Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10–45 % increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174 % in shake-flask and 81 % in bioreactor fermentations with a maximum yield of 454 mg l−1.

An industrial mutant of Streptomyces avermitilis produced avermectin at a high level in industrial complex culture medium. However, almost no avermectin was detected in the cultures of tryptone soya broth (TSB). Its wild-type strain could not synthesize avermectin. To elucidate the regulatory mechanism about avermectin production, proteomic analysis of S. avermitilis was carried out. Results showed that during avermectin biosynthesis, fatty acid metabolism and TCA cycle were repressed. Partial enrichment of glycolytic pathway indicated the critical role of glucose catabolism during avermectin biosynthesis. Some enriched enzymes in amino acid metabolic pathways (glnA, leuC) confirmed the leucine as the possible precursor of avermectin. Highly expressed stress or stress-related proteins indicated a global regulation mechanism at the onset of avermectin production. And highly expressed morphology control proteins revealed an association between hyphal morphology and avermectin production. Further, this study proofed strengthened capability to utilize carbon and nitrogen source in the industrial strain. Some stress or stress-related proteins (eshA, clpC, dnaK and grpE) expressed at low level in the industrial strain cultivated in non-production medium (lower than that in the wild-type strain), but these highly expressed at the onset of avermectin production. More sensible response to environmental stress may be responsible for it.To elucidate the regulatory mechanism about avermectin, proteomic comparison of the responses of S. avermitilis during avermectin production was carried out (show as follows).Highlights► Two strains of Streptomyces avermitilis were used for comparison. ► A global protein abundance analysis using 2DE-based proteomics were carried out. ► Significant protein modulation occurred in the comparison group In-P vs In-NP. ► The comparison group In-NP vs W-NP revealed some information about the mutation.

The coumarate:coenzyme A ligase 1 gene (4CL1) was isolated from secondary developing xylem tissues of Pinus massoniana Lamb (Pm4CL1). The Pm4CL1 gene was inserted into a pQE30 expression vector in both sense and antisense orientations to form a pQE30-4CL1-anti4CL1. The expression level of the Pm4CL1 gene in Escherichia coli cells was inhibited by the antisense mRNA of the Pm4CL1 gene. The results of reverse transcriptase polymerase chain reaction and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the average mRNA and protein expression levels of the Pm4CL1 gene in E. coli cells were decreased by 43 and 46%, respectively. These results are an important basis for further investigation.

The effects of vitamins (nicotinic acid, vitamin B1 and biotin) on the growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated by batch culture in this study. The results showed that nicotinic acid, as a precursor of NAD+/NADH, plays a crucial role in effectively enhancing the phototrophic hydrogen synthesis during photo-fermentation process. Lack of nicotinic acid in hydrogen production medium resulted in the failure of photo-hydrogen production. In addition, though vitamin B1 and biotin do not have direct impact on photo-hydrogen production, they are still essential and must exist in either growth medium or hydrogen production medium. Without either of them, photo-hydrogen production decreased seriously, regardless of the existence of nicotinic acid.Highlights► Vitamins in medium are important factors making effects on photo-H2 production. ► Nicotinic acid plays a crucial role in enhancing the phototrophic hydrogen synthesis. ► Without either of vitamin B1 and biotin, photo-H2 production decreases seriously.

In this study, a new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process by Rhodobacter sphaeroides ZX-5. In order to obtain the high hydrogen yield, photo-hydrogen production by fed-batch culture with on-line oxidation–reduction potential (ORP) feedback control was investigated. Meanwhile, the effects of feeding malic acid concentration and pH adjustment on the growth and hydrogen production of R. sphaeroides ZX-5 were studied. In the entire fed-batch culture, biomass (i.e., OD660) rapidly increased up to 1.79 within 18 h, and then OD660 value stayed constant within a range of 1.85–2.18 until the end of the photo-fermentation. The cumulative hydrogen volumes in each phase of fed-batch process were 2339, 1439, 1328, and 510 ml H2/l-culture, respectively. Throughout the entire repeated fed-batch photo-fermentation, the maximum substrate conversion efficiency of 73.03% was observed in the first fed-batch process, obviously higher than that obtained from batch culture process (59.81%). In addition, compared to the batch culture, a much higher maximum hydrogen production rate (102.33 ml H2/l h) was achieved during fed-batch culture. The results demonstrated that photo-hydrogen production using fed-batch operation based on ORP feedback control is a favorable choice of sustainable and feasible strategy to improve phototrophic hydrogen production efficiency.Highlights► The fed-batch culture mode based on ORP level has proved to be capable of attaining high-rate and high-yield phototrophic H2 production. ► The feeding time controlled by computer is conducive to future large-scale photo-hydrogen production. ► A new outer-cycle flat-panel photobioreactor was designed for an anaerobic, photo-fermentation process.

