Improving glucose oxidase catalysis in Aspergillus niger via Vitreoscilla hemoglobin fusion protein.

Oxygen is crucial for converting glucose to gluconic acid catalyzed by glucose oxidase (Gox). However, industrial gluconic acid production faces oxygen supply limitations. To enhance Gox efficiency, Vitreoscilla hemoglobin (VHb) has been considered as an efficient oxygen transfer carrier. This study identified GoxA, a specific isoform of Gox in the industrial gluconic acid-producing strain of Aspergillus niger. Various forms of VHb expression in A. niger were tested to improve GoxA's catalytic efficiency. Surprisingly, the expression of free VHb, both intracellularly and extracellularly, did not promote gluconic acid production during shake flask fermentation. Then, five fusion proteins were constructed by linking Gox and VHb using various methods. Among these, VHb-GS1-GoxA, where VHb's C-terminus connected to GoxA's N-terminus via the flexible linker GS1, demonstrated a significantly higher Kcat/Km value (96% higher) than GoxA. Unfortunately, the expression of VHb-GS1-GoxA in A. niger was limited, resulting in a low gluconic acid production of 3.0 g/L. To overcome the low expression problem, single- and dual-strain systems were designed with tools of SpyCatcher/SpyTag and SnoopCatcher/SnoopTag. In these systems, Gox and VHb were separately expressed and then self-assembled into complex proteins. Impressively, the single-strain system outperformed the GoxA overexpression strain S1971, resulting in 23% and 9% higher gluconic acid production under 0.6 vvm and 1.2 vvm aeration conditions in the bioreactor fermentation, respectively. The successful construction of Gox and VHb fusion or complex proteins, as proposed in this study, presents promising approaches to enhance Gox catalytic efficiency and lower aerodynamic costs in gluconic acid production. KEY POINTS: • Overexpressing free VHb in A. niger did not improve the catalytic efficiency of Gox • The VHb-GS1-GoxA showed an increased Kcat/Km value by 96% than GoxA • The single-strain system worked better in the gluconic acid bioreactor fermentation.

Engineering a Phosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in Aspergillus niger Enables a Significant Increase in Citric Acid Production.

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Effective production of kojic acid in engineered Aspergillus niger.

BACKGROUND: Kojic acid (KA) is a widely used compound in the cosmetic, medical, and food industries, and is typically produced by Aspergillus oryzae. To meet increasing market demand, it is important to optimize KA production through seeking alternatives that are more economic than current A. oryzae-based methods. RESULTS: In this study, we achieved the first successful heterologous production of KA in Aspergillus niger, an industrially important fungus that does not naturally produce KA, through the expression of the kojA gene from A. oryzae. Using the resulting KA-producing A. niger strain as a platform, we identified four genes (nrkA, nrkB, nrkC, and nrkD) that negatively regulate KA production. Knocking down nrkA or deleting any of the other three genes resulted in a significant increase in KA production in shaking flask cultivation. The highest KA titer (25.71 g/L) was achieved in a pH controlled batch bioreactor using the kojA overexpression strain with a deletion of nrkC, which showed a 26.7% improvement compared to the KA titer (20.29 g/L) that was achieved in shaking flask cultivation. CONCLUSION: Our study demonstrates the potential of using A. niger as a platform for studying KA biosynthesis and regulation, and for the cost-effective production of KA in industrial strain development.

Improved genetic transformation by disarmament of type II Restriction-Modification system in Streptococcus zooepidemicus.

Streptococcus zooepidemicus, group C Streptococci, is currently used for the industrial production of hyaluronic acid (HA). However, genetic manipulation of S. zooepidemicus is severely limited by its low transformation efficiency, which might be in part due to the Restriction-Modification (R-M) systems. The complete genome sequence of S. zooepidemicus ATCC39920 revealed the presence of two putative R-M systems, type I and type II. The putative type I R-M system is encoded by three closely linked genes: hsdR (SeseC_01315), hsdS, hsdM (SeseC_01318), and the putative type II R-M system consists of two closely linked genes: SeseC_02360 and yhdJ (SeseC_02362). Inactivation of hsdR, encoding the restriction endonuclease (REase) of the type I R-M system, showed no apparent effects on transformation efficiency, implying that disarmament of the type I R-M system alone is not sufficient for increasing transformation efficiency. However, inactivation of SeseC_02360, encoding the REase of the type II R-M system, improved transformation efficiency by 4.97 folds, indicating that type II R-M system is the major barrier that restricts genetic transformation in S. zooepidemicus. Furthermore, S. zooepidemicus strains lacking either of the two R-M systems are phenotypically indistinguishable from the wild-type in terms of cell growth and HA production. In summary, our study revealed that the type II R-M system is the main barrier to genetic transformation in S. zooepidemicus ATCC39920, and that the deletion of the type II R-M system renders S. zooepidemicus more transformable, thus facilitating metabolic engineering of this industrially important microorganism. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03227-x.

