antifungal therapy with improved efficacy and safety.

De novo transcriptome assembly and metabolomic analysis of three tissue types in Cinnamomum cassia / 中草药·英文版

OBJECTIVE@#The barks, leaves, and branches of Cinnamomum cassia have been historically used as a traditional Chinese medicine, spice, and food preservative, in which phenylpropanoids are responsible compounds. However phenylpropanoid biosynthesis pathways are not clear in C. cassia. We elucidated the pathways by descriptive analyses of differentially expressed genes related to phenylpropanoid biosynthesis as well as to identify various phenylpropanoid metabolites.@*METHODS@#Chemical analysis, metabolome sequencing, and transcriptome sequencing were performed to investigate the molecular mechanisms underlying the difference of active components content in the barks, branches and leaves of C. cassia.@*RESULTS@#Metabolomic analysis revealed that small amounts of flavonoids, coumarine, and cinnamaldehyde accumulated in both leaves and branches. Transcriptome analysis showed that genes associated with phenylpropanoid and flavonoid biosynthesis were downregulated in the leaves and branches relative to the barks. The observed differences in essential oil content among the three tissues may be attributable to the differential expression of genes involved in the phenylpropanoid and flavonoid metabolic pathways.@*CONCLUSION@#This study identified the key genes in the phenylpropanoid pathway controling the flavonoid, coumarine, and cinnamaldehyde contents in the barks, branches and leaves by comparing the transcriptome and metabolome. These findings may be valuable in assessing phenylpropanoid and flavonoid metabolites and identifying specific candidate genes that are related to the synthesis of phenylpropanoids and flavonoids in C. cassia.

Advances in physiological activities and synthesis of β-nicotinamide mononucleotide / 生物工程学报

Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and β isomer is its active form. Studies have shown that β-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of β-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize β-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of β-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of β-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of β-NMN.

Research progress on triterpenoids of Betula plants / 药学学报

The secondary metabolites of plants are important sources of natural drugs. Betula plants have abundant pharmacological value, complex mechanism and wide applications, which are closely related to the triterpenoids of theirs. Triterpenoids in Betula species are mainly divided into dammarane-type, ocotillol-type, oleanane-type, lupane-type and cycloaltunane-type. The extracts of Betula species have varieties of activities such as anti-tumor, anti-inflammatory, anti-oxidant, anti-bacterial, etc. And the biosynthetic pathways of triterpenoids after 2,3-oxidosqualene are split into four branches of dammarenediol-II, lupeol, cycloartenol and amyrin according to the different oxidosqualene cyclases. This review summarizes the chemical constituents, pharmacological activities and biosynthetic pathways of triterpenoids in Betula plants. It provides a reference for the research and development of new drugs and the production of these triterpenoids in microbial cell factories by synthetic biology methods.

Pyridine pigments from functional Monascus rice / 药学学报

The trace chemical components in functional Monascus rice were studied to explore the potential bioactive substances. MCI column, Sephadex LH-20 gel, and preparative liquid chromatography were used to purified the ethyl acetate extract from functional Monascus rice. Two novel pyridine Monascus pigments were isolated and identified, named monascopyridine G (1) and monascopyridine H (2), respectively based on extensive mass spectrometry (MS), infrared radiation (IR), and nuclear magnetic resonance (NMR) analysis. The molecular docking experiments between compounds 1 and 2 and peroxisome proliferators-activated receptor-gamma (PPARγ) showed that they exhibited obvious binding force with the receptor protein. Besides, the biosynthetic pathways of the two compounds were proposed, which provide a valuable reference for the selective production of these potential bioactive substances.

SmHPPR1 from Salvia miltiorrhiza regulated the biosynthesis of salvianolic acids / 药学学报

