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Heme, an iron-containing tetrapyrrole in hemoproteins, including: hemoglobin, myoglobin, catalase, cytochrome c, and cytochrome P450, plays critical physiological roles in different organisms. Heme-derived chemicals, such as biliverdin, bilirubin, and phycocyanobilin, are known for their antioxidant and anti-inflammatory properties and have shown great potential in fighting viruses and diseases. Therefore, more and more attention has been paid to the biosynthesis of hemoproteins and heme derivatives, which depends on the adequate heme supply in various microbial cell factories. The enhancement of endogenous biosynthesis and exogenous uptake can improve the intracellular heme supply, but the excess free heme is toxic to the cells. Therefore, based on the heme-responsive regulators, several sensitive biosensors were developed to fine-tune the intracellular levels of heme. In this review, recent advances in the: biosynthesis, acquisition, regulation, and upcycling of heme were summarized to provide a solid foundation for the efficient production and application of high-value-added hemoproteins and heme derivatives. [ABSTRACT FROM AUTHOR]

There is a growing interest in the creation of engineered condensates formed via liquid–liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities. Biomolecular condensates can serve as synthetic organelle-like compartments within prokaryotes. Here, the authors report a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins, and demonstrate control of metabolic flux and protein translation in Bacillus subtilis. [ABSTRACT FROM AUTHOR]

Hemoglobins, with heme as a cofactor, are functional proteins that have extensive applications in the fields of artificial oxygen carriers and foods. Although Saccharomyces cerevisiae is an ideal host for hemoglobin synthesis, it lacks a suitable transport system to utilize additional heme for active expression of hemoglobins, resulting in the cellular aggregation and degradation of the latter. Here, an effective heme importer, heme‐responsive gene 4 (Hrg‐4), was selected from six candidates through the comparison of effects on the growth rates of Δhem1 S. cerevisiae strain and the activities of various hemoglobins when supplemented with 5 mg·L−1 exogenous heme. Additionally, to counter the instability of plasmid‐based expression and the metabolic burden introduced from overexpressing Hrg‐4, a series of hrg‐4 integrated strains were constructed and the best engineered strain with five copies of hrg‐4 was chosen. We found that this engineered strain was associated with an increased binding rate of heme in monomeric leghemoglobin and multimeric human hemoglobin (76.3% and 16.5%, respectively), as well as an enhanced expression of both hemoglobins (52.8% and 17.0%, respectively). Thus, the engineered strain with improved heme uptake can be used to efficiently synthesize other heme‐binding proteins and enzymes in S. cerevisiae. [ABSTRACT FROM AUTHOR]

Biosynthesis is the application of enzymes in microbial cell factories and has emerged as a promising alternative to chemical synthesis. However, natural enzymes with limited catalytic performance often need to be engineered to meet specific needs through a time‐consuming trial‐and‐error process. This study presents a quantum mechanics (QM)‐incorporated design–build–test–learn (DBTL) framework to rationally design phosphatase BT4131, an enzyme with an ambiguous substrate spectrum involved in N‐acetylglucosamine (GlcNAc) biosynthesis. First, mutant M1 (L129Q) is designed using force field‐based methods, resulting in a 1.4‐fold increase in substrate preference (kcat/Km) toward GlcNAc‐6‐phosphate (GlcNAc6P). QM calculations indicate that the shift in substrate preference is caused by a 13.59 kcal mol−1 reduction in activation energy. Furthermore, an iterative computer‐aided design is conducted to stabilize the transition state. As a result, mutant M4 (I49Q/L129Q/G172L) with a 9.5‐fold increase in kcat‐GlcNAc6P/Km‐GlcNAc6P and a 59% decrease in kcat‐Glc6P/Km‐Glc6P is highly desirable compared to the wild type in the GlcNAc‐producing chassis. The GlcNAc titer increases to 217.3 g L−1 with a yield of 0.597 g (g glucose)−1 in a 50‐L bioreactor, representing the highest reported level. Collectively, this DBTL framework provides an easy yet fascinating approach to the rational design of enzymes for industrially viable biocatalysts. [ABSTRACT FROM AUTHOR]

