Heterologous Gene Research Articles

Host-derived Lactobacillus plantarum alleviates hyperuricemia by improving gut microbial community and hydrolase-mediated degradation of purine nucleosides.

The gut microbiota is implicated in the pathogenesis of hyperuricemia (HUA) and gout. However, it remains unclear whether probiotics residing in the host gut, such as Lactobacillus, can prevent HUA development. Herein, we isolated Lactobacillus plantarum SQ001 from the cecum of HUA geese and conducted in vitro assays on uric acid (UA) and nucleoside co-culture. Metabolomics and genome-wide analyses, revealed that this strain may promote nucleoside uptake and hydrolysis through its nucleoside hydrolase gene. The functional role of iunH gene was confirmed via heterologous expression and gene knockout studies. Oral administration of L. plantarum SQ001 resulted in increased abundance of Lactobacillus species and reduced serum UA levels. Furthermore, it downregulated hepatic xanthine oxidase, a key enzyme involved in UA synthesis, as well as renal reabsorption protein GLUT9, while enhancing the expression of renal excretion protein ABCG2. Our findings suggest that L. plantarum has potential to ameliorate gut microbial dysbiosis with HUA, thereby offering insights into its potential application as a probiotic therapy for individuals with HUA or gout.

Production and characterization of novel/chimeric sophorose–rhamnose biosurfactants by introducing heterologous rhamnosyltransferase genes into Starmerella bombicola

Glycolipid biosurfactant, sophorolipids (SLs) and rhamnolipids (RLs) can be widely used in agriculture, food and chemical industries. The different physicochemical properties of SLs and RLs, such as hydrophilic lipophilic value (HLB) and critical micelle concentration (CMC), determine they have different application focus. Researchers are still hoping to obtain new glycolipid surfactants with unique surface activities. In this study, we successfully transformed two rhamnosyltransferase genes rhlA and rhlB from Pseudomonas aeruginosa to the sophorolipid-producing Starmerella bombicola CGMGG 1576 to obtain a recombinant strain was SbrhlAB. Two novel components with molecular weight of 554 (C26H50O12) and 536 (C26H48O11) were identified with the ASB C18 column from the fermentation broth of SbrhlAB, the former was a non-acetylated acidic C14:0 glycolipid containing one glucose and one rhamnose, and the latter was an acidic C14:1 glycolipid containing two rhamnoses. With the Venusil MP C18 column, one new glycolipid component was identified as an acidic C18:3 glycolipid with one rhamnose (C24H40O7), which has not been reported before. Our present study demonstrated that novel glycolipids can be synthesized in vivo by reasonable genetic engineering. The results will be helpful to engineer sophorolipid-producing yeast to produce some specific SLs or their derivatives in more rational and controllable way.

Strategies to enhance the hydrolytic activity of Escherichia coli BL21 penicillin G acylase based on heterologous expression and targeted mutagenesis

Penicillin G acylase (PGA) serves as a critical biocatalyst for the hydrolysis of penicillin G, yielding 6-aminopenicillanic acid, a vital precursor for β-lactam semi-synthetic antibiotics. The catalytic efficiency of PGA, however, remains suboptimal in native Escherichia coli strains. To improve this, E. coli BL21 was engineered as a microbial cell factory via heterologous expression and site-directed mutagenesis to enhance PGA activity. The heterologous pga gene from Providencia rettgeri was integrated into E. coli BL21 (DE3) for the biosynthesis of PGA, achieving a PGA activity of 253 ± 2 U/mL after 16 hours of fermentation. The N167 site underwent mutation, producing the sites N167A and N167I. Plasmids carrying these mutations were introduced into E. coli BL21(DE3), and the enzymatic activities were recorded as 293 ± 3 U/mL for the N167A mutant and 238 ± 2 U/mL for the N167I mutant. This study not only introduces a novel approach to enhancing PGA activity but also illustrates the potential for catalytic optimization through targeted modifications of the enzyme's active site.

Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.

