Effect of the initial pH of the culture medium on the nutrient consumption pattern of Bifidobacterium animalis subsp. lactis Bb12 and the improvement of acid resistance by purine and pyrimidine compounds.

AIMS: During fermentation, the accumulation of acidic products can induce media acidification, which restrains the growth of Bifidobacterium animalis subsp. lactis Bb12 (Bb12). This study investigated the nutrient consumption patterns of Bb12 under acid stress and effects of specific nutrients on the acid resistance of Bb12. METHODS AND RESULTS: Bb12 was cultured in chemically defined medium (CDM) at different initial pH values. Nutrient consumption patterns were analyzed in CDM at pH 5.3, 5.7, and 6.7. The patterns varied with pH: Asp + Asn had the highest consumption rate at pH 5.3 and 5.7, while Ala was predominant at pH 6.7. Regardless of the pH levels (5.3, 5.7, or 6.7), ascorbic acid, adenine, and Fe2+ were vitamins, nucleobases, and metal ions with the highest consumption rates, respectively. Nutrients whose consumption rates exceeded 50% were added individually in CDM at pH 5.3, 5.7, and 6.7. It was demonstrated that only some of them could promote the growth of Bb12. Mixed nutrients that could promote the growth of Bb12 were added to three different CDM. In CDM at pH 5.3, 5.7, and 6.7, it was found that the viable cell count of Bb12 was the highest after adding mixed nutrients, which were 8.87, 9.02, and 9.10 log CFU ml-1, respectively. CONCLUSIONS: The findings suggest that the initial pH of the culture medium affects the nutrient consumption patterns of Bb12. Specific nutrients can enhance the growth of Bb12 under acidic conditions and increase its acid resistance.

Protein rational design and modification of erythrose reductase for the improvement of erythritol production in Yarrowia lipolytica.

Erythritol is a natural non-caloric sweetener, which is produced by fermentation and extensively applied in food, medicine and chemical industries. The final step of the erythritol synthesis pathway is involved in erythritol reductase, whose activity and NADPH-dependent become the limiting node of erythritol production efficiency. Herein, we implemented a strategy combining molecular docking and thermal stability screening to construct an ER mutant library. And we successfully obtained a double mutant ERK26N/V295M (ER*) whose catalytic activity was 1.48 times that of wild-type ER. Through structural analysis and MD analysis, we found that the catalytic pocket and the enzyme stability of ER* were both improved. We overexpressed ER* in the engineered strain ΔKU70 to obtain the strain YLE-1. YLE-1 can produce 39.47 g/L of erythritol within 144 h, representing a 35% increase compared to the unmodified strain, and a 10% increase compared to the strain overexpressing wild-type ER. Considering the essentiality of NADPH supply, we further co-expressed ER* with two genes from the oxidative phase of PPP, ZWF1 and GND1. This resulted in the construction of YLE-3, which exhibited a significant increase in production, producing 47.85 g/L of erythritol within 144 h, representing a 63.90% increase compared to the original chassis strain. The productivity and the yield of the engineered strain YLE-3 were 0.33 g/L/h and 0.48 g/g glycerol, respectively. This work provided an ER mutation with excellent performance, and also proved the importance of cofactors in the process of erythritol synthesis, which will promote the industrial production of erythritol by metabolic engineering of Y. lipolytica.

Nutrient consumption patterns of Lactobacillus acidophilus.

BACKGROUND: One of the greatest challenges in using Lactobacillus acidophilus as a probiotic is acid stress. The current research aimed to identify substances that help L. acidophilus resist acid stress; this was achieved through assessing its nutrient consumption patterns under various pH conditions. RESULTS: The consumption rates of alanine, uracil, adenine, guanine, niacin, and manganese were consistently higher than 60% for L. acidophilus LA-5 cultured at pH 5.8, 4.9, and 4.4. The consumption rates of glutamic acid + glutamine and thiamine increased with decreasing pH and were higher than 60% at pH 4.9 and 4.4. The viable counts of L. acidophilus LA-5 were significantly increased under the corresponding acidic stress conditions (pH 4.9 and 4.4) through the appropriate addition of either alanine (3.37 and 2.81 mmol L-1), glutamic acid + glutamine (4.77 mmol L-1), guanine (0.13 and 0.17 mmol L-1), niacin (0.02 mmol L-1), thiamine (0.009 mmol L-1), or manganese (0.73 and 0.64 mmol L-1) (P < 0.05). The viable counts of L. acidophilus LA-5 cultured in a medium supplemented with combined nutritional factors was 1.02-1.03-fold of the counts observed in control medium under all acid conditions (P < 0.05). CONCLUSION: Alanine, glutamic acid + glutamine, guanine, niacin, thiamine, and manganese can improve the growth of L. acidophilus LA-5 in an acidic environment in the present study. The results will contribute to optimizing strategies to enhance the acid resistance of L. acidophilus and expand its application in the fermentation industry. © 2024 Society of Chemical Industry.