Sulfonation as one of the most important modification reactions in nature is essential for many biological macromolecules to function. Development of green sulfonate group donor regeneration systems to efficiently sulfonate compounds of interest is always attractive. Here, we design and engineer two different sulfonate group donor regeneration systems to boost the biosynthesis of sulfated compounds. First, we assemble three modules to construct a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration system and demonstrate its applicability for living cells. After discovering adenosine 5'-phosphosulfate (APS) as another active sulfonate group donor, we engineer a more simplified APS regeneration system that couples specific sulfotransferase. Next, we develop a rapid indicating system for characterizing the activity of APS-mediated sulfotransferase to rapidly screen sulfotransferase variants with increased activity towards APS. Eventually, the active sulfonate group equivalent values of the APS regeneration systems towards trehalose and p-coumaric acid reach 3.26 and 4.03, respectively. The present PAPS and APS regeneration systems are environmentally friendly and applicable for scaling up the biomanufacturing of sulfated products. Sufficient supply of sulfonate group donor is critical to biomanufacturing of the sulfate containing compounds. Here, the authors engineer two sulfonate group donor regeneration systems, including 3'-phosphoadenosine-5'-phosphosulfate and the newly discovered 5'-phosphosulfate, to boost biosynthesis of sulfated compounds. [ABSTRACT FROM AUTHOR]

Microbial synthesis of valuable hemoproteins has become a popular research topic, and Pichia pastoris is a versatile platform for the industrial production of recombinant proteins. However, the inadequate supply of heme limits the synthesis of high‐active hemoproteins. Here a strategy for enhancing intracellular heme biosynthesis to improve the titers and functional activities of hemoproteins is reported. After selecting a suitable expressional strategy for globins, the efficient heme‐supply P. pastoris chassis is established by removing the spatial segregation during heme biosynthesis, optimizing precursor synthesis, assembling rate‐limiting enzymes using protein scaffolds, and inhibiting heme degradation. This robust chassis produces several highly active hemoproteins, including porcine myoglobin, soy hemoglobin, Vitreoscilla hemoglobin, and P450‐BM3, which can be used in the development of artificial meat, high‐cell‐density fermentation, and whole‐cell catalytic synthesis of high‐value‐added compounds. Furthermore, the engineered chassis strain has great potential for producing and applying other hemoproteins with high activities in various fields. [ABSTRACT FROM AUTHOR]

Food proteins are key nutrients, which play an important role in our diet. However, the acquisition of food protein mainly depends on the agroindustry, resulting in environmental pollution, high land usage and the consumption of water resources. Here, we describe a chemo-biocascade catalysis (CBCC) system that combines spatially separated CO2 thermo-catalysis with yeast fermentation to efficiently convert CO2 to lacto-proteins. First, by uncovering and breaking the rate limiting step, we constructed a Pichia pastoris strain that efficiently synthesized human lactoferrin (hLF), able to reach a production of 5.4 g L−1 in bioreactor, the highest titer reported so far. Then, CuZnAl catalyst was used to stably synthesize the methanol from CO2 in a continuous-flow reactor. The collected liquid-phase products consisted of high-purity methanol of 99% in water with a yield of 6.7% (mol/mol). The hLF synthesis titer by P. pastoris using thermally generated methanol as the sole carbon source reached 56.2 mg L−1, with a methanol conversion rate of 1.1 mg g−1, and a carbon atoms conversion rate of 0.17%. Additionally, the developed CBCC was also used to produce other dairy proteins including osteopontin and lactalbumin. These results illustrate the potential of tandem chemocatalysis and biocatalysis for the conversion of renewable resources into food proteins. This methodology paves the way towards the development of sustainable alternatives for protein manufacturing that may allow us to meet the growing demand. [ABSTRACT FROM AUTHOR]