Plasmid-free production of the plant lignan pinoresinol in growing Escherichia coli cells

BackgroundThe high-value aryl tetralin lignan (+)-pinoresinol is the main precursor of many plant lignans including (-)-podophyllotoxin, which is used for the synthesis of chemotherapeutics. As (-)-podophyllotoxin is traditionally isolated from endangered and therefore limited natural sources, there is a particular need for biotechnological production. Recently, we developed a reconstituted biosynthetic pathway from (+)-pinoresinol to (-)-deoxypodophyllotoxin, the direct precursor of (-)-podophyllotoxin, in the recombinant host Escherichia coli. However, the use of the expensive substrate (+)-pinoresinol limits its application from the economic viewpoint. In addition, the simultaneous expression of multiple heterologous genes from different plasmids for a multi-enzyme cascade can be challenging and limits large-scale use.ResultsIn this study, recombinant plasmid-free E. coli strains for the multi-step synthesis of pinoresinol from ferulic acid were constructed. To this end, a simple and versatile plasmid toolbox for CRISPR/Cas9-assisted chromosomal integration has been developed, which allows the easy transfer of genes from the pET vector series into the E. coli chromosome. Two versions of the developed toolbox enable the efficient integration of either one or two genes into intergenic high expression loci in both E. coli K-12 and B strains. After evaluation of this toolbox using the fluorescent reporter mCherry, genes from Petroselinum crispum and Zea mays for the synthesis of the monolignol coniferyl alcohol were integrated into different E. coli strains. The product titers achieved with plasmid-free E. coli W3110(T7) were comparable to those of the plasmid-based expression system. For the subsequent oxidative coupling of coniferyl alcohol to pinoresinol, a laccase from Corynebacterium glutamicum was selected. Testing of different culture media as well as optimization of gene copy number and copper availability for laccase activity resulted in the synthesis of 100 mg/L pinoresinol using growing E. coli cells.ConclusionsFor efficient and simple transfer of genes from pET vectors into the E. coli chromosome, an easy-to-handle molecular toolbox was developed and successfully tested on several E. coli strains. By combining heterologous and endogenous enzymes of the host, a plasmid-free recombinant E. coli growing cell system has been established that enables the synthesis of the key lignan pinoresinol.

Proteomic analysis reveals molecular changes following genetic engineering in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.

Complete sequences of pIJ101-based Streptomyces-Escherichia coli shuttle vectors.

High-copy-number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. The development of genetic tools for the industrially relevant Actinobacteria is of special interest, given their utility in producing keratolytic enzymes and biologically active natural products. Within the Actinobacteria, Streptomyces-Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces-E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these shuttle vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.

Probing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli

RNA recognition motifs (RRMs) are widespread RNA-binding protein domains in eukaryotes, which represent promising synthetic biology tools due to their compact structure and efficient activity. Yet, their use in prokaryotes is limited and their functionality poorly characterized. Recently, we repurposed a mammalian Musashi protein containing two RRMs as a translation regulator in Escherichia coli. Here, employing high-throughput RNA sequencing, we explored the impact of Musashi expression on the transcriptomic and translatomic profiles of E. coli, revealing certain metabolic interference, induction of post-transcriptional regulatory processes, and spurious protein-RNA interactions. Engineered Musashi protein mutants displayed compromised regulatory activity, emphasizing the importance of both RRMs for specific and sensitive RNA binding. We found that a mutation known to impede allosteric regulation led to similar translation control activity. Evolutionary experiments disclosed a loss of function of the synthetic circuit in about 40 generations, with the gene coding for the Musashi protein showing a stability comparable to other heterologous genes. Overall, this work expands our understanding of RRMs for post-transcriptional regulation in prokaryotes and highlight their potential for biotechnological and biomedical applications.

Engineering arabinose-to-arabitol conversion in industrial Saccharomyces cerevisiae for sugar beet pulp valorization

Arabitol, a promising low-calorie sweetener for the food industry, faces production challenges due to the high costs and the environmental impact of conventional chemical methods, as well as the inefficiency of biological approaches using native arabitol-producing yeasts.The Saccharomyces cerevisiae GRE3 gene encodes an NADPH-dependent aldose reductase, capable of converting various aldoses to their respective sugar alcohols. By leveraging the enzyme’s broad substrate specificity, the simultaneous conversion of xylose and arabinose into xylitol and arabitol, respectively, was achieved. This study showcases the feasibility of using engineered industrial yeast strains, overexpressing the native GRE3 gene, for the one-step conversion of arabinose to arabitol. Engineered strains (PE2–2-pGRE3, PE2–2-pGRE3-XII5, CA11-pGRE3, CA11-pGRE3-XII5, CAT1- pGRE3 and CAT1- pGRE3-XII5) exhibited successful arabinose-to-arabitol conversion, with a 3.5-fold variation in arabitol concentrations across strains. Further enhancement of the best-performing strain through overexpression of the GAL2 gene significantly improved arabinose transport, resulting in a 1.95-fold increase in arabitol productivity. This engineered strain, free of heterologous genes, also demonstrated its potential by converting arabinose-rich sugar-beet pulp hydrolysate into arabitol, reaching a titer of 15 g/L with a yield of 1 g/g. This study represents the first example of arabitol production from agro-food waste using genome-edited S. cerevisiae strains, which contain no foreign genes, as a versatile and efficient host.