Cell factories for biosynthesis of D-glucaric acid: a fusion of static and dynamic strategies.

D-glucaric acid is an important organic acid with numerous applications in therapy, food, and materials, contributing significantly to its substantial market value. The biosynthesis of D-glucaric acid (GA) from renewable sources such as glucose has garnered significant attention due to its potential for sustainable and cost-effective production. This review summarizes the current understanding of the cell factories for GA production in different chassis strains, from static to dynamic control strategies for regulating their metabolic networks. We highlight recent advances in the optimization of D-glucaric acid biosynthesis, including metabolic dynamic control, alternative feedstocks, metabolic compartments, and so on. Additionally, we compare the differences between different chassis strains and discuss the challenges that each chassis strain must overcome to achieve highly efficient GA productions. In this review, the processes of engineering a desirable cell factory for highly efficient GA production are just like an epitome of metabolic engineering of strains for chemical biosynthesis, inferring general trends for industrial chassis strain developments.

Multiplex modification of Yarrowia lipolytica for enhanced erythritol biosynthesis from glycerol through modularized metabolic engineering.

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66 g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631 g/g and 1.23 g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol.

Recent advances in metabolic regulation and bioengineering of gibberellic acid biosynthesis in Fusarium fujikuroi.

The plant growth hormone gibberellic acid (GA3), as one of the representative secondary metabolites, is widely used in agriculture, horticulture and brewing industry. GA3 is detected in both plants and several fungi with the ability to stimulate plant growth. Currently, the main mode of industrial production of GA3 is depended on the microbial fermentation via long-period submerged fermentation using Fusarium fujikuroi as the only producing strain, qualified for its natural productivity. However, the demand of large-sale industrialization of GA3 was still restricted by the low productivity. The biosynthetic route of GA3 in F. fujikuroi is now well-defined. Furthermore, the multi-level regulation mechanisms involved in the whole network of GA3 production have also been gradually unveiled by the past two decades based on the identification and characterization of several global regulators and their mutual functions. Combined with the quick development of genetic manipulation techniques, the rational modification of producing strain F. fujikuroi development become practical for higher productivity achievement. Herein, we review the latest advances in the molecular regulation of GA3 biosynthesis in F. fujikuroi and conclude a comprehensive network involving nitrogen depression, global regulator, histone modification and G protein signaling pathway. Correspondingly, the bioengineering strategies covering conventional random mutation, genetic manipulating platform development, metabolic edition and fermentation optimization were also systematically proposed.

Local use of dexamethasone in the treatment of ocular myasthenia gravis.

BACKGROUND: At present, patients with ocular myasthenia gravis (OMG) are typically treated with systemic drugs. We investigated the use of dexamethasone injected in the peribulbar region or extraocular muscle to treat patients with OMG. METHODS: Patients with OMG were given dexamethasone via peribulbar injection or direct injection into the main paralyzed extraocular muscles, once a week, for 4-6 weeks. The severity of diplopia, blepharoptosis, eye position, and eye movement were evaluated before and after treatment. The duration of follow-up time was ≥6 months. RESULTS: Among the 14 patients with OMG who received this treatment, mean age was 38.7 ± 29.7 years. After treatment, symptoms were relieved in 12 patients (85.7%), 1 patient (7.1%) had partial response to treatment, and 1 patient (7.1%) had no response. Two patients (14.2%) experienced symptom recurrence during the follow-up period. CONCLUSIONS: Dexamethasone peribulbar or extraocular muscle injection is effective in the treatment of patients with OMG and may replace systemic drug therapy. TRIAL REGISTRATION: Chinese Clinical Trial Registry, ChiCTR2000038863 , October 7, 2020.Retrospectively registered.

Efficient biosynthesis of L-phenylglycine by an engineered Escherichia coli with a tunable multi-enzyme-coordinate expression system.

