A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5′-UTR complexes (PUTR) from E. coli were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the PBAD promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5′-UTRs. Finally, two combinatorial PUTRs (PssrA-UTRrpsT and PdnaKJ-UTRrpsT) and two cascade PUTRs (PUTRssrA-PUTRinfC-rplT and PUTRalsRBACE-PUTRinfC-rplT) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the PBAD promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products.Keywords: 5′-UTR; metabolic engineering; promoter engineering; RNA-seq; RT-qPCR; synthetic biology;

Ubiquitination can significantly affect the endocytosis and degradation of plasma membrane proteins. Here, the ubiquitination of a Saccharomyces cerevisiae urea plasma membrane transporter (Dur3p) was altered. Two potential ubiquitination sites, lysine residues K556 and K571, of Dur3p were predicted and replaced by arginine, and the effects of these mutations on urea utilization and formation under different nitrogen conditions were investigated. Compared with Dur3p, the Dur3pK556R mutant showed a 20.1% decrease in ubiquitination level in yeast nitrogen base medium containing urea and glutamine. It also exhibited a >75.8% decrease in urea formation in yeast extract–peptone–dextrose medium and 41.3 and 55.4% decreases in urea and ethyl carbamate formation (a known carcinogen), respectively, in a model rice wine system. The results presented here show that the mutation of Dur3p ubiquitination sites could significantly affect urea utilization and formation. Modifying the ubiquitination of specific transporters might have promising applications in rationally engineering S. cerevisiae strains to efficiently use specific nitrogen sources.Keywords: nitrogen catabolite repression; nitrogen sources; post-translational modifications; site-directed mutagenesis; urea transporter;

Production of useful chemicals by industrial microorganisms has been attracting more and more attention. Microorganisms screened from their natural environment usually suffer from low productivity, low stress resistance, and accumulation of by-products. In order to overcome these disadvantages, rational engineering of microorganisms to achieve specific industrial goals has become routine. Rapid development of metabolic engineering and synthetic biology strategies provide novel methods to improve the performance of industrial microorganisms. Rational regulation of gene expression by specific promoters is essential to engineer industrial microorganisms for high-efficiency production of target chemicals. Identification, modification, and application of suitable promoters could provide powerful switches at the transcriptional level for fine-tuning of a single gene or a group of genes, which are essential for the reconstruction of pathways. In this review, the characteristics of promoters from eukaryotic, prokaryotic, and archaea microorganisms are briefly introduced. Identification of promoters based on both traditional biochemical and systems biology routes are summarized. Besides rational modification, de novo design of promoters to achieve gradient, dynamic, and logic gate regulation are also introduced. Furthermore, flexible application of static and dynamic promoters for the rational engineering of industrial microorganisms is highlighted. From the perspective of powerful promoters in industrial microorganisms, this review will provide an extensive description of how to regulate gene expression in industrial microorganisms to achieve more useful goals.

Candida glabrata is one of the most promising pyruvate producers. A series of experiments was conducted to enhance pyruvate production by C. glabrata via metabolic and genetic engineering. Here, a novel screening strategy, which combined atmospheric and room temperature plasma-based random mutagenesis and high-throughput screening (HTS), was used to screen for high pyruvate-producing mutants that could use cheap industrial raw materials as nitrogen sources. A high-titer pyruvate producer (H6) was obtained form 30,000 mutants after 30 rounds of mutagenesis and HTS. Compared with a wild-type strain, pyruvate production by the H6 mutant was 32.2 and 35.4% higher in 500-mL shake flasks and 3-L fermenter, respectively, when cheap peptone was used as the nitrogen source in the seed culture stage. The HTS process significantly improved the screening efficiency and reduced fermentation cost. This procedure could also easily be applied to screen for strains that produce high titers of similar organic acids.

In the phenylpropanoid production process, p-coumaric acid is the most important intermediate metabolite. It is generally accepted that the activity of tyrosine ammonia-lyase (TAL), which converts l-tyrosine to p-coumaric acid, represents the rate-limiting step. Therefore, an error-prone PCR-based random mutagenesis strategy was utilized for screening variants with higher catalytic activity. After rounds of screening, three variant enzymes were obtained, exhibiting improved production rates of 41.2, 37.1, and 38.0 %, respectively. Variants associated with increased expression level (S9N), improved catalytic efficiency (A11T), and enhanced affinity between TAL and L-tyrosine (E518V) were identified as beneficial amino acid substitutions by site-directed mutagenesis. Combining all of the beneficial amino acid substitutions, a variant, MT-S9N/-A11T/-E518V, exhibiting the highest catalytic activity was obtained. The Km value of MT-S9N/-A11T/-E518V decreased by 25.4 % compare to that of wild-type, while the activity, kcat/Km, and p-coumaric-acid yield were improved by 36.5, 31.2, and 65.9 %, respectively. Furthermore, the secondary structure of the 5′-end of MT-S9N mRNA and the three-dimensional protein structure of MT-E518V were modeled. Therefore, two potential mechanisms were speculated: (1) a simplified mRNA 5′-end secondary structure promotes TAL expression and (2) anchoring the flexible loop region (Glu325–Arg336) to maintain the active-site pocket opening ensures easy access by the l-tyrosine to the active site and thus improves p-coumaric acid yields.

