A comprehensive evaluation of stable expression "hot spot" in the ScltI gene of Chinese hamster ovary cells.

The Chinese hamster ovary (CHO) cell is the most widely used biopharmaceutical expression system, but its long-term expression is unstable. This issue can be effectively addressed by site-specific integration of exogenous genes into the genome. Therefore, exogenous protein sites with stable expression in the CHO cell genome must be identified. CRISPR/Cas9 technology was used in this study to integrate various exogenous genes into the ScltI site as a "hot spot" at the CHO-K1 cell genome NW_003614095.1, and the stability and adaptability of exogenous genes expressed at the site were investigated. Flow cytometry sorting technology was used to obtain positive monoclonal cell lines that expressed either intracellular protein green fluorescent protein (EGFP) or secretory protein human serum albumin (HSA). For 60 passages, the positive monoclonal cell lines' cell growth cycles and exogenous protein expression were both observed. The results demonstrated that integrating the gene encoding exogenous proteins into the ScltI site had no effect on cell growth. The fluorescence intensity of EGFP was similar after 60 passages, and the expression of HSA increased slightly. Additionally, the super-monomeric protein VWF hydrolase (ADAMTS13) (190 kDa), human coagulation factor VII (FVII) (55 kDa), and interferon α2b (12 kDa) were integrated into the ScltI site for expression. In conclusion, the site located in the first exon of the ScltI gene within the CHO-K1 cell genome NW_003614095.1 is an ideal "hot spot" for the stable expression of various exogenous proteins. KEY POINTS: • The site-specific integration strategy of an exogenous gene in CHO cells was established for the ScltI site. • The genes for EGFP and HSA were site-directed integrated and stably expressed at the ScltI site. • The ScltI site fulfills the expression of exogenous proteins of different molecular weight sizes (15-190 kDa).

Genome-wide analysis of cell-Free DNA methylation profiling with MeDIP-seq identified potential biomarkers for colorectal cancer.

BACKGROUND: Colorectal cancer is the most common malignancy and the third leading cause of cancer-related death worldwide. This study aimed to identify potential diagnostic biomarkers for colorectal cancer by genome-wide plasma cell-free DNA (cfDNA) methylation analysis. METHODS: Peripheral blood from colorectal cancer patients and healthy controls was collected for cfDNA extraction. Genome-wide cfDNA methylation profiling, especially differential methylation profiling between colorectal cancer patients and healthy controls, was performed by methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq). Logistic regression models were established, and the accuracy of this diagnostic model for colorectal cancer was verified using tissue-sourced data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) due to the lack of cfDNA methylation data in public datasets. RESULTS: Compared with the control group, 939 differentially methylated regions (DMRs) located in promoter regions were found in colorectal cancer patients; 16 of these DMRs were hypermethylated, and the remaining 923 were hypomethylated. In addition, these hypermethylated genes, mainly PRDM14, RALYL, ELMOD1, and TMEM132E, were validated and confirmed in colorectal cancer by using publicly available DNA methylation data. CONCLUSIONS: MeDIP-seq can be used as an optimal approach for analyzing cfDNA methylomes, and 12 probes of four differentially methylated genes identified by MeDIP-seq (PRDM14, RALYL, ELMOD1, and TMEM132E) could serve as potential biomarkers for clinical application in patients with colorectal cancer.

A multi-conformational virtual screening approach based on machine learning targeting PI3Kγ.

Nowadays, more and more attention has been attracted to develop selective PI3Kγ inhibitors, but the unique structural features of PI3Kγ protein make it a very big challenge. In the present study, a virtual screening strategy based on machine learning with multiple PI3Kγ protein structures was developed to screen novel PI3Kγ inhibitors. First, six mainstream docking programs were chosen to evaluate their scoring power and screening power; CDOCKER and Glide show satisfactory reliability and accuracy against the PI3Kγ system. Next, virtual screening integrating multiple PI3Kγ protein structures was demonstrated to significantly improve the screening enrichment rate comparing to that with an individual protein structure. Last, a multi-conformational Naïve Bayesian Classification model with the optimal docking programs was constructed, and it performed a true capability in the screening of PI3Kγ inhibitors. Taken together, the current study could provide some guidance for the docking-based virtual screening to discover novel PI3Kγ inhibitors.

Structural insights to heterodimeric cis-prenyltransferases through yeast dehydrodolichyl diphosphate synthase subunit Nus1.

The polyprenoid glycan carriers are produced by cis-prenyltransferases (cis-PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista and archaea. Heterodimeric cis-PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis-PT shows low sequence similarity to known cis-PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis-PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis-PTs.