Whole-cell catalysis with co-expression of two or more enzymes in a single host as a simple low-cost biosynthesis method has been widely studied and applied but hardly with regulation of multi-enzyme expression. Here we developed an efficient whole-cell catalyst for biosynthesis of L-phenylglycine (L-Phg) from benzoylformic acid through co-expression of leucine dehydrogenase from Bacillus cereus (BcLeuDH) and NAD+-dependent mutant formate dehydrogenase from Candida boidinii (CbFDHA10C) in Escherichia coli with tunable multi-enzyme-coordinate expression system. By co-expressing one to four copies of CbFDHA10C and optimization of the RBS sequence of BcLeuDH in the expression system, the ratio of BcLeuDH to CbFDH in E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was finally regulated to 2:1, which was the optimal one determined by enzyme-catalyzed synthesis. The catalyst activity of E. coli BL21/pETDuet-rbs 4 leudh-3fdh A10C was 28.4 mg L-1 min-1 g-1 dry cell weight for L-Phg production using whole-cell transformation, it's was 3.7 times higher than that of engineered E. coli without enzyme expression regulation. Under optimum conditions (pH 8.0 and 35 °C), 60 g L-1 benzoylformic acid was completely converted to pure chiral L-Phg in 4.5 h with 10 g L-1 dry cells and 50.4 g L-1 ammonium formate, and with enantiomeric excess > 99.9%. This multi-enzyme-coordinate expression system strategy significantly improved L-Phg productivity and demonstrated a novel low-cost method for enantiopure L-Phg production.

Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase.

NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances. IMPORTANCE: FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

Heterologous expression and characterization of a new heme-catalase in Bacillus subtilis 168.

Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784 U/mg expressed in Bacillus subtilis 168 and 98.097 µmol/min/mg peroxidatic activity, the apparent K m of catalytic activity was 59.6 ± 13 mM with higher turnover rate (K cat = 322.651 × 10(3) s(-1)). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40 °C and the recombinant heme-catalase exhibited a strong Fe(2+) preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

Improving the acidic stability of Staphylococcus aureus α-acetolactate decarboxylase in Bacillus subtilis by changing basic residues to acidic residues.

The α-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus α-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of K m and V max were 17.7 µM and 2.06 mM min(-1), respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDCN43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDCN43D-K52D exhibited a high yield of 135.8 U mL(-1) of SaALDC activity, about 320 times higher comparing to 0.42 U mL(-1) of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host.

[Clinical control of different sequential order of three-dimensional conformal radiotherapy combined with transcatheter arterial chemoembolization for portal vein tumor thrombus in patients with hepatocellular carcinoma].

OBJECTIVE: To study the influence of the sequence of three-dimensional conformal radiotherapy (3DCRT) and transcatheter arterial chemoembolization (TACE) on the efficacy and toxicity of treatment in patients with hepatocellular carcinoma (HCC) with portal vein tumor thrombus (PVTT). METHODS: A total of 65 patients who were diagnosed with primary HCC with PVTT were enrolled in the study from November 2008 to March 2012 and were randomly divided into the following two groups:group A,32 patients treated with 3DCRT followed by TACE; group B,33 patients treated with TACE followed by 3DCRT. RESULTS: The total efficacy rates of groups A and B were 68.8% and 69.7% (x² =0.232, P < 0.793). The survival rates,effective percentage of PVTT and AFP remission rates were not significantly different between group A and group B.The exacerbation rate of liver function was significantly higher for group B than for group A (P < 0.05). No serious complication was found in the follow-up period for either group. CONCLUSION: The combination of 3DCRT and TACE is a relatively effective local treatment for patients with primary HCC and PVTT.Compared with TACE followed by 3DCRT, 3DCRT followed by TACE may have a negative influence on liver function.

Efficient carbon flux allocation towards D-pantothenic acid production via growth-decoupled strategy in Escherichia coli.

For industrial strain construction, rational allocation of carbon flux is of paramount importance especially for decoupling cell growth and chemical productions to get maximum titer, rate, yield (TRY), which become Gordian Knot. Here, a temperature-sensitive switch and genetic circuits was used for effectively decoupling cell growth from D-pantothenic acid (DPA) production, along with systematically metabolic engineering including blocking redundant pathways of pyruvate and enhancing DPA driving force. Afterwards, rapid biomass accumulation only happened during growth stage, and subsequent high-efficient DPA production was initiated with reducing fermentation temperature. Finally, 97.20 g/L DPA and 0.64 g/g glucose conversion rate were achieved in 5-liter fed-batch fermentation. These undisputedly represent a milestone for the biosynthesis of DPA. With using strategies for decoupling cell growth from chemical productions, it would serve as "Alexander's sword" to cut Gordian Knot to get industrial chassis cells with excellent TRY for de novo biosynthesis of valuable chemicals.