Natural products, a chemically and structurally diverse class of molecules, possess a wide spectrum of biological activities, have been used therapeutically for millennia, and have provided many lead compounds for the development of synthetic drugs. Cytochrome P450 enzymes (P450s, CYP) are widespread in nature and are involved in the biosynthesis of many natural products. P450s are heme-containing enzymes that use molecular oxygen and the hydride donor NAD(P)H (coupled via enzymic redox partners) to catalyze the insertion of oxygen into C-H bonds in a regio- and stereo-selective manner, effecting hydroxylation and several other reactions. With the rapid development of systems biology, numerous novel P450s have been identified for the biosynthesis of natural products, but there are still several challenges to the efficient heterologous expression of active P450s. This review covers recent developments in P450 research and development, including the properties and functions of P450s, discovery and mining of novel P450s, modification and screening of P450 mutants, improved heterologous expression of P450s in microbial hosts, efficient whole-cell transformation with P450s, and current applications of P450s for the biosynthesis of natural products. This resource provides a solid foundation for the application of highly active and stable P450s in microbial cell factories to biosynthesize natural products. [ABSTRACT FROM AUTHOR]

By exploiting versatile P450 enzymes, whole‐cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole‐cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate‐limiting enzymes using DNA‐guided scaffolds. The intracellular heme level is fine‐tuned by the combined use of mutated heme‐sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca‐2, and CYP105D7, are enhanced through fine‐tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3′,4′‐trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine‐tuned heme biosynthesis will be generally useful for developing whole‐cell biocatalysts involving hemoproteins. [ABSTRACT FROM AUTHOR]

Menthol, a high-value commodity monoterpenoid chemical, holds an important market share commercially because of its distinct functions. The menthol on the market mainly originates from plant extraction, which is facing challenges such as the seasonal fluctuations and long growth cycle of plants. Therefore, this study attempted to realize the de novo synthesis of menthol through microbial fermentation. First, through heterologous expression and subcellular localization observation, a synthetic route from glucose to (−)-menthol was successfully designed and constructed in Saccharomyces cerevisiae. Then, the mevalonate (MVA) pathway was enhanced, and the expression of farnesyl diphosphate synthase (ERG20) was dynamically regulated to improve the synthesis of D-limonene, a key precursor of (−)-menthol. Shake flask fermentation results showed that the D-limonene titer of the recombinant strain reached 459.59 mg/L. Next, the synthesis pathway from D-limonene to (−)-menthol was strengthened, and the fermentation medium was optimized. The (−)-menthol titer of 6.28 mg/L was obtained, implying that the de novo synthesis of menthol was successfully realized for the first time. This study provides a good foundation for the synthesis of menthol through microbial fermentation. [ABSTRACT FROM AUTHOR]

High-sugar diet causes health problems, many of which can be addressed with the use of sugar substitutes. Rubusoside and rebaudiosides are interesting molecules, considered the next generation of sugar substitutes due to their low-calorie, superior sweetness and organoleptic properties. However, their low abundance in nature makes the traditional plant extraction process neither economical nor environmental-friendly. Here we engineer baker's yeast Saccharomyces cerevisiae as a chassis for the de novo production of rubusoside and rebaudiosides. In this process, we identify multiple issues that limit the production, including rate-liming steps, product stress on cellular fitness and unbalanced metabolic networks. We carry out a systematic engineering strategy to solve these issues, which produces rubusoside and rebaudiosides at titers of 1368.6 mg/L and 132.7 mg/L, respectively. The rubusoside chassis strain here constructed paves the way towards a sustainable, large-scale fermentation-based manufacturing of diverse rebaudiosides. Rubusoside and rebaudiosides are considered the next generation of sugar substitutes. In this article, the authors report the engineering of Saccharomyces cerevisiae, remodelling the complex metabolic networks by a modular engineering approach, obtaining rubusoside and rebaudiosides at titers of around 1.4 g/L and 100 mg/L, respectively. [ABSTRACT FROM AUTHOR]

With the pursuit of natural non-calorie sweeteners, steviol glycosides (SGs) have become one of the most popular natural sweeteners in the market. The SGs in Stevia are a mixture of SGs synthesized from steviol (a terpenoid). SGs are diterpenoids. Different SGs depend on the number and position of sugar groups on the core steviol backbone. This diversity comes from the processing of glycoside steviol by various glycosyltransferases. Due to the differences in glycosylation, each SG has unique sensory properties. At present, it is more complicated to extract high-quality SGs from plants, so the excavation of the metabolic pathways of engineered microorganisms to synthesize SGs has been extensively studied. Specifically, the expression of different glycosyltransferases in microbes is key to the synthesis of various SGs by engineered microorganisms. To trigger more researches on the functional characterization of the enzymes encoded by these genes, this review describes the latest research progresses of the related enzymes involved in SG biosynthesis and metabolic engineering. Key points • Outlines the research progress of key enzymes in the biosynthetic pathway of SGs • Factors affecting the catalytic capacity of stevia glucosyltransferase • Provide guidance for the efficient synthesis of SGs in microbial cell factories [ABSTRACT FROM AUTHOR]

