Strain For Industrial Production Research Articles

Polygenic Analysis in Absence of Major Effector ATF1 Unveils Novel Components in Yeast Flavor Ester Biosynthesis.

Flavor production in yeast fermentation is of paramount importance for industrial production of alcoholic beverages. Although major enzymes of flavor compound biosynthesis have been identified, few specific mutations responsible for strain diversity in flavor production are known. The ATF1-encoded alcohol acetyl coenzyme A (acetyl-CoA) transferase (AATase) is responsible for the majority of acetate ester biosynthesis, but other components affecting strain diversity remain unknown. We have performed parallel polygenic analysis of low production of ethyl acetate, a compound with an undesirable solvent-like off-flavor, in strains with and without deletion of ATF1 We identified two unique causative mutations, eat1K179fs and snf8E148*, not present in any other sequenced yeast strain and responsible for most ethyl acetate produced in absence of ATF1EAT1 encodes a putative mitochondrial ethanol acetyl-CoA transferase (EATase) and its overexpression, but not that of EAT1K179fs , and strongly increases ethyl acetate without affecting other flavor acetate esters. Unexpectedly, a higher level of acetate esters (including ethyl acetate) was produced when eat1K179fs was present together with ATF1 in the same strain, suggesting that the Eat1 and Atf1 enzymes are intertwined. On the other hand, introduction of snf8E148* lowered ethyl acetate levels also in the presence of ATF1, and it affected other aroma compounds, growth, and fermentation as well. Engineering of snf8E148* in three industrial yeast strains (for production of wine, saké, and ale beer) and fermentation in an application-relevant medium showed a high but strain-dependent potential for flavor enhancement. Our work has identified EAT1 and SNF8 as new genetic elements determining ethyl acetate production diversity in yeast strains.IMPORTANCE Basic research with laboratory strains of the yeast Saccharomyces cerevisiae has identified the structural genes of most metabolic enzymes, as well as genes encoding major regulators of metabolism. On the other hand, more recent work on polygenic analysis of yeast biodiversity in natural and industrial yeast strains is revealing novel components of yeast metabolism. A major example is the metabolism of flavor compounds, a particularly important property of industrial yeast strains used for the production of alcoholic beverages. In this work, we have performed polygenic analysis of production of ethyl acetate, an important off-flavor compound in beer and other alcoholic beverages. To increase the chances of identifying novel components, we have used in parallel a wild-type strain and a strain with a deletion of ATF1 encoding the main enzyme of acetate ester biosynthesis. This revealed a new structural gene, EAT1, encoding a putative mitochondrial enzyme, which was recently identified as an ethanol acetyl-CoA transferase in another yeast species. We also identified a novel regulatory gene, SNF8, which has not previously been linked to flavor production. Our results show that polygenic analysis of metabolic traits in the absence of major effector genes can reveal novel structural and regulatory genes. The mutant alleles identified can be used to affect the flavor profile in industrial yeast strains for production of alcoholic beverages in more subtle ways than by deletion or overexpression of the already known major effector genes and without significantly altering other industrially important traits. The effect of the novel variants was dependent on the genetic background, with a highly desirable outcome in the flavor profile of an ale brewing yeast.

Purification, characterization and gene identification of a membrane-bound glucose dehydrogenase from 2-keto-d-gluconic acid industrial producing strain Pseudomonas plecoglossicida JUIM01

The membrane-bound glucose dehydrogenase (mGDH) is a rate-limiting enzyme for the industrial production of 2-keto-d-gluconic acid (2KGA) from glucose. In this study, mGDH was firstly purified from a 2KGA industrial producing strain Pseudomonas plecoglossicida JUIM01. The purified mGDH exhibited a specific activity of 16.85 U/mg and was identified as monomeric membrane-bound PQQ-dependent dehydrogenase with a molecular mass of ~87 kDa. The Km and Vmax value of d-glucose were 0.042 mM and 14.620 μM/min, and the optimal pH and temperature were of 6.0 and 35 °C with favorable acid resistance and poor heat tolerance. Ca2+/Mg2+ showed a significantly positive effect on mGDH activity with 20% increase, whereas EDTA/EGTA had a negative influence, and Ca2+ was essential for enzyme activity. Furthermore, a 2412 bp-length gcd was amplified by genome walking technique and heterologously expressed in Escherichia coli. Bioinformatics analysis and heterologous expression further confirmed it as a mGDH encoding gene. mGDH contained binding sites of Ca2+, cofactor PQQ and polypeptide binding sites concluded from alignment results of mGDHs from different genera. This study would lay the foundation for improving 2KGA productivity through further strain modification.

Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique.

BackgroundRalstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative.ResultsAn efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains.ConclusionsWe present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina

BackgroundArachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment.ResultsThe obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L− 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively.ConclusionsThis study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Isolation and Evaluation of Bacillus Strains for Industrial Production of 2,3-Butanediol.

Biologically produced 2,3-butanediol (2,3-BDO) has diverse industrial applications. In this study, schematic isolation and screening procedures were designed to obtain generally regarded as safe (GRAS) and efficient 2,3-BDO producers. Over 4,000 candidate strains were isolated by pretreatment and enrichment, and the isolated Bacillus strains were further screened by morphological, biochemical, and genomic analyses. The screened strains were then used to test the utilization of the most common carbon (glucose, xylose, fructose, sucrose) and nitrogen (yeast extract, corn steep liquor) sources for the economical production of 2,3-BDO. Two-stage fed-batch fermentation was finally carried out to enhance 2,3-BDO production. In consequence, a newly isolated Bacillus licheniformis GSC3102 strain produced 92.0 g/l of total 2,3-BDO with an overall productivity and yield of 1.40 g/l/h and 0.423 g/g glucose, respectively, using a cheap and abundant nitrogen source. These results strongly suggest that B. licheniformis, which is found widely in nature, can be used as a host strain for the industrial fermentative production of 2,3-BDO.

Engineering the Salt-Inducible Ectoine Promoter Region of Halomonas elongata for Protein Expression in a Unique Stabilizing Environment.

It has been firmly established that organic osmolytes (compatible solutes) of halophilic Bacteria and Archaea have positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strain Halomonas elongata accumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins. For the construction of broad hoast range vector systems with fluorescent reporter proteins, we chose the salt-inducible promoter region of the ectoine gene cluster (promA). A closer inspection of the genetic background revealed that its combination of sigma 38 (σ38) and sigma 70 (σ70) promoters was followed by a weak ribosomal binding site (RBS). This inspired a systematic approach for the construction of a promA-based vector series with a synthetic RBS region using the RBS Calculator v2.0, which resulted in a greatly improved salt-dependent expression—even in a deletion construct lacking the σ38 promoter. To expand the application range of this expression system, we looked further into the possible export of recombinant proteins into the periplasm. Both sec and tat leader sequences from H. elongata proved to be suitable for directed periplasmic transport into an extreme environment of freely selectable ionic strength.

A Novel 2-Keto-D-Gluconic Acid High-Producing Strain Arthrobacter globiformis JUIM02.

2-Keto-D-gluconic acid (2KGA) is mainly used for industrial production of erythorbic acid, a food antioxidant. In this study, a 2KGA producing strain JUIM02 was firstly identified as Arthrobacter globiformis by morphological observation and 16S rDNA sequencing. The 2KGA synthetic capacity of A. globiformis JUIM02 was evaluated by both fermentation and bioconversion, with 180g/L dextrose monohydrate as substrates, in shake flasks and 5L fermenters. For fermentation, 2KGA titer, yield, molar yield, and productivity of JUIM02 reached 159.05g/L, 0.97g/g, 90.18%, and 6.63g/L/h in 24h. For non-sterile and buffer-free bioconversion by free resting cells (~ 3.2g/L dry cell weight) of JUIM02, these data were 172.96g/L, 1.06g/g, 98.07%, and 5.41g/L/h in 32h. Moreover, JUIM02 resting cells could be repeatedly used. Resting cells stored at 4°C within 30days showed stable bioconversion capacity, with 2KGA titers ≥ 171.50g/L, yields ≥ 1.04g/g, and molar yields ≥ 97.24%. The 2KGA synthetic pathway in A. globiformis, which was rarely reported, was also speculated similar to Pseudomonas and verified preliminarily. In conclusion, A. globiformis JUIM02 is a promising 2KGA industrial-producing strain suitable for various production methods and a suitable object for 2KGA metabolism research of A. globiformis.