Enhanced heterologous gene expression in Trichoderma reesei by promoting multicopy integration

Trichoderma reesei displays a high capability to produce extracellular proteins and therefore is used as a platform for the expression of heterologous genes. In a previous study, an expression cassette with the constitutive tef1 promoter and the cbh1 terminator compatible with flow cytometry analysis was developed. Independent transformants obtained by a random integration into the genome of a circular plasmid containing the expression cassette showed a wide range of fluorescence levels. Whole genome sequencing was conducted on eight of the transformed strains using two next-generation sequencing (NGS) platforms: Illumina paired-end sequencing and Oxford Nanopore. In all strains, the expression plasmid was inserted at the same position in the genome, i.e., upstream of the tef1 gene, indicating an integration by homologous recombination. The different levels of fluorescence observed correspond to different copy numbers of the plasmid. Overall, the integration of a circular plasmid with the green fluorescence protein (egfp) transgene under the control of tef1 promoter favors multicopy integration and allows over-production of this heterologous protein on glucose. In conclusion, an expression system based on using the tef1 promotor could be one of the building blocks for improving high-value heterologous protein production by increasing the copy number of the encoding genes into the genome of the platform strain.Key points• Varied eGFP levels from tef1 promoter and cbh1 terminator expression.• Whole genome sequencing on short and long reads platforms reveals various plasmid copy numbers in strains.• Plasmids integrate at the same genomic site by homologous recombination in all strains.

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of Escherichia coli.

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of Escherichia coli from 3 aspects. First, the integration site slmA with the highest expression activity was screened out of 18 sites in the ORI region of the E. coli BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in E. coli and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL-1, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.

Synthetic redesign of Escherichia coli W for faster metabolism of sugarcane molasses

BackgroundSugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes.ResultsIn this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP.ConclusionsDespite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications.

Genetic Manipulation of Neisseria gonorrhoeae and Commensal Neisseria Species.

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

Harnessing native-cryptic plasmids for stable overexpression of heterologous genes in Clostridium butyricum DSM 10702 for industrial and medical applications

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids’ potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Metabolic engineering of the substrate utilization pathway in Escherichia coli increases L-lysine production

L-lysine is an essential amino acid with broad applications in the animal feed, human food, and pharmaceutical industries. The fermentation production of L-lysine by Escherichia coli has limitations such as poor substrate utilization efficiency and low saccharide conversion rates. We deleted the global regulatory factor gene mlc and introduced heterologous genes, including the maltose phosphotransferase genes (malAP) from Bacillus subtilis, to enhance the use efficiency of disaccharides and trisaccharides. The engineered strain E. coli XC3 demonstrated improved L-lysine production, yield, and productivity, which reached 160.00 g/L, 63.78%, and 4.44 g/(L‧h), respectively. Furthermore, we overexpressed the glutamate dehydrogenase gene (gdhA) and assimilated nitrate reductase genes (BsnasBC) from B. subtilis, along with nitrite reductase genes (EcnirBD) from E. coli, in strain E. coli XC3. This allowed the construction of E. coli XC4 with a nitrate assimilation pathway. The L-lysine production, yield, and productivity of E. coli XC4 were elevated to 188.00 g/L, 69.44%, and 5.22 g/(L‧h), respectively. After optimization of the residual sugar concentration and carbon to nitrogen ratio, the L-lysine production, yield, and productivity were increased to 204.00 g/L, 72.32%, and 5.67 g/(L‧h), respectively, in a 5 L fermenter. These values represented the increases of 40.69%, 20.03%, and 40.69%, respectively, compared with those of the starting strain XC1. By engineering the substrate utilization pathway, we successfully constructed a high-yield L-lysine producing strain, laying a solid foundation for the industrial production of L-lysine.