α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle and has broad applications. The mitochondrial ketoglutarate dehydrogenase (KGDH) complex catalyzes the oxidation of α-KG to succinyl-CoA. Disruption of KGDH, which may enhance the accumulation of α-KG theoretically, was found to be lethal to obligate aerobic cells. In this study, individual overexpression of dihydrolipoamide succinyltransferase (DLST), which serves as the inner core of KGDH, decreased overall activity of the enzyme complex. Furthermore, two conserved active site residues of DLST, His419, and Asp423 were identified. In order to determine whether these residues are engaged in enzyme reaction or not, these two conserved residues were individually mutated. Analysis of the kinetic parameters of the enzyme variants provided evidence that the catalytic reaction of DLST depended on residues His419 and Asp423. Overexpression of mutated DLST not only impaired balanced assembly of KGDH, but also disrupted the catalytic integrity of the enzyme complex. Replacement of the Asp423 residue by glutamate increased extracellular α-KG by 40 % to 50 g L−1 in mutant strain. These observations uncovered catalytic roles of two conserved active site residues of DLST and provided clues for effective metabolic strategies for rational carbon flux control for the enhanced production of α-KG and related bioproducts.

During bioproduction of short-chain carboxylates, a shift in pH is a common strategy for enhancing the biosynthesis of target products. Based on two-dimensional gel electrophoresis, comparative proteomics analysis of general and mitochondrial protein samples was used to investigate the cellular responses to environmental pH stimuli in the α-ketoglutarate overproducer Yarrowia lipolytica WSH-Z06. The lower environmental pH stimuli tensioned intracellular acidification and increased the level of reactive oxygen species (ROS). A total of 54 differentially expressed protein spots were detected, and 11 main cellular processes were identified to be involved in the cellular response to environmental pH stimuli. Slight decrease in cytoplasmic pH enhanced the cellular acidogenicity by elevating expression level of key enzymes in tricarboxylic acid cycle (TCA cycle). Enhanced energy biosynthesis, ROS elimination, and membrane potential homeostasis processes were also employed as cellular defense strategies to compete with environmental pH stimuli. Owing to its antioxidant role of α-ketoglutarate, metabolic flux shifted to α-ketoglutarate under lower pH by Y. lipolytica in response to acidic pH stimuli. The identified differentially expressed proteins provide clues for understanding the mechanisms of the cellular responses and for enhancing short-chain carboxylate production through metabolic engineering or process optimization strategies in combination with manipulation of environmental conditions.

•Membrane proteomes of E. coli under different phenylpropanoids were analyzed.•16 differentially expressed membrane proteins were identified by MALDI-TOF/TOF MS.•Effect of 14 proteins on E. coli were studied by gene overexpression and silencing.Phenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. These compounds exist in plants in low amounts. Production of them in genetically engineered microorganisms has many advantages. A majority of functional phenylpropanoids are toxic to microbial hosts. Export of these compounds may relieve the cellular toxicity and increase the yield. However, proteins and mechanisms involved in phenylpropanoids transport and tolerance remain poorly understood. In this study, 16 membrane proteins that were differentially expressed in Escherichia coli in response to three typical phenylpropanoids (resveratrol, naringenin and rutin) were identified using a membrane proteomics approach. These proteins included outer membrane proteins OmpA, OmpF, OmpW, FadL, TolC, LamB, and YaeT, peripheral membrane proteins AtpD, AtpH, YgaU, OppA, MalK, and MalE, and cytoplasmic membrane proteins OppD, PotG, and ManX. Functions of these proteins were determined by using gene overexpression and silencing. The results suggest that OmpA and FadL may play important roles in the transmembrane export of phenylpropanoids in E. coli. LamB, MalE, MalK and ManX may participate in phenylpropanoid uptake. The role of YgaU in enhancing the tolerance to phenylpropanoids remains to be determined. These results may assist the engineering of microorganisms with enhanced phenylpropanoid producing capabilities.Biological significancePhenylpropanoids are phytochemicals produced by some plants and possess a wide variety of biological activities. Both the tolerance and the transport of phenylpropanoids play important roles in systematic metabolic engineering of microorganisms to produce these phytochemicals. Both specific and non-specific transporters are essential for these functions but remain poorly understood. This research utilized membrane proteomics to identify E. coli BL21 (DE3) membrane proteins that may be involved in phenylpropanoid transport and tolerance. These results may facilitate the construction of more efficient microbial phenylpropanoid producers through genetic engineering of membrane transporter proteins.

