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D-Pantothenic acid, as a momentous vitamin, is extensively applied to feed, medicine, cosmetics and other fields. However, there are still limitations to produce D-pantothenic acid by microbial fermentation at present. In this paper, we constructed a recombinant strain for D-pantothenic acid production by blocking the organic acid pathway, boosting pyruvate biosynthesis, relieving feedback inhibition of acetolactate synthase, improving glucose intake capacity, and modifying essential genes in the metabolic pathway. In addition, a new acetolactate isomeroreductase mutant V412A origin from Escherichia coli(EcAHAIR) encoded by ilvCwas obtained to explore its substrate promiscuity. Compared with the wild type, the variant EcAHAIR-V412A has reduced steric hindrance and enhanced intermolecular forces, resulting in a high affinity for 2-acetolactate. Eventually, the fermentation production of the final strain DPAN19/trc-ilvCV412Areached 4.65 g/L, increased by 192.5% compared with strain DPA8 in shake flask cultivation and produced 62.82 g/L D-pantothenic acid in a 5 L bioreactor. The metabolic engineering strategies and enzyme modification approaches described in this paper provide a particular perspective for the bio-manufacturing of D-pantothenic acid, branched-chain amino acids and its derivates.

The production of echinocandin B (ECB) by Aspergillus nidulansCCTCC M2012300 was improved by integrating the temperature-shift and fed-batch control strategies. The kinetic characteristics of batch cultures were analyzed at different culture temperatures, and then a two-stage temperature control strategy was established. In the first 6 days, the temperature was maintained at 30 °C to obtain the maximal cell growth rate; subsequently, 25 °C was used to gain a high ECB formation rate. On the basis of temperature control, the ECB productivity was increased to 143.3 mg/(L day), which was a 1.3-fold improvement compared with the optimal constant-temperature cultivations. The influences of fed-batch cultures were further investigated. A maximal ECB productivity of 170.8 mg/(L day) was obtained through a three-stage mannitol pulse-feeding strategy, which was another 1.2-fold improvement than that of the batch fermentation. This is the first report of the use of a two-stage temperature control fed-batch strategy in ECB fermentation. This strategy was simple and economical to operate and may provide new guidance for the industrial-scale production of ECB.

Microbial fermentation for L-methionine (L-Met) production based on natural renewable resources is attractive and challenging. In this work, the effects of medium composition and fermentation conditions were investigated to improve L-Met production by genetically engineered Escherichia coliMET-3. Statistical optimization techniques including Plackett–Burman (PB) design and Box–Behnken design (BBD) were adopted first to optimize the culture medium. Results of PB-designed experiments indicated that the culture medium components including glucose, yeast extract, KH2PO4, and MgSO4.7H2O had significant effects on L-Met biosynthesis. With their best-predicted concentration established by BBD (glucose 37.43 g/L, yeast extract 0.95 g/L, KH2PO41.82 g/L, and MgSO4.7H2O 4.51 g/L), L-Met titer was increased to 3.04 g/L from less than 2.0 g/L. For further enhancement of L-Met biosynthesis, the fermentation conditions of batch cultivation carried out in a 5-L fermentor were optimized, and the optimum results were obtained at an agitation rate of 300 rpm, medium pH of 7.0, and induction temperature of 28 °C. Based on the optimization parameters, fed-batch fermentation with the modified medium was conducted. As a result, great improvement of L-Met titer (12.80 g/L) and yield (0.13 mol/mol) were achieved, with an increase of 38.53% and 30.0% compared with those of the basal medium, respectively. Furthermore, higher L-Met productivity of 0.261 g/L/h was obtained, representing 2.13-fold higher in comparison to the original medium. The results may provide a helpful reference for further study on strain improvement and fermentation control.

O-succinyl-l-homoserine (OSH) is a promising platform chemical for the production of C4 chemicals with huge market potential which can be produced by fermentation from glucose. To construct a strain capable of producing OSH with high yield, the metJ(encodes transcriptional repressor) and metI(encodes a subunit of dl-methionine transporter) were deleted in Escherichia coliW3110 to obtain a strain E. coli∆JI. Then, overexpression of metL(encodes bifunctional aspartate kinase/homoserine dehydrogenase II) and inactivation of metB(encodes cystathionine γ-synthase) were implemented in one step, and the OSH titer of the resulting strain E. coli∆JIB*TrcmetLwas dramatically increased to 7.30 g/L. The feedback regulation was further relieved by progressively overexpressing metAfbr(encodes homoserine O-succinyltransferase), yjeH(encodes l-methionine exporter), and thrAfbr(encodes bifunctional aspartate kinase/homoserine dehydrogenase I) to increase the metabolic flux from aspartate to OSH. The 100% rationally designed strain E. coli∆JIB*TrcmetL/pTrc-metAfbr-Trc-thrAfbr-yjeHproduced 9.31 g/L OSH from 20 g/L glucose (0.466 g/g glucose) in batch fermentation, which represents the highest OSH yield from glucose reported to date. The culture profiles of the newly constructed strains were recorded to investigate their productive properties. The effects of l-methionine addition on the fermentation process of the optimal strain were also studied. Our results demonstrate that tuning the expression level of metL, inactivation of metB, and attenuation of feedback resistance of the crucial enzymes in the biosynthetic pathway are the key factors that impact the OSH production in E. coli.