Identification of an industrial microalgal strain for starch production in biorefinery context: The effect of nitrogen and carbon concentration on starch accumulation

The recent trends in microalgal cultures are focused on the biorefinery of the biomass components. Some of them are not completely valorised, for example starch. Since there is a wide market for starch products in food and non-food industries, the exploitation of microalgal starch fractions could improve the economic sustainability of microalgae production. In this perspective, the optimization of nitrogen and carbon source uptake for starch accumulation is a critical point for reducing the nitrogen requirement footprint and to increase CO2 capture. In this study, four robust microalgal strains, already known as starch-accumulating strain, were investigated: Chlorella sorokiniana, Scenedesmus vacuolatus, Dunaliella tertiolecta, and Tetraselmis chuii. C. sorokiniana was selected as the best starch producer in the biorefinery context, and the role nitrogen and CO2 concentration had on the starch production was investigated. For light irradiance of 300μmolm−2s−1 the optimal nitrogen concentration for growth and starch accumulation resulted 32mgL−1. The CO2 concentration clearly does not influence the starch accumulation, but concentrations distant from 2% negatively influence microalgal growth, affecting the final starch productivity. The biomass composition during the batch growth of C. sorokiniana was also analysed in order to explicitly characterise the dynamic of starch accumulation during the different growth phases. Protein content decreased during N-depletion, carbohydrates were mainly produced during the early N-depletion, followed by the accumulation of lipids in the late depletion.

Engineering Bacillus megaterium for production of functional intracellular materials

BackgroundOver the last 10–15 years, a technology has been developed to engineer bacterial poly(3-hydroxybutyrate) (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli, which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals.ResultsIn this study, we have bioengineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled PHB production in B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40–50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads.ConclusionsThis study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.

Transcriptome changes in adaptive evolution of xylose-fermenting industrial Saccharomyces cerevisiae strains with δ-integration of different xylA genes.

It is of utmost importance to construct industrial xylose-fermenting Saccharomyces cerevisiae strains for lignocellulosic bioethanol production. In this study, two xylose isomerase-based industrial S. cerevisiae strains, O7 and P5, were constructed by δ-integration of the xylose isomerase (XI) gene xylA from the fungus Orpinomyces sp. and from the bacterium Prevotella ruminicola, respectively. The xylose consumption of the strains O7 and P5 at 48-h fermentation was 17.71 and 26.10g/L, respectively, in synthetic medium with xylose as the sole sugar source. Adaptive evolution further improved the xylose fermentation capacity of the two strains to 51.0 and 28.9% in average, respectively. The transcriptomes of these two strains before and after evolution were analyzed using RNA-Seq. The expression levels of the genes involved in cell integrity, non-optimal sugar utilization, and stress response to environment were significantly up-regulated after evolution and did not depend on the origin of xylA; the expression levels of the genes involved in transmembrane transport, rRNA processing, cytoplasmic translation, and other processes were down-regulated. The expression of genes involved in central carbon metabolism was fine-tuned after the evolution. The analysis of transcription factors (TFs) indicated that most of the genes with significant differential expression were regulated by the TFs related to cell division, DNA damage response, or non-optimal carbon source utilization. The results of this study could provide valuable references for the construction of efficient xylose-fermenting XI strains.