Crystal structure and proposed mechanism of an enantioselective hydroalkoxylation enzyme from Penicillium herquei.

Hydroalkoxylation is a useful and efficient reaction which generates C-O bond and produces cyclic ethers, the common structural elements of natural products. The dedicative enzyme which can catalyze enantioselective hydroalkoxylation named PhnH was recently identified in the herqueinone biosynthetic gene from Penicillium herquei. It catalyzes addition of a phenol to the terminal olefin on substrate to produce a dihydrobenzofuran. Here, the crystal structure of PhnH is reported and the putative substrate-binding pocket is illustrated. Through docking experiment, possible substrate-binding poses are displayed and the catalytic mechanism is therefore proposed. Our findings form the basis for further studies of enantioselective hydroalkoxylation enzymes.

Fine-tuning the (2S)-naringenin synthetic pathway using an iterative high-throughput balancing strategy.

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry-fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl3 and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.

Site-specific integration of light chain and heavy chain genes of antibody into CHO-K1 stable hot spot and detection of antibody and fusion protein expression level.

Expression cell line constructed by random integration method will often meet with unstable expression problem because target genes may be integrated into unstable region of chromatin. Rational cell line construction can overcome this shortcoming by inserting target gene into stable region of chromatin specifically. Here, we successfully got one knock-in cell line where light chain and heavy chain genes of antibody was site specifically integrated into stable hot spot reported before via homologous dependent recombination method mediated by CRISPR/Cas9. The targeting efficiency was around 1.35%. This cell line together with other three pre-established targeting cell lines (targeting with glucagon-like peptide 1 with human serum albumin fusion protein gene, or NGGH) were all undergoing protein expression level detection. In adherent cell mode, the amount of antibody expressed per cell per day were all around 0.006 pg/cell/day over passage 3, 12, 23, 35 and 50 while the amount of NGGH expressed per cell per day of 3 cell lines were all around 1.2 pg/cell/day over passage 3, 12, 23, 35 and 50. In batch mode, the antibody concentration within supernatant were around 2.5 µg/L over passage 1, 25, and 50 while the NGGH fusion protein concentration within supernatant were around 17 mg/L over passage 1, 25, and 50.

Impact of mycolic acid deficiency on cells of Corynebacterium glutamicum ATCC13869.

Mycolic acid (MA) plays important role in Corynebacterium glutamicum, but the key enzymes in the biosynthetic pathway of MA in C. glutamicum ATCC13869 have not been characterized. Since the locus BBD29_RS14045 in C. glutamicum ATCC13869 shows high similarity to the gene Cgl2871, which encodes Pks13, the key enzyme for synthesizing MA in C. glutamicum ATCC13032, it was deleted, resulting in the mutant WG001. Compared with the wild-type ATCC13869, MA was not synthesized in WG001, but more phosphatidylglycerol and phosphatidylinositol containing longer unsaturated fatty acids were produced. WG001 cells also show hindered cell growth and defective cell separation when compared with ATCC13869 cells. Transcriptomic analysis shows that many genes relevant to the pathways of fatty acids, inositol, phospholipids, cell wall, and cell division were significantly regulated in WG001 cells when compared with ATCC13869 cells. This study demonstrates that the locus BBD29_RS14045 encodes a key enzyme that plays important role for synthesizing MA in C. glutamicum ATCC13869.

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA-IGF-1 reached 100 mg/L. The fusion protein HSA-IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA-IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA-IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA-IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

The application of powerful promoters to enhance gene expression in industrial microorganisms.

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.

Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-ß-endoglucanase.

Eukaryotic 1,4-ß-endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications because they effectively catalyze hydrolysis of cellulose, the main component of the plant cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4-ß-endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris, which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (ß/α)8-TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to -4 - -1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N-glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme.

Effects of co-overexpression of secretion helper factors on the secretion of a HSA fusion protein (IL2-HSA) in pichia pastoris.

Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2-HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2-HSA to ~2.3-, 1.9-, 2.2-, 2.5- and 1.9-fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd.

Trends of imported malaria in China 2010-2014: analysis of surveillance data.