Is hyaluronic acid production transcriptionally regulated? A transcriptional repressor gene deletion study in Streptococcus zooepidemicus.

Hyaluronic acid (HA) is a multiple-function biopolymer that is widely used in food, cosmetic, and biomedical fields. In group C streptococci, the major workhorse of HA production in industry, the HA biosynthetic pathway has been proposed, while how HA synthesis is regulated is unclear. In this study, we identified twenty-five putative transcriptional repressors in S. zooepidemicus and studied whether they regulate HA synthesis or not. The individual gene deletion strain was firstly constructed, and the phenotypic changes of the corresponding deletion strains in stress tolerance and HA production were detected. The hrcA deletion strain is more sensitive to high temperature, and the rex deletion strain is more resistant to the oxidative stress. Three transcriptional repressor deletions resulted significantly decreased transcriptional levels of hasA, among which the scrR deletion strain shows most dramatical decrease in HA production. The regulatory mechanism of how ScrR affects the production of HA was further explored by transcriptional expression analysis of scrA and scrB, two direct target genes of ScrR regulon. Our results indicates that the deficiency of ScrR results in the unbalanced expression of scrA and scrB, which might also partly account for the decreasing production of HA. In agreement with the speculation, overexpression of scrB in ΔscrR genetic background results in 80% improvement in HA production. Taken together, the systemic genetic study of transcriptional repressors expands our understanding for the physiological regulation process of S. zooepidemicus and should help in the development of high-performance industrial strains for the efficient production of HA. KEY POINTS: • Twenty-two transcriptional repressor genes in S. zooepidemicus were deleted individually, and the phenotypes of corresponding mutants on a variety of conditions were characterized. • HrcA deficiency showed inferior cell tolerance to high temperature, and Rex deficiency showed superior cell tolerance to reactive oxygen stress, and four repressors deficiency showed inferior hyaluronic acid synthesis, among which the transcriptional levels of hasA of three mutants decreased significantly. • Optimizing sucrose metabolic flux can enhance hyaluronic acid synthesis significantly.

Development of an efficient pathway construction strategy for rapid evolution of the biodegradation capacity of Pseudomonas putida KT2440 and its application in bioremediation.

In this work, we developed an efficient pathway construction strategy, consisting of DNA assembler-assisted pathway assembly and counterselection system-based chromosomal integration, for the rapid and efficient integration of synthetic biodegradation pathways into the chromosome of Pseudomonas putida KT2440. Using this strategy, we created a novel degrader capable of complete mineralization of γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) by integrating γ-HCH and TCP biodegradation pathways into the chromosome of P. putida KT2440. Furthermore, the chromosomal integration efficiencies of γ-HCH and TCP biodegradation pathways were improved to 50% and 41.6% in P. putida KT2440, respectively, by the inactivation of a type I DNA restriction-modification system. The currently developed pathway construction strategy coupled with the mutant KTUΔhsdRMS will facilitate implantation of heterologous catabolic pathways into the chromosome for rapid evolution of the biodegradation capacity of P. putida. More importantly, the successful removal of γ-HCH (10 mg/kg soil) and TCP (0.2 mM) from soil and wastewater within 14 days, respectively, highlighted the potential of the novel degrader for in situ bioremediation of γ-HCH- and TCP-contaminated sites. Moreover, chromosomal integration of gfp made the degrader to be monitored easily during bioremediation. In the future, this strategy can be expanded to a broad range of bacterial species for widespread applications in bioremediation.

Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production.