•Proteome of Saccharomyces cerevisiae with different nitrogen sources were analyzed.•169 differentially expressed proteins were detected by 2-DE, 121 of them were identified by MALDI-TOF/TOF.•The result provides clues that how yeast adapt to different nutritional conditions.•Proteomics data were compared with previous reported corresponding mRNA dataIn cultures containing multiple sources of nitrogen, Saccharomyces cerevisiae exhibits a sequential use of nitrogen sources through a mechanism known as nitrogen catabolite repression (NCR). To identify proteins differentially expressed due to NCR, proteomic analysis of S. cerevisiae S288C under different nitrogen source conditions was performed using two-dimensional gel electrophoresis (2-DE), revealing 169 candidate protein spots. Among these 169 protein spots, 121 were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF). The identified proteins were closely associated with four main biological processes through Gene Ontology (GO) categorical analysis. The identification of the potential proteins and cellular processes related to NCR offer a global overview of changes elicited by different nitrogen sources, providing clues into how yeast adapt to different nutritional conditions. Moreover, by comparing our proteomic data with corresponding mRNA data, proteins regulated at the transcriptional and post-transcriptional level could be distinguished.Biological significanceIn S. cerevisiae, different nitrogen sources provide different growth characteristics and generate different metabolites. The nitrogen catabolite repression (NCR) process plays an important role for S. cerevisiae in the ordinal utilization of different nitrogen sources. NCR process can result in significant shift of global metabolic networks. Previous works on NCR primarily focused on transcriptomic level. The results obtained in this study provided a global atlas of the proteome changes triggered by different nitrogen sources and would facilitate the understanding of mechanisms for how yeast could adapt to different nutritional conditions.

Yarrowia lipolytica WSH-Z06 harbours a promising capability to oversynthesize α-ketoglutarate (α-KG). Its wide utilization is hampered by the formation of high concentrations of pyruvate. In this study, a metabolic strategy for the overexpression of the α and β subunits of pyruvate dehydrogenase E1, E2 and E3 components was designed to reduce the accumulation of pyruvate. Elevated expression level of α subunit of E1 component improved the α-KG production and reduced the pyruvate accumulation. Due to a reduction in the acetyl-CoA supply, neither the growth of cells nor the synthesis of α-KG was restrained by the overexpression of β subunit of E1, E2 and E3 components. Furthermore, via the overexpression of these thiamine pyrophosphate (TPP)-binding subunits, the dependency of pyruvate dehydrogenase on thiamine was diminished in strains T1 and T2, in which α and β subunits of E1 component were separately overexpressed. In these two recombinant strains, the accumulation of pyruvate was insensitive to variations in exogenous thiamine. The results suggest that α-KG production can be enhanced by altering the dependence on TPP of pyruvate dehydrogenase and that the competition for the cofactor can be switched to ketoglutarate dehydrogenase via separate overexpression of the TPP-binding subunits of pyruvate dehydrogenase. The results presented here provided new clue to improve α-KG production.

The katA gene that encodes catalase (CAT) in Bacillus subtilis WSHDZ-01 was overexpressed in B. subtilis WB600 and B. subtilis WSHDZ-01. The CAT yield in both transformed strains was significantly improved compared to that in the wild-type WSHDZ-01 in shake flask culture. When cultured in a 3-L stirred tank reactor (STR), the recombinant CAT activity in B. subtilis WSHDZ-01 could be improved by 419 %, reaching up to 39,117 U/mL and was 8,149.4 U/mg dry cell weight, which is the highest activity reported in Bacillus sp. However, the recombinant CAT in B. subtilis WB600 cultured in a 3-L STR was not significantly improved by any of the common means for process optimization, and the highest CAT activity was 3,673.5 U/mg dry cell weight. The results suggest that self-cloning of the complete expression cassette in the original strain is a reasonable strategy to improve the yield of wild-type enzymes.

