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• The promoter PbacA was proven as the most efficient promoter for iturin A synthesis. • Deletion of regulator gene abrB improved iturin A synthetase gene cluster transcription and iturin A production. • The titer of 2013.43 mg/L iturin A was produced by optimizing fermentation medium formulas, increased by 392.15 %. • Enhancing the synthetic capability of iturin A was beneficial for the suppression of A. alternate. Brown spot disease, caused by Alternaria alternate , is one of the most destructive leaf spot diseases that made a major impact on tobacco growing. Biocontrol has attracted increasing attentions as its features of environmental friendship, promoting plant growth, etc. Iturin A, with broad spectrum antifungal activity, is a kind of biosurfactant that mainly produced by Bacillus. In this study, the promoter of iturin A synthetase cluster of Bacillus amyloliquefaciens HZ-12 was respectively replaced by promoters P43, P bacA , P srfA and P ylb , our results implied that transcriptional level of gene ituD and iturin A titer showed consistent change trends, and P bacA was proven as the most efficient promoter, iturin A titer of which reached 950.08 ± 19.43 mg/L. Furthermore, regulator gene abrB was deleted to release the repression effect of AbrB on P bacA , ituD transcriptional level and iturin A titer were increased by 133.25 % and 20.88 %, respectively. Then, the maximum iturin A titer reached 2013.43 ± 32.86 mg/L by optimizing fermentation medium, increased by 392.15 % compared to the original (408.97 ± 21.35 mg/L). Finally, our results demonstrated that enhancing iturin A synthetic capability benefited the suppression of A. alternate. Collectively, this study provided a promising strain with an efficient fermentation medium for large-scale industrial production of iturin A. [ABSTRACT FROM AUTHOR]

[Display omitted] • Achieving cost-effective bioproduction from starch by engineered B. licheniformis. • Modular metabolic engineering to improve starch utilization in B. licheniformis. • Starch conversion rate was increased by over 22% in multiple products production. • The highest bacitracin titer of 1252.2 U/mL was achieved in batch culture. Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a multifunctional biopolymer mainly produced by Bacillus. The cofactor specificity of enzymes plays a critical role in regulating metabolic process and metabolite production. Here, we present a novel approach for switching cofactor specificity of glutamate dehydrogenase RocG from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) to improve γ-PGA production. Firstly, 3D structural modeling and molecular docking were performed to predict the binding modes of NADH and NADPH. Several site-specific mutants based on the conventional and Random Accelerated Molecular Dynamics simulations were obtained to alter cofactor specificity. Then, the effects of RocG variants overexpressions on γ-PGA production were evaluated. Compared to the wild-type, the mutant RocGD276E showed highest increase in γ-PGA yield, increased by 40.50%. Meanwhile, yields of main by-products acetoin and 2,3-butandieol were decreased by 21.70% and 16.53%, respectively. Finally, the results of enzymatic properties confirmed that glutamate dehydrogenase mutant RocGD276E exhibited the higher affinity for NADH, caused a shift in coenzyme preference from NADPH to NADH, with a catalytic efficiency comparable with NADPH-dependent RocG. Taken together, this research demonstrated that switching the cofactor preference of glutamate dehydrogenase via rational design was an effective strategy for high-level production of γ-PGA in Bacillus licheniformis. [Display omitted] • Three-dimension structure of B. licheniformis RocG was attained by homology modeling. • Potential mutation sites of RocG were scoped by cMD and RAMD simulations. • The specificity of RocG was altered to NADH-dependent by rational enzyme design. • Cofactor engineering was proven as an effective strategy for increasing γ-PGA production. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a natural and multifunctional biopolymer. Glycerol, especially crude glycerol, was used as fermentation raw material for production of a variety of chemical products. Here, glycerol was used with nitrate addition for γ-PGA production by Bacillus licheniformis WX-02 at a 5 L fermenter scale. However, nitrite accumulation was discovered in batch fermentation, which resulted in low γ-PGA yield (19.58 g/L). Furthermore, based on transcription analysis and intracellular NADH measurement, nitrite accumulation was probably due to the low content of intracellular NADH in glycerol-based medium, which might be resulted from low efficiency of NADH generation rate and quick NADH consumption by nitrate reduction. Accordingly, different nitrate feeding strategies were designed and optimized in γ-PGA fermentation with glycerol, then applied in fermentation with crude glycerol. Finally, 35.63 g/L of γ-PGA was achieved with the constant-rate nitrate feeding of 0.38 g/(L h) by using crude glycerol, while 36.83 g/L of γ-PGA was achieved with pH-feedback controlling. Therefore, this study provided a novel strategy for cost-effective production of γ-PGA from crude glycerol. • Nitrite accumulation observed in glycerol medium resulted in low γ-PGA yield. • Intracellular NADH content was proven as the key factor for nitrite conversion. • 35 g/L γ-PGA was achieved by using optimized constant-rate nitrate feeding. • pH-feedback control reduced nitrate addition by half with 34 g/L γ-PGA production. • The nitrate feeding strategy was applied for γ-PGA production with crude glycerol. [ABSTRACT FROM AUTHOR]