Chondroitin sulfate (CS) is widely applied in the medical, clinical, and nutraceutical fields. However, all commercialized CS is extracted from animal tissues, which is associated with many problems, such as heavy reliance on antibiotics, animal excrement waste and long breeding time. Here, we developed a promising new green route for the de novo biosynthesis of non-animal CS from methanol, a one-carbon compound, using the engineered Pichia pastoris cell factory. Firstly, three exogenous genes, namely kfoC and kfoA from Escherichia coli K4 and tuaD from Bacillus subtilis, were introduced into P. pastoris to construct the chondroitin synthesis pathway, which yielded 5.5 mg L−1 chondroitin. After optimizing the expression of committed enzymes by codon optimization, the production of chondroitin improved by approximately 35-fold to 189.8 mg L−1. Thereafter, 6 kinds of Kozak sequences and 10 different endogenous promoters were compared and applied to achieve the expression of active chondroitin-4-O-sulfotransferase (41.3 U L−1). After integrating the chondroitin biosynthesis and sulfonation modules, the engineered strain, Pp006, successfully produced 182.0 mg L−1 CSA [GlcA-GalNAc (4S)] with a sulfation degree of 1.1%. We further improved the sulfation degree to 2.8% by strengthening the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) supply with the overexpression of adenosine-5′-triphosphate sulfurylase and adenosine 5′-phosphosulfate kinase. In the fed-batch cultivation, the final titer of CSA reached 2.1 g L−1 and the sulfation degree increased to 4.0%. Moreover, the results suggest that the P. pastoris cell factory could be used as a green route to produce other sulfated glycosaminoglycans. [ABSTRACT FROM AUTHOR]

L-Alanyl- l -Glutamine (Ala-Gln) is a kind of functional dipeptide, which is widely used in food and health care. Ala-Gln can be synthesized from l -glutamine and l -alanine methyl ester hydrochloride by α-Amino acid ester acyltransferase (AET). Still, the low catalytic efficiency and conversion rate of AET are key limiting factors in Ala-Gln production. In this study, the mutant library of AET were built based on structural model and semi-rational design. To facilitate the screening of AET mutants, the double enzymes coupled screening method was constructed and optimized. The amino acid residues I115, D203, F211, D300, A298, and R334 were selected as candidate mutation sites by alanine scanning and virtual saturation mutation and the catalytic activity of the optimal mutation A298C increased by 23.7%. Molecular dynamics simulation and molecular docking analysis suggested that the increased flexibility of the catalytic pocket in AET (A298C) and the formation of additional hydrogen bonds with substrate contributed to the improvement of enzyme activity. [ABSTRACT FROM AUTHOR]

flavor liquor is one of the most popular distilled liquors in China that contains various flavor substances produced by a specialized brewing microbiome. Bacillus licheniformis is one of the major bacterial species in the Maotai-flavor liquor-brewing microbiome. However, the whole genome sequence of the B. licheniformis brewing strain has not been reported, which hampers further understanding of its effects on brewing. In this study, whole genome sequencing of B. licheniformis MT-B06 isolated from Maotai Daqu, a fermented wheat starter containing brewing microorganisms, was carried out using the Pacific Bioscience RS II platform. The sequence had a length of 4,440,765 bp with 45.7% GC content, encoding 5023 CDS sequences, 81 tRNAs, and 24 rRNAs. Next, genome annotation was performed based on four high-quality databases, including the COG, KEGG, GO/IPR, and Swiss-Prot databases. The genome sequence obtained was comparatively analyzed with that of B. licheniformis ATCC 14580, which is used for industrial enzyme production, which revealed that 69.95% of the differential genes are metabolically related, suggesting that these genes may be closely related to the flavor compounds biosynthesis. Therefore, the flavor compounds biosynthesis pathways and 26 relevant genes of B. licheniformis MT-B06 was analyzed, including alsS and alsD for 3-hydroxy-2-butanone synthesis, ispD, and ispE for terpenoids synthesis. This is the first report of high-quality whole genome sequencing of B. licheniformis MT-B06. Our results provide an important foundation for investigating metabolic pathways of desired compounds, such as C4 compounds and pyrazines, in Maotai-flavor liquor to improve its taste and quality. [ABSTRACT FROM AUTHOR]

Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and β-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and β-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes. [ABSTRACT FROM AUTHOR]

α-Ketoisocaproate (KIC) is used widely in the pharmaceutical and nutraceutical industries. In previous studies, we achieved a one-step biosynthesis of KIC from -leucine, using an Escherichia coli whole-cell biocatalyst expressing an -amino acid deaminase (-AAD) from Proteus vulgaris. Herein, we report the fine-tuning of -AAD gene expression in E. coli BL21 (DE3) at the transcriptional and translational levels to improve the KIC titer. By optimizing the plasmid origin with different copy numbers, modulating messenger RNA structure downstream of the initiation codon, and designing the sequences at the ribosome binding site, we increased biocatalyst activity to 31.77%, 24.89%, and 30.20%, respectively, above that achieved with BL21/pet28a-lad. The highest KIC titers reached 76.47 g·L-1, 80.29 g·L-1, and 81.41 g·L-1, respectively. Additionally, the integration of these three engineering strategies achieved an even higher KIC production of 86.55 g·L-1 and a higher -leucine conversion rate of 94.25%. The enzyme-engineering strategies proposed herein may be generally applicable to the construction of other biocatalysts. [ABSTRACT FROM AUTHOR]

α-Keto-γ-methylthiobutyric acid (KMTB), a keto derivative of -methionine, has great potential for use as an alternative to -methionine in the poultry industry and as an anti-cancer drug. This study developed an environment friendly process for KMTB production from -methionine by an Escherichia coli whole-cell biocatalyst expressing an engineered -amino acid deaminase (-AAD) from Proteus vulgaris. We first overexpressed the P. vulgaris -AAD in E. coli BL21 (DE3) and further optimized the whole-cell transformation process. The maximal molar conversion ratio of -methionine to KMTB was 71.2% (mol/mol) under the optimal conditions (70 g/L -methionine, 20 g/L whole-cell biocatalyst, 5 mM CaCl2, 40°C, 50 mM Tris-HCl [pH 8.0]). Then, error-prone polymerase chain reaction was used to construct P. vulgaris -AAD mutant libraries. Among approximately 104 mutants, two mutants bearing lysine 104 to arginine and alanine 337 to serine substitutions showed 82.2% and 80.8% molar conversion ratios, respectively. Furthermore, the combination of these mutations enhanced the catalytic activity and molar conversion ratio by 1.3-fold and up to 91.4% with a KMTB concentration of 63.6 g/L. Finally, the effect of immobilization on whole-cell transformation was examined, and the immobilized whole-cell biocatalyst with Ca2+ alginate increased reusability by 41.3% compared to that of free cell production. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of KMTB has an advantage in terms of environmental pollution and thus has great potential for industrial KMTB production. [ABSTRACT FROM AUTHOR]

Vitreoscilla hemoglobin (VHb), the first discovered bacterial hemoglobin, is a soluble heme-binding protein with a faster rate of oxygen dissociation. Since it can enhance cell growth, product synthesis and stress tolerance, VHb has been widely applied in the field of metabolic engineering for microorganisms, plants, and animals. Especially under oxygen-limited conditions, VHb can interact with terminal oxidase to deliver enough oxygen to achieve high-cell-density fermentation. In recent years, with the development of bioinformatics and synthetic biology, several novel physicochemical properties and metabolic regulatory effects of VHb have been discovered and numerous strategies have been utilized to enhance the expression level of VHb in various hosts, which greatly promotes its applications in biotechnology. Thus, in this review, the new information regarding structure, function and expressional tactics for VHb is summarized to understand its latest applications and pave a new way for the future improvement of biosynthesis for other products. [ABSTRACT FROM AUTHOR]