Biohydrogen has gained attention due to its potential as a sustainable alternative to conventional methods for hydrogen production. In this study, the effect of light intensity as well as cultivation method (standing- and shaking-culture) on the cell growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated in 38-ml anaerobic photobioreactor with RCVBN medium. Thus, a novel shaking and extra-light supplementation (SELS) approach was developed to enhance the phototrophic H2 production by R. sphaeroides ZX-5 using malate as the sole carbon source. The optimum illumination condition for shaking-culture by strain ZX-5 increased to 7000–8000 lux, markedly higher than that for standing-culture (4000–5000 lux). Under shaking and elevated illumination (7000–8000 lux), the culture was effective in promoting photo-H2 production, resulting in a 59% and 56% increase of the maximum and average hydrogen production rate, respectively, in comparison with the culture under standing and 4000–5000 lux conditions. The highest hydrogen-producing rate of 165.9 ml H2/l h was observed under the application of SELS approach. To our knowledge, this record is currently the highest hydrogen production rate of non-immobilized purple non-sulphur (PNS) bacteria. This optimal performance of photo-H2 production using SELS approach is a favorable choice of sustainable and economically feasible strategy to improve phototrophic H2 production efficiency.

In order to improve the productivity of vitamin B12 by Pseudomonas denitrificans carried out in a 120-m3 fermenter, the effect of pH on vitamin B12 biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B12. In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B12 production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B12 in a 120-m3 industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15–7.30 during fermentation. Thus, 214.3 μg/mL of vitamin B12 was achieved.

In reduced glutathione (GSH) production by high-cell-density fed-batch cultivation of Saccharomyces cerevisiae T65, a respiratory quotient (RQ) feedback control strategy was applied to determine glucose feeding rate (F) based on on-line off-gas monitoring in a 15-l stirred bioreactor. Glucose feed was manipulated by a classical proportional-integral-derivative (PID) controller to control RQ at its set-point. RQ controlled at 0.65 resulted in the highest GSH productivity (46.9 mg/l/h) and cell productivity (3.5 g/l/h) as well as improved GSH yield and intracellular GSH content. RQ feedback control achieved yeast dry weight (126 g/l), GSH yield (2.1 g/l), GSH content (1.67%) and GSH productivity (55.3 mg/l/h), which improved by 11.5%, 75%, 57.5% and 82.5%, respectively, compared with conventional ethanol feedback control in the GSH industry. Moreover, RQ feedback control reduced ethanol (byproduct) level to below 0.3 g/l. Advantages of RQ feedback control over the conventional ethanol feedback control in the GSH industry include lower byproduct concentration, shorter cultivation period, higher GSH content, and higher GSH yield. Hence, feedback control based on RQ is a promising method that poses as a simple and efficient alternative to conventional feed control techniques presently practiced in the GSH industry.

The methylotrophic yeast Pichia pastoris has become a popular host for the production of recombinant protein under the tight control of methanol-induced alcohol oxidase promoter (PAOX1), which is, however, strongly repressed by glucose at the transcription level. A real-time parameter-based controlled glucose feed strategy (rpcGFS) is developed here to realize Pichia high-cell density culture. It allows for the high-level production of recombinant phytase by P. pastoris YY113 using glucose as the growth substrate instead of glycerol. As a result, high phytase production (2200 FTU phytase ml−1) and yield (0.25 FTU ml−1[phytase] g−1[methanol]) are achieved successfully, respectively, after 100 h methanol induction by this novel simple fermentation strategy.

Oxytetracycline (OTC) is a widely used antibiotic, which is commercially produced by Streptomyces rimosus. The type II minimal polyketide synthases (minimal PKS) genes of the oxytetracycline biosynthesis cluster in S. rimosus, consisting of oxyA, oxyB and oxyC, are involved in catalyzing 19-C chain building by the condensation of eight malonyl-CoA groups to form the starting polyketide. This study aimed to investigate the effects of overexpression of the minimal PKS gene in a model S. rimosus strain (M4018) and in an industrial overproducer (SR16) by introduction of a second copy of the gene into the chromosome. Increased levels of oxyA, oxyB and oxyC gene transcription were monitored using reverse transcription quantitative real-time PCR. Overexpression of the minimal PKS gene elicited retardation of cell growth and a significant improvement in OTC production in corresponding mutants (approximately 51.2% and 32.9% in M4018 and SR16 mutants respectively). These data indicate that the minimal PKS plays an important role in carbon flux redirection from cell growth pathways to OTC biosynthesis pathways.Highlights► Duplication of minimal PKS genes in Streptomyces rimosus. ► Increased transcriptional levels of oxyA, oxyB and oxyC were observed. ► Phenotype changes and growth retardation were observed in mutants. ► Oxytetracycline production in the industrial strain mutant was improved. ► Transcriptional levels of a resistance gene otrB were upregulated in mutants.