italic>Salvia miltiorrhiza Bunge is a traditional Chinese medicinal herb widely used to treat cardiovascular and cerebrovascular diseases at clinic. Its main water-soluble components are rosmarinic acid (RA) and salvianolic acid B (SAB), which are produced by phenylpropanoid pathway. 4-Hydroxyphenylpyruvate reductase (HPPR) is a key enzyme in phenylpropanoid metabolism pathway. SmHPPR1 was cloned from S. miltiorrhiza and was constructed into plant expression vector pJR-SmHPPR1. On this basis, SmHPPR1 transgenic Arabidopsis plants were induced and the content of 4-hydroxyphenyllactic acid (pHPL) was determined. SmHPPR1-overexpressing (SmHPPR1-OE) hairy roots of S. miltiorrhiza were obtained and the concentration of active components and transcriptome analysis were performed. The results showed that the concentration of pHPL in SmHPPR1 transgenic Arabidopsis T1 was 0.594 mg·g-1 dry weight. The concentration of RA, SAB and total salvianolic acid in SmHPPR1-OE-3 hairy roots were 1.09, 1.29, 1.15 times of that in control-3, respectively, and the content of Danshensu was 36.26% of that in control-3. Transcriptomic analysis revealed that overexpression of SmHPPR1 caused the upregulation of other phenylpropanoid pathway genes like SmTAT2. Protein-protein interaction indicated CYT (TR74706_c0_g1), NADP+ (TR26565_c0_g1) and NADP+ (TR68771_c0_g1) is the central node of the network and participated in metabolic process and cellular process. The tracking work in this study proved that SmHPPR1 could catalyze the reduction of 4-hydroxyphenylpyruvic acid to 4-hydroxyphenyllactic acid in SmHPPR1 transgenic Arabidopsis, and SmHPPR1-overexpressing in hairy roots of S. miltiorrhiza could increase the concentration of salvianolic acids through synergistically regulating other pathway genes.

Dihydrofolate reductase-like protein inactivates hemiaminal pharmacophore for self-resistance in safracin biosynthesis

Dihydrofolate reductase (DHFR), a housekeeping enzyme in primary metabolism, has been extensively studied as a model of acid-base catalysis and a clinic drug target. Herein, we investigated the enzymology of a DHFR-like protein SacH in safracin (SAC) biosynthesis, which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance. Furthermore, based on the crystal structure of SacH-NADPH-SAC-A ternary complexes and mutagenesis, we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore. These findings expand the functions of DHFR family proteins, reveal that the common reaction can be catalyzed by distinct family of enzymes, and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.

Insights into the biosynthesis of septacidin l-heptosamine moiety unveils a VOC family sugar epimerase

l-Heptopyranoses are important components of bacterial polysaccharides and biological active secondary metabolites like septacidin (SEP), which represents a group of nucleoside antibiotics with antitumor, antifungal, and pain-relief activities. However, little is known about the formation mechanisms of those l-heptose moieties. In this study, we deciphered the biosynthetic pathway of the l,l-gluco-heptosamine moiety in SEPs by functional characterizing four genes and proposed that SepI initiates the process by oxidizing the 4'-hydroxyl of l-glycero-α-d-manno-heptose moiety of SEP-328 ( 2) to a keto group. Subsequently, SepJ (C5 epimerase) and SepA (C3 epimerase) shape the 4'-keto-l-heptopyranose moiety by sequential epimerization reactions. At the last step, an aminotransferase SepG installs the 4'-amino group of the l,l-gluco-heptosamine moiety to generate SEP-327 ( 3). An interesting phenomenon is that the SEP intermediates with 4'-keto-l-heptopyranose moieties exist as special bicyclic sugars with hemiacetal-hemiketal structures. Notably, l-pyranose is usually converted from d-pyranose by bifunctional C3/C5 epimerase. SepA is an unprecedented monofunctional l-pyranose C3 epimerase. Further in silico and experimental studies revealed that it represents an overlooked metal dependent-sugar epimerase family bearing vicinal oxygen chelate (VOC) architecture.

Functional characterization of a cycloartenol synthase and four glycosyltransferases in the biosynthesis of cycloastragenol-type astragalosides from Astragalus membranaceus

Astragalosides are the main active constituents of traditional Chinese medicine Huang-Qi, of which cycloastragenol-type glycosides are the most typical and major bioactive compounds. This kind of compounds exhibit various biological functions including cardiovascular protective, neuroprotective, etc. Owing to the limitations of natural sources and the difficulties encountered in chemical synthesis, re-engineering of biosynthetic machinery will offer an alternative and promising approach to producing astragalosides. However, the biosynthetic pathway for astragalosides remains elusive due to their complex structures and numerous reaction types and steps. Herein, guided by transcriptome and phylogenetic analyses, a cycloartenol synthase and four glycosyltransferases catalyzing the committed steps in the biosynthesis of such bioactive astragalosides were functionally characterized from Astragalus membranaceus. AmCAS1, the first reported cycloartenol synthase from Astragalus genus, is capable of catalyzing the formation of cycloartenol; AmUGT15, AmUGT14, AmUGT13, and AmUGT7 are four glycosyltransferases biochemically characterized to catalyze 3-O-xylosylation, 3-O-glucosylation, 25-O-glucosylation/O-xylosylation and 2'-O-glucosylation of cycloastragenol glycosides, respectively. These findings not only clarified the crucial enzymes for the biosynthesis and the molecular basis for the structural diversity of astragalosides in Astragalus plants, also paved the way for further completely deciphering the biosynthetic pathway and constructing an artificial pathway for their efficient production.