2-chloronicotinic acid (2-CA) is a key precursor for the synthesis of a series of pesticides and pharmaceuticals. Nitrilase-catalyzed bioprocess is a promising method for 2-CA production from 2-chloronicotinonitrile (2-CN). In this study, a mutant of nitrilase from Rhodococcus zopfii(RzNIT/W167G) was constitutively overexpressed with Escherichia colias host, which exhibited a onefold increase in enzymatic activity compared with inducible expression. Biosynthesis of 2-CA using whole cells harboring nitrilase as biocatalysts were investigated and 318.5 mM 2-CA was produced, which was the highest level for 2-CA production catalyzed by nitrilase to date. 2-CA was recovered from the reaction mixture through a simple acidification step with a recovery yield of 90%. This study developed an efficient bioprocess for 2-CA with great potential for industrial application.

Microbial fermentation for enzyme production and then whole-cell catalysis for l-2-aminobutyric acid (l-ABA) production have huge potential for industrial application, but the catalytic capacities of cells are directly related to the fermentation process. Using a 50 L fermenter, the effects of initial glycerol concentration in the medium and rotating speed on cell catalytic capacity were investigated. Fermentation cells showed the best catalytic activity when the initial glycerol concentration was 12 g/L and the rotating speed was 250 rpm. Furthermore, we studied the difference between glycerol and glycerol mixtures as fed-batch media in pH–stat fed-batch fermentation. Results showed that glycerol had better catalytic activity than the glycerol mixture, and the effect of fed-batch fermentation was better than batch fermentation. Meanwhile, the enzyme activities of leucine dehydrogenase and formate dehydrogenase reached 129.87 U/g DCW and 437.02 U/g DCW, respectively, and the intracellular NAD(H) concentration reached 14.94 μmol/g DCW. Using the optimized fermentation parameters, amplified fermentation was then carried out in a 5000 L fermenter to demonstrate the industrial production of l-ABA by Escherichia coliBL21.

Amphotericin, as an important macrolide antibiotic, is synthesized by Streptomyces nodosus. A high-yield S. nodosusZJB2016050 was obtained by mutagenesis in our lab with the advantages of high yield, short fermentation cycle and few by-products, which was more suitable for industrial production. The fermentation differences in 50-tons bioreactor between S. nodosusATCC14899 and S. nodosusZJB2016050 were compared. The amphotericin B (AmB) yield of S. nodosusZJB2016050 was 9.73 mg/g at 96 h, which was 30% higher than that of S. nodosusATCC14899. The by-product amphotericin A (AmA) production of S. nodosusZJB2016050 was 78% lower than that of S. nodosusATCC14899. By performing whole-genome sequencing of S. nodosusZJB2016050 and comparative genome analysis with the wild-type S. nodosusATCC14899, it was found that the two strains have high synteny, but each has a special gene fragment. The genes functions of fragment were identified in the amino acid transport and metabolism, carbohydrate metabolism and lipid transport and metabolism. The gene functions of SNP (single nucleotide polymorphism) genes were identified in amino acid transport and metabolism, carbohydrate metabolism, coenzyme metabolism and secondary metabolites biosynthesis. The difference in signal-regulation and transcription may be the main reason for the differences between these two strains. Three GntR family egulatory factors of S. nodosusATCC14899 may reduce the synthesis of amphotericin. Based on the analysis of comparative genomes, the effects of corn oil in S. nodosusATCC14899 and S. nodosusZJB2016050 were also compared. The results showed that corn oil can promote the fermentation of S. nodosusZJB2016050. The S. nodosusZJB2016050 may degrade fatty acids faster, and the degraded acyl-coenzyme can be used to synthesize amphotericin.

Different carbon sources lead to differential acarbose production in Actinoplanes. To uncover the underlying differentiation in the context of genes and pathways, we performed transcriptome sequencing of Actinoplanes utahensisZJB-03852 grown on different saccharides, such as glucose, maltose, or the saccharide complex consisting of glucose plus maltose. The differentially expressed genes were classified into GO (gene ontology) terms and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways for functional annotations. Key enriched modules were uncovered. Our data revealed that both maltose and its complex with glucose gave improved acarbose titer. Sugar transportation, cytochrome oxidase, protein synthesis and amino acid metabolism modules were enriched under the saccharide complex condition, while ferritin metabolism gene expressions were enriched in the glucose medium. Our results provided the foundation for uncovering the mechanism of carbon source on acarbose production in A. utahensis.

Gibberellic acid (GA3) is a natural plant growth hormone that has been widely used in agriculture and horticulture. To obtain higher GA3producing strains, the method of screening the strains for resistance to simvastatin was used after treatment with nitrosoguanidine (NTG) and gamma rays. The rationale for the strategy was that mutants showing simvastatin resistance were likely to be high GA3producers, as their activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is relatively more effective. GA3yield of mutant S109 increased by 14.2% than that of the original strain. The GA3production ability in mutant S109 remained relatively stable after ten generations. With the addition of 0.4 g glycerol on the 5th day during the fermentation process in Erlenmeyer flask, maximum GA3production of 2.7 g/L was achieved by this mutant, exhibiting 28.6% increase compared with original strain. Furthermore, we also achieved 2.8 g/L GA3and had a 33.3% increase with addition 20 g glycerol on the 5th day during the fermentation process in a 5-L bioreactor. Our results indicated efficient GA3production could be achieved on the condition that the supply of glycerol at the suitable conditions. This study would lay a foundation for industrial production of GA3.