Bioremediation of lignocellulosic wastes of food industries by Aspergillus flavus as food and feed additive protein by solid-state fermentation process

The lignocellulosic wastes produced in food industries are suitable raw materials for the production of biological products. In this study, the solid state fermentation of Aspergillus flavus on lignocellulosic wastes was evaluated for microbial protein production. The fraction of the full factorial method was applied for experiment design and process optimization. The results analysis was performed through signal to noise statistical index using the Taguchi approach via Qualitek-4 software. Glycine, ammonium sulfate and iron sulfate concentration as well as temperature were considered as effective parameters. The maximum biomass concentration of 45.7 g/kg containing 55.75% (w/w) pure protein was obtained at optimal conditions including 0.5, 0.02, and 2 g/kg of ammonium sulfate, iron sulfate and glycine, respectively, at 25 °C. Ammonium sulfate (33.78% (w/w) contribution) and culture temperature (31.98% contribution) were evaluated as the most effective factors on biomass and microbial protein production. The highest interaction occurred between ammonium sulfate and glycine with an interaction severity index of 50.03%. The low deviation of 3.94% was determined between optimum theoretical biomass concentration (43.9 g/kg) and the experimentally measured one (45.7 g/kg). Due to the high protein content of 55.75% (w/w), Aspergillus flavus was introduced as a suitable strain for industrial protein production.

Molecular evolution of acetohydroxyacid synthase in bacteria.

Acetohydroxyacid synthase (AHAS) is the key enzyme in the biosynthetic pathways of branched chain amino acids in bacteria. Since it does not exist in animal and plant cells, AHAS is an attractive target for developing antimicrobials and herbicides. In some bacteria, there is a single copy of AHAS, while in others there are multiple copies. Therefore, it is necessary to investigate the origin and evolutionary pathway of various AHASs in bacteria. In this study, all the available protein sequences of AHAS in bacteria were investigated, and an evolutionary model of AHAS in bacteria is proposed, according to gene structure, organization and phylogeny. Multiple copies of AHAS in some bacteria might be evolved from the single copy of AHAS, the ancestor. Gene duplication, domain deletion and horizontal gene transfer might occur during the evolution of this enzyme. The results show the biological significance of AHAS, help to understand the functions of various AHASs in bacteria, and would be useful for developing industrial production strains of branched chain amino acids or novel antimicrobials.

GlyA gene knock-out in Escherichia coli enhances L-serine production without glycine addition

In E. coli, glyA encodes for serine hydroxymethyltransferase (SHMT), which converts L-serine to glycine. When engineering L-serine-producing strains, it is therefore favorable to inactivate glyA to prevent L-serine degradation. However, most glyA knockout strains exhibit slow cell growth because of the resulting lack of glycine and C1 units. To overcome this problem, we overexpressed the gcvTHP genes of the glycine cleavage system (GCV), to increase the C1 supply before glyA was knocked out. Subsequently, the kbl and tdh genes were overexpressed to provide additional glycine via the L-threonine degradation pathway, thus restoring normal cell growth independent of glycine addition. Finally, the plasmid pPK10 was introduced to overexpress pgk, serA Δ197 , serC and serB, and the resulting strain E4G2 (pPK10) accumulated 266.3 mg/L of L-serine in a semi-defined medium without adding glycine, which was 3.18-fold higher than the production achieved by the control strain E3 (pPK10). This strategy can accordingly be applied to disrupt the L-serine degradation pathway in industrial production strains without causing negative side-effects, ultimately making L-serine production more efficient.

Developing elite Neurospora crassa strains for cellulosic ethanol production using fungal breeding

The demand for renewable and sustainable energy has generated considerable interest in the conversion of cellulosic biomass into liquid fuels such as ethanol using a filamentous fungus. While attempts have been made to study cellulose metabolism through the use of knock-out mutants, there have been no systematic effort to characterize natural variation for cellulose metabolism in ecotypes adapted to different habitats. Here, we characterized natural variation in saccharification of cellulose and fermentation in 73 ecotypes and 89 laboratory strains of the model fungus Neurospora crassa. We observed significant variation in both traits among natural and laboratory generated populations, with some elite strains performing better than the reference strain. In the F1 population N345, 15% of the population outperformed both parents with the top performing strain having 10% improvement in ethanol production. These results suggest that natural alleles can be exploited through fungal breeding for developing elite industrial strains for bioethanol production.