BACKGROUND: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. RESULTS: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. CONCLUSIONS: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications.

Deletion of genomic islands in the Pseudomonas putida KT2440 genome can create an optimal chassis for synthetic biology applications.

BACKGROUND: Genome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation. RESULTS: In this study, the identified GIs in P. putida KT2440 accounting for 4.12% of the total genome size were deleted to generate a series of genome-reduced strains. The mutant KTU-U13 with the largest deletion was advantageous over the original strain KTU in several physiological characteristics evaluated. The mutant KTU-U13 showed high plasmid transformation efficiency and heterologous protein expression capacity compared with the original strain KTU. The metabolic phenotype analysis showed that the types of carbon sources utilized by the mutant KTU-U13 and the utilization capabilities for certain carbon sources were increased greatly. The polyhydroxyalkanoate (PHA) yield and cell dry weight of the mutant KTU-U13 were improved significantly compared with the original strain KTU. The chromosomal integration efficiencies for the γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) biodegradation pathways were improved greatly when using the mutant KTU-U13 as the recipient cell and enhanced degradation of γ-HCH and TCP by the mutant KTU-U13 was also observed. The mutant KTU-U13 was able to stably express a plasmid-borne zeaxanthin biosynthetic pathway, suggesting the excellent genetic stability of the mutant. CONCLUSIONS: These desirable traits make the GIs-deleted mutant KTU-U13 an optimum chassis for synthetic biology applications. The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.

Metabolic engineering of Bacillus amyloliquefaciens LL3 for enhanced poly-γ-glutamic acid synthesis.

Poly-γ-glutamic acid (γ-PGA) is a biocompatible and biodegradable polypeptide with wide-ranging applications in foods, cosmetics, medicine, agriculture and wastewater treatment. Bacillus amyloliquefaciens LL3 can produce γ-PGA from sucrose that can be obtained easily from sugarcane and sugar beet. In our previous work, it was found that low intracellular glutamate concentration was the limiting factor for γ-PGA production by LL3. In this study, the γ-PGA synthesis by strain LL3 was enhanced by chromosomally engineering its glutamate metabolism-relevant networks. First, the downstream metabolic pathways were partly blocked by deleting fadR, lysC, aspB, pckA, proAB, rocG and gudB. The resulting strain NK-A6 synthesized 4.84 g l-1 γ-PGA, with a 31.5% increase compared with strain LL3. Second, a strong promoter PC 2up was inserted into the upstream of icd gene, to generate strain NK-A7, which further led to a 33.5% improvement in the γ-PGA titre, achieving 6.46 g l-1 . The NADPH level was improved by regulating the expression of pgi and gndA. Third, metabolic evolution was carried out to generate strain NK-A9E, which showed a comparable γ-PGA titre with strain NK-A7. Finally, the srf and itu operons were deleted respectively, from the original strains NK-A7 and NK-A9E. The resulting strain NK-A11 exhibited the highest γ-PGA titre (7.53 g l-1 ), with a 2.05-fold improvement compared with LL3. The results demonstrated that the approaches described here efficiently enhanced γ-PGA production in B. amyloliquefaciens fermentation.

Enhanced production of antifungal lipopeptide iturin A by Bacillus amyloliquefaciens LL3 through metabolic engineering and culture conditions optimization.

BACKGROUND: Iturins, which belong to antibiotic cyclic lipopeptides mainly produced by Bacillus sp., have the potential for application in biomedicine and biocontrol because of their hemolytic and antifungal properties. Bacillus amyloliquefaciens LL3, isolated previously by our lab, possesses a complete iturin A biosynthetic pathway as shown by genomic analysis. Nevertheless, iturin A could not be synthesized by strain LL3, possibly resulting from low transcription level of the itu operon. RESULTS: In this work, enhanced transcription of the iturin A biosynthetic genes was implemented by inserting a strong constitutive promoter C2up into upstream of the itu operon, leading to the production of iturin A with a titer of 37.35 mg l-1. Liquid chromatography-mass spectrometry analyses demonstrated that the strain produced four iturin A homologs with molecular ion peaks at m/z 1044, 1058, 1072 and 1086 corresponding to [C14 + 2H]2+, [C15 + 2H]2+, [C16 + 2H]2+ and [C17 + 2H]2+. The iturin A extract exhibited strong inhibitory activity against several common plant pathogens. The yield of iturin A was improved to 99.73 mg l-1 by the optimization of the fermentation conditions using a response surface methodology. Furthermore, the yield of iturin A was increased to 113.1 mg l-1 by overexpression of a pleiotropic regulator DegQ. CONCLUSIONS: To our knowledge, this is the first report on simultaneous production of four iturin A homologs (C14-C17) by a Bacillus strain. In addition, this study suggests that metabolic engineering in combination with culture conditions optimization may be a feasible method for enhanced production of bacterial secondary metabolites.

Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01.

Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA.

Morphology engineering for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01.

Polyhydroxyalkanoates (PHAs) can be produced by microorganisms from renewable resources and are regarded as promising bioplastics to replace petroleum-based plastics. A medium-chain-length PHAs (mcl-PHA)-producing strain Pseudomonas mendocina NK-01 was isolated previously by our lab and its whole-genome sequence is currently available. Morphology engineering of manipulating cell morphology-related genes has been applied for enhanced accumulation of the intracellular biopolymer short-chain-length PHAs (scl-PHA). However, it has not yet been reported to improve the yield of mcl-PHA by morphology engineering so far. In this work, several well-characterized cell morphology-related genes, including the cell fission ring (Z-ring) location genes minCD, peptidoglycan degradation gene nlpD, actin-like cytoskeleton protein gene mreB, Z-ring formation gene ftsZ, and FtsZ inhibitor gene sulA, were intensively investigated for their impacts on the cell morphology and mcl-PHA accumulation by gene knockout and overexpression in P. mendocina NKU, a upp knockout mutant of P. mendocina NK-01. For a minCD knockout mutant P. mendocina NKU-∆minCD, the average cell length was obviously increased and the mcl-PHA production was improved. However, the nlpD knockout mutant had a shorter cell length and lower mcl-PHA yield compared with P. mendocina NKU. Overexpression of mreB in P. mendocina NKU resulted in spherical cells. When ftsZ was overexpressed in P. mendocina NKU, the cell division was accelerated and the mcl-PHA titer was improved. Furthermore, mreB, ftsZ, or sulA was overexpressed in P. mendocina NKU-∆minCD. Consequently, the mcl-PHA titers were all increased compared with P. mendocina NKU-∆minCD carrying the empty vector. The multiple fission pattern was finally achieved in ftsZ-overexpressing NKU-∆minCD. In this work, improved production of mcl-PHA in P. mendocina NK-01 has been achieved by morphology engineering. This work provides an alternative strategy to enhance mcl-PHA accumulation in mcl-PHA-producing strains.

An order-aware scheme for robust direction of arrival estimation in the spherical harmonic domain.

Direction of arrival (DOA) estimation of sound sources using a spherical microphone array is usually performed in the spherical harmonic (SH) domain. In a non-noisy environment, it suffices to use only the zeroth- and first-order spherical harmonic beams (SHBs) in the SH domain for DOA estimation. One such method is based on the pseudo-intensity vector (PIV), which is attractive due to its low computational complexity. To improve the performance of the PIV method in reverberant environments, some methods have been proposed recently to further exploit high-order SHBs. However, these methods ignore the effect of noise on high-order SHBs, which may lead to poor performance in low signal-to-noise ratio (SNR) environments. To address the problem, this paper proposes an order-aware scheme that is able to select the high-order SHBs reliable for robust DOA estimation of multiple speech sources. Simulation and real-world experimental results demonstrate that the order-aware scheme based methods outperform their existing counterparts with less computational complexity in terms of both accuracy and robustness of DOA estimation. Moreover, the performance improvement is more significant in low SNR environment and in a scenario with small angular separation of sources.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

BACKGROUND: Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. RESULTS: For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. CONCLUSIONS: The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP+ ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

Construction of energy-conserving sucrose utilization pathways for improving poly-γ-glutamic acid production in Bacillus amyloliquefaciens.