[Advances in efficient biosynthesis of erythritol by metabolic engineering of Yarrowia lipolytica].

Erythritol is a novel 4-carbon sugar alcohol produced by microbes in the presence of hyper-osmotic stress. It has excellent potential to serve as an alternative sugar for people with diabetes and also a platform compound for synthesizing various C4 compounds, such as 1, 3-butadiene, 1, 4-butanediol, 2, 5-dihydrofuran and so on. Compared with other polyols, the fermentative production of erythritol is more challenging. Yarrowia lipolytica is the preferred chassis of erythritol biosynthesis for its high-titer and high-productivity. At present, there are still some bottlenecks in the production of erythritol by Y. lipolytica, such as weak metabolic activity, abundant by-products, and low industrial attributes. Progress has been made in tailoring high version strains according to industrial needs. For example, the highest titer of erythritol produced by the metabolically engineered Y. lipolytica reached 196 g/L and 150 g/L, respectively, by using glucose or glycerol as the carbon sources. However, further improving its production performance becomes challenging. This review summarizes the research progress in the synthesis of erythritol by Y. lipolytica from the perspectives of erythritol producing strains, metabolic pathways, modular modifications, and auxiliary strategies to enhance the industrial properties of the engineered strain. Key nodes in the metabolic pathway and their combination strategies are discussed to guide the research on promoting the production of erythritol by Y. lipolytica.

Synthetic biology for Monascus: From strain breeding to industrial production.

Traditional Chinese food therapies often motivate the development of modern medicines, and learning from them will bring bright prospects. Monascus, a conventional Chinese fungus with centuries of use in the food industry, produces various metabolites, including natural pigments, lipid-lowering substances, and other bioactive ingredients. Recent Monascus studies focused on the metabolite biosynthesis mechanisms, strain modifications, and fermentation process optimizations, significantly advancing Monascus development on a lab scale. However, the advanced manufacture for Monascus is lacking, restricting its scale production. Here, the synthetic biology techniques and their challenges for engineering filamentous fungi were summarized, especially for Monascus. With further in-depth discussions of automatic solid-state fermentation manufacturing and prospects for combining synthetic biology and process intensification, the industrial scale production of Monascus will succeed with the help of Monascus improvement and intelligent fermentation control, promoting Monascus applications in food, cosmetic, agriculture, medicine, and environmental protection industries.

Development of a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in Fusarium fujikuroi.

Iterative metabolic engineering of Fusarium fujikuroi has traditionally been hampered by its low homologous recombination efficiency and scarcity of genetic markers. Thus, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas9) system has emerged as a promising tool for precise genome editing in this organism. Some integrated CRISPR/Cas9 strategies have been used to engineer F. fujikuroi to improve GA3 production capabilities, but low editing efficiency and possible genomic instability became the major obstacle. Herein, we developed a marker recyclable CRISPR/Cas9 system for scarless and multigene editing in F. fujikuroi. This system, based on an autonomously replicating sequence, demonstrated the capability of a single plasmid harboring all editing components to achieve 100%, 75%, and 37.5% editing efficiency for single, double, and triple gene targets, respectively. Remarkably, even with a reduction in homologous arms to 50 bp, we achieved a 12.5% gene editing efficiency. By employing this system, we successfully achieved multicopy integration of the truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase gene (tHMGR), leading to enhanced GA3 production. A key advantage of our plasmid-based gene editing approach was the ability to recycle selective markers through a simplified protoplast preparation and recovery process, which eliminated the need for additional genetic markers. These findings demonstrated that the single-plasmid CRISPR/Cas9 system enables rapid and precise multiple gene deletions/integrations, laying a solid foundation for future metabolic engineering efforts aimed at industrial GA3 production.

Protein multi-level structure feature-integrated deep learning method for mutational effect prediction.