α-Cyclodextrin glycosyltransferase is a key enzyme in the cyclodextrin industry. The Gram-positive bacterium Bacillus megaterium was chosen for production of recombinant α-CGTase for safety concerns. Successful production of heterologous α-CGTase was achieved by adapting the original α-cgt gene to the codon usage of B. megaterium by systematic codon optimization. This balanced the tRNA pool and reduced ribosomal traffic jams. Protein expression and secretion was ensured by using the strong inducible promoter Pxyl and the signal peptide SPLipA. The impact of culture medium composition and induction strategies on α-CGTase production was systematically analyzed. Production and secretion at 32 °C for 24 h using modified culture medium was optimal for α-CGTase yield. Batch- and simple fed-batch fermentation was applied to achieve a high yield of 48.9 U·mL–1, which was the highest activity reported for a Bacillus species, making this production system a reasonable alternative to Escherichia coli.

•A simple procedure used for the fast detection of protein ubiquitination was constructed.•The optimal fluorescent condition and spectrum of reassociated EGFP was determined.•The reliability of this procedure was verified by an independent method.We established a simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae. Enhanced green fluorescent protein (EGFP) was split into two parts, an N-terminal (GN) and a C-terminal (GC) region. The fusion fragments GN-UBI3 and multi-cloning site (MCS)-GC were inserted into the vector pY26-TEF/GPD, resulting in pUbDetec16. pUbDetec16 was designed for use in detecting protein ubiquitination. Any gene of interest can be inserted into the MCS and the recombinant plasmid can be transferred into a Δura3 auxotrophic S. cerevisiae strain. Protein ubiquitination can then be detected using a fluorescence microscope. The ubiquitination of a protein can be determined based on a fluorescence signal. To validate the reliability of this procedure, Gap1p, a protein known to be ubiquitinated, was used as a positive control. A triple mutant of Gap1p, Gap1pK9R,K16R,K76R, which did not contain any ubiquitination site, was used as a negative control. pUbDetec16-GAP1 and pUbDetec16-GAP1K9R,K16R,K76R were constructed and transferred into the Δura3 auxotrophic S. cerevisiae strain CEN.PK2-1D. Transformants of pUbDetec16-GAP1 emitted fluorescence, while the pUbDetec16-GAP1K9R,K16R,K76R transformants did not. The ubiquitination of Gap1p and Gap1pK9R, K16R, K76R was further verified using classical SDS–PAGE analysis. This procedure significantly simplifies manipulation involving ubiquitination detection using the BiFC approach, particularly on a large scale.

•Production of plant natural products by fermentation processes.•Metabolic engineering of microorganisms to produce more diverse natural products.•Synthetic biology devices as tools for manufacturing plant natural products.•Novel technologies that could be applied to improve the processes.Microbial production of plant natural products (PNPs), such as terpenoids, flavonoids from renewable carbohydrate feedstocks offers sustainable and economically attractive alternatives to their petroleum-based production. Rapid development of metabolic engineering and synthetic biology of microorganisms shows many advantages to replace the current extraction of these useful high price chemicals from plants. Although few of them were actually applied on a large scale for PNPs production, continuous research on these high-price chemicals and the rapid growing global market of them, show the promising future for the production of these PNPs by microorganisms with a more economic and environmental friendly way. Introduction of novel pathways and optimization of the native cellular processes by metabolic engineering of microorganisms for PNPs production are rapidly expanding its range of cell-factory applications. Here we review recent progress in metabolic engineering of microorganisms for the production of PNPs. Besides, factors restricting the yield improvement and application of lab-scale achievements to industrial applications have also been discussed.Download high-res image (292KB)Download full-size image

•Single colonies of R. delemar could be formed by addition of sodium deoxycholate.•Transformation of R. delemar could be achieved by using of fresh germinated spores.•Pretreatment of spores with LiAc and DTT improved the transformation efficiency.High efficient transformation of mycelial fungi is essential to both metabolic engineering and physiological analysis of these industrially important microorganisms. However, transformation efficiencies for mycelial fungi are highly restricted by difficulties in colony formation and competent cell preparation. In this work, an innovative transformation procedure that could significantly improve the efficiency of colony formation and transformation process has been established for a typical mycelial fungus, Rhizopus delemar. Single colonies of R. delemar were obtained with the addition of sodium deoxycholate. Fresh germinated spores of R. delemar were successfully transformed by electroporation. In addition, by pretreatment of the germinated spores with 0.05 M lithium acetate (LiAc) and 20 mM dithiothreitol (DTT) before electroporation, the transformation efficiency was further improved by 9.5-fold. The final transformation efficiency at optimal conditions reached 1239 transformants/μg DNA. The method described here would facilitate more efficient metabolic engineering and investigation of physiological functions in R. delemar or other similar mycelial fungi.