Engineering Halomonas bluephagenesis TD01 for non-sterile production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), short as P(3HB-co-4HB), was successfully produced by engineered Halomonas bluephagenesis TD01 grown in glucose and γ-butyrolactone under open non-sterile conditions. Gene orfZ encoding 4HB-CoA transferase of Clostridium kluyveri was integrated into the genome to achieve P(3HB-co-4HB) accumulation comparable to that of strains encoding orfZ on plasmids. Fed-batch cultivations conducted in 1-L and 7-L fermentors, respectively, resulted in over 70g/L cell dry weight (CDW) containing 63% P(3HB-co-12mol% 4HB) after 48h under non-sterile conditions. The processes were further scaled up in a 1000-L pilot fermentor to reach 83g/L CDW containing 61% P(3HB-co-16mol% 4HB) in 48h, with a productivity of 1.04g/L/h, again, under non-sterile conditions. The elastic P(3HB-co-16mol% 4HB) shows an elongation at break of 1022±43%. Results demonstrate that the engineered Halomonas bluephagenesis TD01 is a suitable industrial strain for large scale production under open non-sterile conditions.

Improvement of lincomycin production by mutant selection and metabolic regulation

ABSTRACTLincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136 u/ml under the optimal condition as compared to original strain S. lincolnensis 07–5 (6634 u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.

Improvement of paromomycin production by Streptomyces rimosus subsp paromomycinus NRRL 2455 using gamma irradiation mutagenesis

Streptomyces(S.) rimosus NRRL 2455 produces paromomycin, a 2-deoxystreptamine aminocyclitol aminoglycoside antibiotic (2DOS-ACAGA) with broad-spectrum activity against most of the Gram-positive and Gram-negative bacteria as well as protozoa. The mutation has become one of the beneficial methods used in enhancing the microbial production. Improvement of the paromomycin production by S. rimosus NRRL 2455 was achieved via irradiation mutagenesis using gamma (ˠ) radiation. The culture of S. rimosus was irradiated with different doses of gamma radiation (3, 4 and 5 KiloGray (KGy) to find out the best dose for the mutation that gave 99.99% killing. The optimum dose was found to be 4 KGy. Six morphologically changed colony types appeared on tryptic soy agar plates. These colonies were bio-assayed for their antimicrobial activity against standard Staphylococcus aureus ATCC 25923 using agar well diffusion technique. A mutant coded 5M showed about 1.44, and 2 fold increase in its activity as compared with the wild-type when cultivated in basal culture or optimized media (soybean meal 30 g/L, NH4CL 4 g/L, CaCO3 5 g/L and glycerol 40 ml/L), respectively. Moreover, high genetic stability was observed upon subsequent culturing of 5M-mutant. Therefore, S. rimosus mutant-5M can be used as a potential industrial strain for paromomycin production.

Engineering Escherichia coli BL21 genome to improve the heptanoic acid tolerance by using CRISPR-Cas9 system

Acid tolerance is one of the critical factors to determine the quality of the industrial production strains. Therefore, we have investigated the introduction of the acid tolerance genes into the genome of Escherichia coli BL21 by using CRISPR-Cas9 system. The dsrA and rcsB genes of E. coli K-12, which are involved in the heptanoic acid tolerance, were inserted into the genome of E. coli BL21 without scar. The native transcription unit (TU) of dsrA and the synthetic TU of rcsB were integrated in E. coli BL21 genome. We found that the position of genomic coordinate of 1,300,270 was more efficient to integrate dsrA and rcsB than genomic coordinate of 3,876,428. Furthermore, the rcsB was successfully expressed in the resulting engineered strains (i.e., rcsB+ or dsrA+rcsB+ strains). The engineered strains expressing dsrA and/or rcsB showed the higher survival rate and specific growth rate under n-heptanoic acid stress than wild-type E. coli BL21. These results indicate that the newly introduced acid-tolerance systems were active in the E. coli BL21 strain.