Through iterative rounds of mutation and selection, proteins can be engineered to enhance their desired biological functions. Nevertheless, identifying optimal mutation sites for directed evolution remains challenging due to the vastness of the protein sequence landscape and the epistatic mutational effects across residues. To address this challenge, we introduce MLSmut, a deep learning-based approach that leverages multi-level structural features of proteins. MLSmut extracts salient information from protein co-evolution, sequence semantics, and geometric features to predict the mutational effect. Extensive benchmark evaluations on 10 single-site and two multi-site deep mutation scanning datasets demonstrate that MLSmut surpasses existing methods in predicting mutational outcomes. To overcome the limited training data availability, we employ a two-stage training strategy: initial coarse-tuning on a large corpus of unlabeled protein data followed by fine-tuning on a curated dataset of 40-100 experimental measurements. This approach enables our model to achieve satisfactory performance on downstream protein prediction tasks. Importantly, our model holds the potential to predict the mutational effects of any protein sequence. Collectively, these findings suggest that our approach can substantially reduce the reliance on laborious wet lab experiments and deepen our understanding of the intricate relationships between mutations and protein function.

Balancing the AspC and AspA Pathways of Escherichia coli by Systematic Metabolic Engineering Strategy for High-Efficient l-Homoserine Production.

l-Homoserine is a promising C4 platform compound used in the agricultural, cosmetic, and pharmaceutical industries. Numerous works have been conducted to engineer Escherichia coli to be an excellent l-homoserine producer, but it is still unable to meet the industrial-scale demand. Herein, we successfully engineered a plasmid-free and noninducible E. coli strain with highly efficient l-homoserine production through balancing AspC and AspA synthesis pathways. First, an initial strain was constructed by increasing the accumulation of the precursor oxaloacetate and attenuating the organic acid synthesis pathway. To remodel the carbon flux toward l-aspartate, a balanced route prone to high yield based on TCA intensity regulation was designed. Subsequently, the main synthetic pathway and the cofactor system were strengthened to reinforce the l-homoserine synthesis. Ultimately, under two-stage DO control, strain HSY43 showed 125.07 g/L l-homoserine production in a 5 L fermenter in 60 h, with a yield of 0.62 g/g glucose and a productivity of 2.08 g/L/h. The titer, yield, and productivity surpassed the highest reported levels for plasmid-free strains in the literature. The strategies adopted in this study can be applied to the production of other l-aspartate family amino acids.

Integrating shotgun metagenomics and metabolomics to elucidate the dynamics of microbial communities and metabolites in fine flavor cocoa fermentation in Hainan.

The aim of this study was to investigate the dynamic profile of microorganisms and metabolites in Hainan Trinitario cocoa during a six-day spontaneous box fermentation process. Shotgun metagenomic and metabolomic approaches were employed for this investigation. The potential metabolic functions of microorganisms in cocoa fermentation were revealed through a joint analysis of microbes, functional genes, and metabolites. During the anaerobic fermentation phase, Hanseniaspora emerged as the most prevalent yeast genus, implicated in pectin decomposition and potentially involved in glycolysis and starch and sucrose metabolism. Tatumella, possessing potential for pyruvate kinase, and Fructobacillus with a preference for fructose, constituted the primary bacteria during the pre-turning fermentation stage. Upon the introduction of oxygen into the fermentation mass, acetic acid bacteria ascended to dominant within the microflora. The exponential proliferation of Acetobacter resulted in a decline in taxonomic richness and abundance. Moreover, the identification of novel species within the Komagataeibacter genus suggests that Hainan cocoa may serve as a valuable reservoir for the discovery of unique cocoa fermentation bacteria. The KEGG annotation of metabolites and enzymes also highlighted the significant involvement of phenylalanine metabolism in cocoa fermentation. This research will offer a new perspective for the selection of starter strains and the formulation of mixed starter cultures.

Highly efficient production of rhamnolipid in P. putida using a novel sacB-based system and mixed carbon source.

Pseudomonas putida KT2440, a GRAS strain, has been used for synthesizing bulk and fine chemicals. However, the gene editing tool to metabolically engineer KT2440 showed low efficiency. In this study, a novel sacB-based system pK51mobsacB was established to improve the efficiency for marker-free gene disruption. Then the rhamnolipid synthetic pathway was introduced in KT2440 and genes of the competitive pathways were deleted to lower the metabolic burden based on pK51mobsacB. A series of endogenous and synthetic promoters were used for fine tuning rhlAB expression. The limited supply of dTDP-L-rhamnose was enhanced by heterologous rmlBDAC expression. Cell growth and rhamnolipid production were well balanced by using glucose/glycerol as mixed carbon sources. The final strain produced 3.64 g/L at shake-flask and 19.77 g/L rhamnolipid in a 5 L fermenter, the highest obtained among metabolically engineered KT2440, which implied the potential of KT2440 as a promising microbial cell factory for industrial rhamnolipid production.