Engineering a non-oxidative glycolysis pathway in escherichia coli for high-level citramalate production

BackgroundMethyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation.ResultsThis study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively.ConclusionThis is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

Production of (R)-citramalate by engineered Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, has a high tolerance to organic acids and alcohols, and thus grows well under toxic concentrations of various compounds in the culture medium, potentially allowing for highly efficient compound production. (R)-citramalate is a raw material for methyl methacrylate and can be used as a metabolic intermediate in the biosynthesis of higher alcohols. (R)-citramalate is synthesized from pyruvate and acetyl-CoA. Unlike Escherichia coli, S. cerevisiae has organelles, and its intracellular metabolites are compartmentalized, preventing full use of intracellular acetyl-CoA. Therefore, in this study, to increase the amount of cytosolic acetyl-CoA for highly efficient production of (R)-citramalate, we inhibited the transport of cytosolic acetyl-CoA and pyruvate to the mitochondria. We also constructed a heterologous pathway to supply cytosolic acetyl-CoA. Additionally, we attempted to export (R)-citramalate from cells by expressing a heterologous dicarboxylate transporter gene. We evaluated the effects of these approaches on (R)-citramalate production and constructed a final strain by combining these positive approaches. The resulting strain produced 16.5 mM (R)-citramalate in batch culture flasks. This is the first report of (R)-citramalate production by recombinant S. cerevisiae, and the (R)-citramalate production by recombinant yeast achieved in this study was the highest reported to date.

Functional plasticity of HCO3– uptake and CO2 fixation in Cupriavidus necator H16

Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of β-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3– uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.

The Tet-on system for controllable gene expression in the rock-inhabiting black fungus Knufia petricola

Knufia petricola is a black fungus that colonizes sun-exposed surfaces as extreme and oligotrophic environments. As ecologically important heterotrophs and biofilm-formers on human-made surfaces, black fungi form one of the most resistant groups of biodeteriorating organisms. Due to its moderate growth rate in axenic culture and available protocols for its transformation and CRISPR/Cas9-mediated genome editing, K.petricola is used for studying the morpho-physiological adaptations shared by extremophilic and extremotolerant black fungi. In this study, the bacteria-derived tetracycline (TET)-dependent promoter (Tet-on) system was implemented to enable controllable gene expression in K. petricola. The functionality i.e., the dose-dependent inducibility of TET-regulated constructs was investigated by using GFP fluorescence, pigment synthesis (melanin and carotenoids) and restored uracil prototrophy as reporters. The newly generated cloning vectors containing the Tet-on construct, and the validated sites in the K. petricola genome for color-selectable or neutral insertion of expression constructs complete the reverse genetics toolbox. One or multiple genes can be expressed on demand from different genomic loci or from a single construct by using 2A self-cleaving peptides, e.g., for localizing proteins and protein complexes in the K.petricola cell or for using K. petricola as host for the expression of heterologous genes.

Construction of an amylolytic Saccharomyces cerevisiae strain with high copies of α-amylase and glucoamylase genes integration for bioethanol production from sweet potato residue.

Sweet potato residue (SPR) is the by-product of starch extraction from fresh sweet potatoes and is rich in carbohydrates, making it a suitable substrate for bioethanol production. An amylolytic industrial yeast strain with co-expressing α-amylase and glucoamylase genes would combine enzyme production, SPR hydrolysis, and glucose fermentation into a one-step process. This consolidated bioprocessing (CBP) shows great application potential in the economic production of bioethanol. In this study, a convenient heterologous gene integration method was developed. Eight copies of a Talaromyces emersonii α-amylase expression cassette and eight copies of a Saccharomycopsis fibuligera glucoamylase expression cassette were integrated into the genome of industrial diploid Saccharomyces cerevisiae strain 1974. The resulting recombinant strains exhibited clear transparent zones in the iodine starch plates, and SDS-PAGE analysis indicated that α-amylase and glucoamylase were secreted into the culture medium. Enzymatic activity analysis demonstrated that the optimal temperature for α-amylase and glucoamylase was 60-70°C, and the pH optima for α-amylase and glucoamylase was 4.0 and 5.0, respectively. Initially, soluble corn starch with a concentration of 100 g/L was initially used to evaluate the ethanol production capability of recombinant amylolytic S. cerevisiae strains. After 7 days of CBP fermentation, the α-amylase-expressing strain 1974-temA and the glucoamylase-expressing strain 1974-GA produced 33.03 and 28.37 g/L ethanol, respectively. However, the 1974-GA-temA strain, which expressed α-amylase and glucoamylase, produced 42.22 g/L ethanol, corresponding to 70.59% of the theoretical yield. Subsequently, fermentation was conducted using the amylolytic strain 1974-GA-temA without the addition of exogenous α-amylase and glucoamylase, which resulted in the production of 32.15 g/L ethanol with an ethanol yield of 0.30 g/g. The addition of 20% glucoamylase (60 U/g SPR) increased ethanol concentration to 50.55 g/L, corresponding to a theoretical yield of 93.23%, which was comparable to the ethanol production observed with the addition of 100% α-amylase and glucoamylase. The recombinant amylolytic strains constructed in this study will facilitate the advancement of CBP fermentation of SPR for the production of bioethanol.