BACKGROUND: Sucrose is an naturally abundant and easily fermentable feedstock for various biochemical production processes. By now, several sucrose utilization pathways have been identified and characterized. Among them, the pathway consists of sucrose permease and sucrose phosphorylase is an energy-conserving sucrose utilization pathway because it consumes less ATP when comparing to other known pathways. Bacillus amyloliquefaciens NK-1 strain can use sucrose as the feedstock to produce poly-γ-glutamic acid (γ-PGA), a highly valuable biopolymer. The native sucrose utilization pathway in NK-1 strain consists of phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-P hydrolase and consumes more ATP than the energy-conserving sucrose utilization pathway. RESULTS: In this study, the native sucrose utilization pathway in NK-1 was firstly deleted and generated the B. amyloliquefaciens 3Δ strain. Then four combination of heterologous energy-conserving sucrose utilization pathways were constructed and introduced into the 3Δ strain. Results demonstrated that the combination of cscB (encodes sucrose permease) from Escherichia coli and sucP (encodes sucrose phosphorylase) from Bifidobacterium adolescentis showed the highest sucrose metabolic efficiency. The corresponding mutant consumed 49.4% more sucrose and produced 38.5% more γ-PGA than the NK-1 strain under the same fermentation conditions. CONCLUSIONS: To our best knowledge, this is the first report concerning the enhancement of the target product production by introducing the heterologous energy-conserving sucrose utilization pathways. Such a strategy can be easily extended to other microorganism hosts for reinforced biochemical production using sucrose as substrate.

Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum.

BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production. RESULTS: In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain. CONCLUSIONS: We proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.

Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements.

Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5 g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4 g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102 g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.

Mutations in genes encoding antibiotic substances increase the synthesis of poly-γ-glutamic acid in Bacillus amyloliquefaciens LL3.

Poly-γ-glutamic acid (γ-PGA) is an important natural biopolymer that is used widely in fields of foods, medicine, cosmetics, and agriculture. Several B. amyloliquefaciens LL3 mutants were constructed to improve γ-PGA synthesis via single or multiple marker-less in-frame deletions of four gene clusters (itu, bae, srf, and fen) encoding antibiotic substances. γ-PGA synthesis by the Δsrf mutant showed a slight increase (4.1 g/L) compared with that of the wild-type strain (3.3 g/L). The ΔituΔsrf mutant showed increased γ-PGA yield from 3.3 to 4.5 g/L, with an increase of 36.4%. The γ-PGA yield of the ΔituΔsrfΔfen and ΔituΔsrfΔfenΔbae mutants did not show a further increase. The four gene clusters' roles in swarming motility and biofilm formation were also studied. The Δsrf and Δbae mutant strains were both significantly defective in swarming, indicating that bacillaene and surfactin are involved in swarming motility of B. amyloliquefaciens LL3. Furthermore, Δsrf and Δitu mutant strains were obviously defective in biofilm formation; therefore, iturin and surfactin must play important roles in biofilm formation in B. amyloliquefaciens LL3.

Effects of MreB paralogs on poly-γ-glutamic acid synthesis and cell morphology in Bacillus amyloliquefaciens.

Actin-like MreB paralogs play important roles in cell shape maintenance, cell wall synthesis and the regulation of the D,L-endopeptidases, CwlO and LytE. The gram-positive bacteria, Bacillus amyloliquefaciens LL3, is a poly-γ-glutamic acid (γ-PGA) producing strain that contains three MreB paralogs: MreB, Mbl and MreBH. In B. amyloliquefaciens, CwlO and LytE can degrade γ-PGA. In this study, we aimed to test the hypothesis that modulating transcript levels of MreB paralogs would alter the synthesis and degradation of γ-PGA. The results showed that overexpression or inhibition of MreB, Mbl or MreBH had distinct effects on cell morphology and the molecular weight of the γ-PGA products. In fermentation medium, cells of mreB inhibition mutant were 50.2% longer than LL3, and the γ-PGA titer increased by 55.7%. However, changing the expression level of mbl showed only slight effects on the morphology, γ-PGA molecular weight and titer. In the mreBH inhibition mutant, γ-PGA production and its molecular weight increased by 56.7% and 19.4%, respectively. These results confirmed our hypothesis that suppressing the expression of MreB paralogs might reduce γ-PGA degradation, and that improving the cell size could strengthen γ-PGA synthesis. This is the first report of enhanced γ-PGA production via suppression of actin-like MreB paralogs.