Late-stage cascade of oxidation reactions during the biosynthesis of oxalicine B in Penicillium oxalicum

Oxalicine B ( 1) is an α-pyrone meroterpenoid with a unique bispirocyclic ring system derived from Penicillium oxalicum. The biosynthetic pathway of 15-deoxyoxalicine B ( 4) was preliminarily reported in Penicillium canescens, however, the genetic base and biochemical characterization of tailoring reactions for oxalicine B ( 1) has remained enigmatic. In this study, we characterized three oxygenases from the metabolic pathway of oxalicine B ( 1), including a cytochrome P450 hydroxylase OxaL, a hydroxylating Fe(II)/α-KG-dependent dioxygenase OxaK, and a multifunctional cytochrome P450 OxaB. Intriguingly, OxaK can catalyze various multicyclic intermediates or shunt products of oxalicines with impressive substrate promiscuity. OxaB was further proven via biochemical assays to have the ability to convert 15-hydroxdecaturin A ( 3) to 1 with a spiro-lactone core skeleton through oxidative rearrangement. We also solved the mystery of OxaL that controls C-15 hydroxylation. Chemical investigation of the wild-type strain and deletants enabled us to identify 10 metabolites including three new compounds, and the isolated compounds displayed potent anti-influenza A virus bioactivities exhibiting IC50 values in the range of 4.0-19.9 μmol/L. Our studies have allowed us to propose a late-stage biosynthetic pathway for oxalicine B ( 1) and create downstream derivatizations of oxalicines by employing enzymatic strategies.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica / 中西医结合学报

OBJECTIVE@#AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.@*METHODS@#To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.@*RESULTS@#One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses.@*CONCLUSION@#This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

A Comprehensive Review on Cytoskeletal Organization and Its Interaction with Polysaccharides and Enzymes during Primary Cell Wall Development

A well-coordinated process is required to construct a complicated structure like the cell wall, which consists of several elements that must be joined appropriately from various sources inside the cell. In order to successfully moderate dynamic responses to developmental and environmental signals, further complexity is necessary. The plasma membrane is continually and actively transporting sugars, enzymes, and other cell wall elements throughout diffused development. Actin filaments and microtubules make up the cytoskeletal pathways used to transport cell wall elements in vesicles during cell division. In addition to these elements, other proteins, vesicles and lipids are transported from and to the cell plate while cytokinesis occurs. Adding additional cell wall material or building a new cell wall requires a rearrangement of the cytoskeleton, which we examine in this review first. We next look at the commonalities between these two processes. Our next topic is the transport of cell wall-building polysaccharides and enzymes via motor proteins and other interactions with the cytoskeleton. Final thoughts on cytokinesis-generated cell walls include a look at some of their unique properties.

Improving the production of conjugated linoleic acid from sunflower oil by lactic acid bacteria spp: Effect of calcium carbonate supplementation in fermentation medium

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid with various positional and geometric isomers of linoleic acid (LA) present mainly in food items and produced endogenously in non-ruminants and humans or through fermentation process. It is associated with health-beneficial effects and subject to more research on its natural sources (ruminant-derived foods) and strategies to increase the content in various foodstuffs. Although several studies have reported the most common intake value of 0.8 g/day (0.6 to 3.0 g/day), research for raising in situ concentration should focus on strategies such as in vitro bioconversion of its precursors by bacteria and supplementation of LA rich-oils in foods fermentation process. In this study, the ability of some lactic acid bacteria (LAB) from diverse samples to produce CLA from sunflower oil and the effect on production yield of fermentation medium supplementation with carbonate calcium were investigated. Results showed that ten Limosilactobacillus fermentum and Enterococcus faecium produced trans-10, cis-12-CLA isomer accounted for at least 85% of total CLA ranging from 4.64 to 5.22 µg/mL. Despite the fermentation medium supplementation with CaCO3 enhanced the production yield, the residual LA inhibitory effect on bacteria growth governing CLA biosynthesis process was not mitigated. So, although our LAB strains can produce CLA, the more the LA concentration goes up, the more the conversion rate downgrades. Further studies on strains behavior in a wide range of LA concentrations will help establish a stable relationship between bacteria and LA in the presence of CaCO3.