English

This work aimed to develop an efficient L-leucine industrial production strain of Corynebacterium glutamicum by using metabolic engineering. A recombinant C. glutamicum strain was constructed by expressing a feedback-resistant leuA-encoded 2-isopropylmalate synthase (IPMS) that carries three amino acid exchanges (R529H, G532D and L535V) from the mutant strain C. glutamicum ML1-9 which was obtained by screening for structural analogues. In order to improve the expression of IPMS, a strong promoter (tac promoter) was used to ensure efficient expression of the rate-limiting enzyme. In addition, reasonable metabolic modifications on the central carbon metabolic pathway and competitive metabolic pathways to optimize the L-leucine biosynthesis pathway by redistribution of various types of precursors and repression of negative regulation were used aimed for increased L-leucine production. The modifications involved (1) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (2) deletion of the gene encoding the AlaT to decrease the concentration of extracellular L-alanine, and increased availability of pyruvate for L-leucine formation, (3) deletion of the gene encoding the threonine dehydratase to abolish L-isoleucine synthesis and to eliminate the intermediate precursor of L-isoleucine biosynthesis competing with L-leucine biosynthesis, (4) inactivation of the pantothenate synthetase to increase α- ketoisovalerate formation, and to enable its further conversion to L-leucine, and (5) inactivation of lactate dehydrogenase to decrease lactate production and its pyruvate consumption, concomitant to decreased glucose consumption rates and prevention of lactic acid to restrict cell growth. The production performance of the engineered strain MDLeu-19/pZ8-1leuAr was characterized with cultivations in a bioreactor. Under fed-batch conditions in a 50-L automated fermentor, the best producer strain accumulated 38.1 g L-1 of L-leucine; the molar product yield being 0.42 mol L-leucine per mole of glucose (glucose conversion rate attained 26.4%). Moreover, during large-scale fermentation using a 150-m3 fermentor, this strain produced more than 37.5 g L-1 L-leucine and the glucose conversion rate was 25.8%, making this process potentially viable for industrial production. Key words: Corynebacterium glutamicum, L-leucine, metabolic engineering, fermentation, industrial production.

Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi

BackgroundClustered regularly interspaced short palindromic repeats interference (CRISPRi) has provided an efficient approach for targeted gene inhibition. A non-model microorganism Halomonas species TD01 has been developed as a promising industrial producer of polyhydroxyalkanoates (PHA), a family of biodegradable polyesters accumulated by bacteria as a carbon and energy reserve compound. A controllable gene repression system, such as CRISPRi, is needed for Halomonas sp. TD01 to regulate its gene expression levels.ResultsFor the first time CRISPRi was successfully used in Halomonas sp. TD01 to repress expression of ftsZ gene encoding bacterial fission ring formation protein, leading to an elongated cell morphology with typical filamentous shape similar to phenomenon observed with Escherichia coli. CRISPRi was employed to regulate expressions of prpC gene encoding 2-methylcitrate synthase for regulating 3-hydroxyvalerate monomer ratio in PHBV copolymers of 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV). Percentages of HV in PHBV copolymers were controllable ranging from less than 1 to 13%. Furthermore, repressions on gltA gene encoding citrate synthase channeled more acetyl-CoA from the tricarboxylic acid (TCA) cycle to poly(3-hydroxybutyrate) (PHB) synthesis. The PHB accumulation by Halomonas sp. TD01 with its gltA gene repressed in various intensities via CRISPRi was increased by approximately 8% compared with the wild type control containing the CRISPRi vector without target.ConclusionsIt has now been confirmed that the CRISPRi system can be applied to Halomonas sp. TD01, a promising industrial strain for production of various PHA and chemicals under open and continuous fermentation process conditions. In details, the CRISPRi system was successfully designed in this study to target genes of ftsZ, prpC and gltA, achieving longer cell sizes, channeling more substrates to PHBV and PHB synthesis, respectively. CRISPRi can be expected to use for more metabolic engineering applications in non-model organisms.