Metabolic engineering facilitates chemical biosynthesis by rewiring cellular resources to produce target compounds. However, an imbalance between cell growth and bioproduction often reduces production efficiency. Genetic code expansion (GCE)-based orthogonal translation systems incorporating non-canonical amino acids (ncAAs) into proteins by reassigning non-canonical codons to ncAAs qualify for balancing cellular metabolism. Here, GCE-based cell growth and biosynthesis balance engineering (GCE-CGBBE) is developed, which is based on titrating expression of cell growth and metabolic flux determinant genes by constructing ncAA-dependent expression patterns. We demonstrate GCE-CGBBE in genome-recoded Escherichia coli Δ321AM by precisely balancing glycolysis and N-acetylglucosamine production, resulting in a 4.54-fold increase in titer. GCE-CGBBE is further expanded to non-genome-recoded Bacillus subtilis to balance growth and N-acetylneuraminic acid bioproduction by titrating essential gene expression, yielding a 2.34-fold increase in titer. Moreover, the development of ncAA-dependent essential gene expression regulation shows efficient biocontainment of engineered B. subtilis to avoid unintended proliferation in nature. An imbalance between cell growth and bioproduction of engineered microbes often reduces production efficiency. Here, the authors report genetic code expansion-based cell growth and biosynthesis balance engineering to achieve high levels production of N-acetylglucosamine in E. coli and N-acetylneuraminic acid in B. subtilis. [ABSTRACT FROM AUTHOR]

Lactic acid is a major organic acid and precursor of ethyl lactate, which confers the important flavor of Chinese Maotai -flavor liquor. Moreover, lactic acid helps modulate the microbiome pH, which coordinates the microbial community. Lactobacillus panis L7 is the major lactic acid producer, however, the whole genome sequence is not available and the important genes for lactic acid biosynthesis have not been unidentified, which limits understanding of lactic acid biosynthesis. Whole genome sequencing of L. panis L7 was undertaken, obtaining 127 genome sequence scaffolds with an N50 of 33,743 bp. The genome total length was 2,026,099 bp encoding 1932 genes with a GC content of 47.0%. Heterolactic acid fermentation pathways and the involved genes were identified, including multiple copies of lactate dehydrogenase encoding genes. To identify important genes for lactic acid production, L. panis L7 was genetically perturbed using atmospheric and room temperature plasma followed by high-throughput screening of mutants with improved or reduced lactic acid production. Comparative genomic analysis of L. panis L7 and its mutants identified genes encoding for glutamate racemase, methylated-DNA-protein-cysteine methyltransferase, fumarate reductase flavoprotein subunit, and amidophosphoribosyltransferase as potentially important factors to improve lactic acid biosynthesis; genes encoding for ribokinase, fructokinase, argininosuccinate lyase, and large subunit ribosomal protein L30 as potentially important factors for reduced lactic acid biosynthesis. The results improved the understanding of the mechanism of lactic acid metabolism by L. panis for the benefit of the Chinese liquor brewing industry, and will, hopefully, help develop effective measures to modulate lactic acid content during brewing. [ABSTRACT FROM AUTHOR]

This work aimed to develop a whole-cell biotransformation process for the production of α-ketoisocaproate from L-leucine. A recombinant Escherichia coli strain was constructed by expressing an L-amino acid deaminase from Proteus vulgaris. To enhance α-ketoisocaproate production, the reaction conditions were optimized as follows: whole-cell biocatalyst 0.8 g/L, leucine concentration 13.1 g/L, temperature 35 °C, pH 7.5, and reaction time 20 h. Under the above conditions, the α-ketoisocaproate titer reached 12.7 g/L with a leucine conversion rate of 97.8%. In addition, different leucine feeding strategies were examined to increase the α-ketoisocaproate titer. When 13.1 g/L leucine was added at 2-h intervals (from 0 to 22 h, 12 addition times), the α-ketoisocaproate titer reached 69.1 g/L, while the leucine conversion rate decreased to 50.3%. We have developed an effective process for the biotechnological production of α-ketoisocaproate that is more environmentally friendly than the traditional petrochemical synthesis approach. [ABSTRACT FROM AUTHOR]