Effects of Cholesterol-lowering Agents on Proliferation, Invasion and Neutrophil Extracellular Trap Formation in Liver Cancer Cells / 肿瘤防治研究

Objective To investigate the effects of cholesterol-lowering agents on the proliferation, stemness characters, migration, invasion, and neutrophil extracellular traps formation (NETs) formation in liver cancer cells. Methods ASPP2 or HMGCR gene was knocked down in mouse liver cancer cell Hepa1-6 to establish cells with high or low cholesterol, respectively. Simvastatin and berberine were used to reduce cholesterol synthesis. CCK-8 and plate cloning assays were conducted to detect the proliferation ability of liver cancer cells. Sphere formation assay and qRT-PCR were used to analyze the stemness character and expression of related genes. Wound-healing assay and Transwell assay were used to analyze the ability of cell migration and invasion. Immunofluorescence staining was carried out to analyze the effect of lipid-lowering agent on NETs formation. Results Cholesterol-lowering agents significantly inhibited the proliferation and stemness-related gene expression of Hepa1-6 cells (P < 0.001), significantly inhibited the migration and invasion of Hepa1-6 cells (P < 0.001), and significantly inhibited the neutrophil-induced invasion and formation of NETs (P < 0.001). Conclusion Cholesterol-lowering agents suppress the proliferation and invasion via inhibiting the stemness characters and NETs formation in liver cancer cells. It is a potential strategy for the treatment of liver cancer metastasis.

Construction of an l-cysteine hyper-producing strain of Escherichia coli based on a balanced carbon and sulfur module strategy / 生物工程学报

l-cysteine is an important sulfur-containing α-amino acid. It exhibits multiple physiological functions with diverse applications in pharmaceutical cosmetics and food industry. Here, a strategy of coordinated gene expression between carbon and sulfur modules in Escherichia coli was proposed and conducted for the production of l-cysteine. Initially, the titer of l-cysteine was improved to (0.38±0.02) g/L from zero by enhancing the biosynthesis of l-serine module (serAf, serB and serCCg) and overexpression of CysB. Then, promotion of l-cysteine transporter, increased assimilation of sulfur, reduction or deletion of l-cysteine and l-serine degradation pathway and enhanced expression of cysEf (encoding serine acetyltransferase) and cysBSt (encoding transcriptional dual regulator CysB) were achieved, resulting in an improved l-cysteine titer (3.82±0.01) g/L. Subsequently, expressions of cysM, nrdH, cysK and cysIJ genes that were involved in sulfur module were regulated synergistically with carbon module combined with utilization of sulfate and thiosulfate, resulting in a strain producing (4.17±0.07) g/L l-cysteine in flask shake and (11.94±0.1) g/L l-cysteine in 2 L bioreactor. Our results indicated that efficient biosynthesis of l-cysteine could be achieved by a proportional supply of sulfur and carbon in vivo. This study would facilitate the commercial bioproduction of l-cysteine.

Advances on microbial synthesis of L-proline and trans-4-hydroxy-L-proline / 生物工程学报

L-proline (L-Pro) is the only imino acid among the 20 amino acids that constitute biological proteins, and its main hydroxylated product is trans-4-hydroxy-L-proline (T-4-Hyp). Both of them have unique biological activities and play important roles in biomedicine, food and beauty industry. With the in-depth exploration of the functions of L-Pro and T-4-Hyp, the demand for them is gradually increasing. Traditional methods of biological extraction and chemical synthesis are unable to meet the demand of "green, environmental protection and high efficiency". In recent years, synthetic biology has developed rapidly. Through the intensive analysis of the synthetic pathways of L-Pro and T-4-Hyp, microbial cell factories were constructed for large-scale production, which opened a new chapter for the green and efficient production of L-Pro and T-4-Hyp. This paper reviews the application and production methods of L-Pro and T-4-Hyp, the metabolic pathways for microbial synthesis of L-Pro and T-4-Hyp, and the engineering strategies and advances on microbial production of L-Pro and T-4-Hyp, aiming to provide a theoretical basis for the "green bio-manufacturing" of L-Pro and T-4-Hyp and promote their industrial production.