BACKGROUND: To describe the epidemiologic profile and trends of imported malaria, and to identify the populations at risk of malaria in China during 2010-2014. METHODS: This is a descriptive analysis of laboratory confirmed malaria cases during 2010-2014. Data were obtained from surveillance reports in the China Information System for Disease Control and Prevention (CISDCP). The distribution of imported malaria cases over the years was analysed with X(2) for trend analysis test. All important demographic and epidemiologic variables of imported malaria cases were analysed. RESULTS: Malaria incidence in general reduced greatly in China, while the proportion of Plasmodium falciparum increased threefold from 0.08 to 0.21 per 100,000 population during the period 2010-2014. Of a total 17,725 malaria cases reported during the study period, 11,331 (64%) were imported malaria and included an increasing trend: 292 (6%), 2103 (63%), 2151 (84%), 3881 (96%), 2904 (97%), respectively, (X(2) = 2110.70, p < 0.01). The majority of malaria cases (imported and autochthonous) were adult (16,540, 93%), male (15,643, 88%), and farming as an occupation (11,808, 66%). Some 3027 (94%) of imported malaria cases had labour-related travel history during the study period; 90% (6340/7034) of P. falciparum infections were imported into China from Africa, while 77% of Plasmodium vivax infections (2440/3183) originated from Asia. CONCLUSIONS: Malaria elimination in China faces the challenge of imported malaria, especially imported P. falciparum. Malaria prevention activities should target exported labour groups given the increasing number of workers returning from overseas.

Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis.

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 K m value of MT-S9N/-A11T/-E518V decreased by 25.4 % compare to that of wild-type, while the activity, k cat/K m, 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.

Expression of a glucagon-like peptide-1 analogue, as a therapeutic agent for type II diabetes, with enhanced bioactivity and increased N-terminal homogeneity in Pichia pastoris.

OBJECTIVE: To improve the bioactivity and increase the N-terminal homogeneity of a glucagon-like peptide-1 (GLP-1) analogue expressed in Pichia pastoris. RESULTS: The GLP-1 analogue. GGH, consisting of two tandem mutant GLP-1 (GLP-1[A2G]) fused with the N-terminus of human serum albumin (HSA), was expressed in P. pastoris. We also designed and expressed the novel GLP-1 analogue NGGH, which had a His-tag fused with the N-terminus of GGH and an enterokinase (EK) cleavage site at the fusion junction. The His-tag was removed by EK digestion to yield GGH2, which was subsequently compared with GGH expressed in P. pastoris. The purification recovery of GGH2 was 35 % compared with 23 % for GGH. Furthermore, the bioactivity of GGH2 was 605 % higher than GGH, and N-terminal homogeneity was also improved. CONCLUSIONS: A simple method for the preparation of GGH2 with a cleavable His-tag was developed, and the resultant protein possessed improved bioactivity and N-terminal homogeneity.

[Impact on prevalence of schitosomiasis after runs of Three Gorges Reservoir Project in the section of Anhui province].

OBJECTIVE: To investigate the changes of water level and the distribution of snails in Anhui province before and after runs of the Three Gorges Reservoir Project, and to determine the relationship between the two factors and schistosomiasis transmission. METHODS: The hydrologic data of Datong hydrologic station and the data of snail status and schistosomiasis morbidity in Anhui Province were collected. The data from 1991 to 2002 and 2003 to 2012 were considered as before and after the impoundment of the Three Gorges Reservoir Project. Based on the prevalence of schistosomiasis, the cases of people and cattle were speculated, and the average infection rate of people and cattle were calculated. The t-test was used to compare the difference of snail area and the density of living snails before and after the impoundment of the Three Gorges Project. The pearson method was used to analyze the relationship between water level and snail area. The spearman method was used to analyze the relationship between the water level and the distribution of snails. RESULTS: From 1991 to 2012, the range of the highest water level, the lowest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons, the mean in the dry water seasons, and the difference between the abundant water seasons and the dry water seasons was 11.40-16.30, 3.68-5.20, 6.70-12.12, 9.92-14.40, 4.77-7.64 and 4.13-8.93 m, respectively. The snail areas was (28 613 ± 362) hm² and (29 477 ± 918) hm² (t = -3.00, P = 0.007), the density of living snails was 1.51 (1.15-2.43) and 0.43 (0.29-1.10) snails/0.11 m² (H = 4.28, P < 0.001) before and after the impoundment of the Three Gorges Project, respectively. The average infection rate of people and cattle was 1.68% (99 482/5 935 147) and 4.62% (13 923/3 011 33), and the average number of acute schistosomiasis cases was 328, before the impoundment of the Three Gorges Project; 0.60% (39 747/6 649 380), 1.65% (1 291/783 224) and 71 after the impoundment of the Three Gorges Reservoir Project, respectively. The snail areas had negative correlation with the highest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons (r value was -0.514, -0.509 and -0.477; P value was 0.014, 0.015 and 0.025, respectively). The infection rate of people had positive correlation with the highest water level, the difference between the highest and lowest water level, the mean in the abundant water seasons (r value was 0.532, 0.587 and 0.446; P value was 0.011, 0.004 and 0.038, respectively). The infection rate of cattle had positive correlation with the highest water level, the difference between the highest and lowest water level (r value was 0.507 and 0.553; P value was 0.016 and 0.008, respectively). The number of acute schistosomiasis cases had positive correlation with the highest water level, the difference between the highest and lowest water level (r value was 0.481 and 0.486; P value was 0.023 and 0.022, respectively). CONCLUSION: Following the runs of the Three Gorges Reservoir Project, the change of water level in the section of Anhui Province affected the distribution of snails and the infection of people and cattle to some extent. The snail areas showed an upward trend, and the density of living snails, the infection rate of people and cattle showed a downward trend. The runs of Three Gorges Reservoir Project has certain role to reduce flood and helpful for schistosomiasis control.