[Display omitted] • The key gene 4kas causing browning has been discovered. • Reconstructed the proportion of intracellular glucose metabolism in G. oxydans. • Engineering strains produce 2-KLG through co-utilization of glucose and sorbitol. • Obtained the highest reported 2-KLG yield of 38.62 g/L by one-step fermentation. As the highest-demand vitamin, the development of a one-step vitamin C synthesis process has been slow for a long time. In previous research, a Gluconobacter oxydans strain (GKLG9) was constructed that can directly synthesize 2-keto-L-gulonic acid (2-KLG) from glucose, but carbon source utilization remained low. Therefore, this study first identified the gene 4kas (4-keto-D-arabate synthase) to reduce the loss of extracellular carbon and inhibit the browning of fermentation broth. Then, promoter engineering was conducted to enhance the intracellular glucose transport pathway and concentrate intracellular glucose metabolism on the pentose phosphate pathway to provide more reducing power. Finally, by introducing the D-sorbitol pathway, the titer of 2-KLG was increased to 38.6 g/L within 60 h in a 5-L bioreactor, with a glucose-to-2-KLG conversion rate of about 46 %. This study is an important step in the development of single-bacterial one-step fermentation to produce 2-KLG. [ABSTRACT FROM AUTHOR]

Highlights: [•] The specificity of CGTase towards maltose was improved by site-saturation engineering of lysine 47. [•] The mechanism for enhanced specificity was clarified by structure modeling of the CGTase. [•] Enzymatic synthesis of AA-2G with maltose as glycosyl donor was optimized. [•] The obtained CGTase mutants have great potential in the large-scale production of AA-2G. [Copyright &y& Elsevier]

Highlights: [•] This is the first report about the biosynthesis of homoeriodictyol by enzymatic transformation. [•] The flavone 3′-O-methyltransferase ROMT-9 gene from rice was synthesized and expressed in Y. lipolytica. [•] The ROMT-9 was purified and characterized in terms of optimal pH, temperature, and catalytic kinetics. [•] The maximum amount of homoeriodictyol reached 110mg/L with a transformation ratio of 52.4% at 16h. [•] The biotransformation has less environmental pollution compared with the traditional chemical synthesis. [ABSTRACT FROM AUTHOR]

• Overexpression of menA , menG , and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module resulted in 8.1 ± 0.2 mg/L of MK-4. • By knocking out hepT gene, the titer of MK-4 in the resultant strain BY03 was increased by 3.8-fold. • The MK-4 titer reached 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor, respectively. Menaquinone-4 (MK-4), one form of vitamin K, plays an important role in cardiovascular and bone health. Menaquinone-4 (MK-4) is a valuable vitamin K2 that is difficult to synthesize organically, and now is mainly produced by microbial fermentation. Herein we significantly improved the synthesis efficiency of MK-4 by combinatorial pathway engineering in Bacillus subtilis 168, a model industrial strain widely used for production of nutraceuticals. The metabolic networks related to MK-4 synthesis include four modules, namely, MK-4 biosynthesis module, methylerythritol phosphate (MEP) module, mevalonate-dependent (MVA) isoprenoid module, and menaquinone module. Overexpression of menA , menG , and crtE genes from Synechocystis sp. PCC 6803 in MK-4 synthesis module with strong constitutive promoter P 43 resulted in 8.1 ± 0.2 mg/L of MK-4 (No MK-4 was detected in the wild-type B. subtilis 168). MK-4 titer was further increased by 3.8-fold to 31.53 ± 0.95 mg/L by knockout of hepT gene, which catalyzes the conversion of Farnesyl diphosphate to Heptaprenyl diphosphate. In addition, simultaneous overexpression of dxs, dxr , and ispD-ispF genes in MEP module with strong promoter P 43 increased the titer of MK-4 to 78.1 ± 1.6 mg/L. Moreover, expression of the heterogeneous MVA module genes (mvaK1, mvaK2 , mvaD, mvaS, and mvaA) resulted in 90.1 ± 1.7 mg/L of MK-4. Finally, in order to further convert the enhanced carbon metabolism flux to MK-4, simultaneous overexpression of the genes crtE, menA , and menG in menaquinone pathway with strong promoter P 43 increased the titer of MK-4 to 120.1 ± 0.6 mg/L in shake flask and 145 ± 2.8 mg/L in a 3-L fed-batch bioreactor. Herein the engineered B. subtilis strain may be used for the industrial production of MK-4 in the future. [ABSTRACT FROM AUTHOR]