•The mechanisms of formation of EC in alcoholic beverages were illuminated.•This traditional methods as well as several new methods of EC elimination were introduced.•The advantages and disadvantages of mentioned methods were compared.•Various issues are addressed relating to the development of measures to eliminate EC.Ethyl carbamate (EC) is a potentially carcinogenic compound that is widely found in alcoholic beverages. Because of its toxicity, carcinogenicity, and universality, EC is currently one of the biggest challenges in the alcoholic beverages industry. Many methods for reducing the EC level have been investigated, incorporating physical, chemical, enzymatic, and metabolic engineering technologies. This review focuses on the traditional methods as well as several new methods. Furthermore, by comparing the advantages and disadvantages of these methods, various issues are addressed relating to the development of measures to eliminate EC in alcoholic beverages on a laboratory scale and an industrial scale.

•The polyphenolic compound resveratrol was produced from l-tyrosine in Escherichia coli.•A multivariate modular metabolic strategy was employed to balance the overall pathway for resveratrol production.•Current fermentation strategies relying on two separate step culture protocols were developed for using a single medium to perform resveratrol production.Microbial fermentations and bioconversion promise to revolutionize the conventional extraction of resveratrol from natural plant sources. However, the development of efficient and feasible microbial processes remains challenging. Current fermentation strategies often require supplementation of expensive phenylpropanoic precursors and two separate fermentation protocols, which are significantly more difficult and expensive to undertake when migrating to large-scale fermentation processes. In this study, an Escherichia coli fermentation system, consisting of tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), stilbene synthase (STS), malonate synthetase, and malonate carrier protein, was developed to produce resveratrol from l-tyrosine. Multivariate modular metabolic engineering, which redefined the overall pathway as a collection of distinct modules, was employed to assess and alleviate pathway bottlenecks. Using this strategy, the optimum strain was capable of producing 35.02 mg/L of resveratrol from l-tyrosine in a single medium. The strategy described here paves the way to the development of a simple and economical process for microbial production of resveratrol and other similar stilbene chemicals.Download full-size image

•High-throughput screening combined with random mutagenesis screened varied mutants.•Comparative genomic analysis was performed to identify gene variation.•Partial mechanisms of α-KG accumulation were demonstrated in Y. lipolytica mutants.Yarrowia lipolytica is one of the most intensively investigated α-ketoglutaric acid (α-KG) producers, and metabolic engineering has proven effective for enhancing production. However, regulation of α-KG metabolism remains poorly understood. Genetic engineering of new strains is accompanied by potential safety concerns in some countries and regions. A series of mutants with varied capacity for α-KG production were obtained using random mutagenesis of Y. lipolytica WSH-Z06. Comparative genomics analysis was implemented to identify genes candidates associated with α-KG production. Manipulation of genes regulating mitochondrial biogenesis and energy metabolism could improve α-KG production, while genes involved in regulating transformation between keto acids and amino acids may decrease production. One gene associated with cell cycle control well represented in all mutants, whereas this gene involved in cell concentration do not appear to influence α-KG production. The results shed light on α-KG production in eukaryotic cells, and pave the way for a high-throughput screening and random mutagenesis method for enhancing α-KG production.

•An efficient de novo one-step production process of (2S)-pinocembrin was established.•A stepwise modular engineering approach was developed to optimize the pathway.•Reducing mRNA structure can be used to improve heterologous gene expression.Developing efficient microbial processes to produce flavonoids has been a metabolic engineering goal over the past decade due to their important functions. Previously, the de novo production of the main flavonoid precursor (2S)-pinocembrin was achieved. However, low productivity and two separate fermentation steps made it inappropriate for industrial scale low-cost production. Here, a stepwise modular engineering approach was introduced to systematically identify and eliminate metabolic pathway bottlenecks. The overall pathway was firstly divided into four modules and analysis then revealed that efficient conversion of l-phenylalanine to (2S)-pinocembrin is the major limiting factor. Therefore, the pathway from l-phenylalanine to (2S)-pinocembrin was re-cast into three modules to alleviate this bottleneck by modifying both the gene copy number and promoter strength. Furthermore, the expression of the rate-limiting enzyme PAL was found to be correlated with 5′ region of mRNA structure. The efficiency of the synthetic pathway was then improved by customizing the PAL expression level based on modification of the mRNA secondary structure. Fed-batch cultivation of engineered strains in a 3-L fermentor resulted in a final (2S)-pinocembrin production of 432.4 mg/L. The results presented here pave the way for the development of an economical and simple process for microbial production of flavonoids.