The rebalanced pathway significantly enhances acetoin production by disruption of acetoin reductase gene and moderate-expression of a new water-forming NADH oxidase in Bacillus subtilis.

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639g/(lh).

Surveillance systems for neglected tropical diseases: global lessons from China's evolving schistosomiasis reporting systems, 1949-2014.

Though it has been a focus of the country's public health surveillance systems since the 1950s, schistosomiasis represents an ongoing public health challenge in China. Parallel, schistosomiasis-specific surveillance systems have been essential to China's decades-long campaign to reduce the prevalence of the disease, and have contributed to the successful elimination in five of China's twelve historically endemic provinces, and to the achievement of morbidity and transmission control in the other seven. More recently, an ambitious goal of achieving nation-wide transmission interruption by 2020 has been proposed. This paper details how schistosomiasis surveillance systems have been structured and restructured within China's evolving public health system, and how parallel surveillance activities have provided an information system that has been integral to the characterization of, response to, and control of the disease. With the ongoing threat of re-emergence of schistosomiasis in areas previously considered to have achieved transmission control, a critical examination of China's current surveillance capabilities is needed to direct future investments in health information systems and to enable improved coordination between systems in support of ongoing control. Lessons drawn from China's experience are applied to the current global movement to reduce the burden of helminthiases, where surveillance capacity based on improved diagnostics is urgently needed.

Moderate expression of the transcriptional regulator ALsR enhances acetoin production by Bacillus subtilis.

Acetoin, a major extracellular catabolic product of Bacillus subtilis cultured on glucose, is widely used to add flavor to food and also serves as a precursor for chemical synthesis. The biosynthesis of acetoin from pyruvate requires the enzymes α-acetolactate synthase (ALS) and α-acetolactate decarboxylase (ALDC), both of which are encoded by the alsSD operon. The transcriptional regulator ALsR is essential for the expression of alsSD. Here we focused on enhancing the production of acetoin by B. subtilis using different promoters to express ALsR. The expression of reporter genes was much higher under the control of the HpaII promoter than under control of the P bdhA promoter. Although the HpaII promoter highly enhanced transcription of the alsSD operon through overexpression of ALsR, the production of acetoin was not significantly increased. In contrast, moderate enhancement of ALsR expression using the P bdhA promoter significantly improved acetoin production. Compared with the wild-type, the enzyme activities of ALS and ALDC in B. subtilis harboring P bdhA were increased by approximately twofold, and the molar yield of acetoin from glucose was improved by 62.9 % in shake flask fermentation. In a 5-L fermentor, the engineered B. subtilis ultimately yielded 41.5 g/L of acetoin. Based on these results, we conclude that enhanced expression of ALDC and ALS by moderately elevated expression of the transcriptional regulator ALsR could increase acetoin production in recombinant B. subtilis.

Mutation breeding of acetoin high producing Bacillus subtilis blocked in 2,3-butanediol dehydrogenase.

Bacillus subtilis mutants were obtained after the wild strain JNA 3-10 was mutagenized by UV irradiation coupled with diethyl sulfate. A visual filter assay was employed for the qualitative identification of 2,3-butanediol dehydrogenase (BDH) blocked B. subtilis. Selected mutants were tested for the activities of acetoin reductase (AR) and BDH. According to further batch fermentation, one mutant named JNA-UD-6 that produced 24.3 % more acetoin than JNA 3-10 with the corresponding byproducts of 2,3-butanediol decreased by 39.8 % was isolated. A nonsense mutation (p.Tyr118X) that precluded the synthesis of a full-length functional AR/BDH within the bdhA gene of JNA-UD-6 was detected. Acetoin production of JNA-UD-6 was further improved to about 53.9 g/L in a 5-L fermentor with 150 g/L glucose consumed. However,a small amount of 2,3-butanediol was found in late phase of JNA-UD-6 fermentation, and it was due to the existence of a putative gene that encoding a minor AR. This work proved a strategy to efficiently breeding an acetoin high producing strain by traditional mutation methods.